Biomedical subjects

D L Boyle

Publications and source records attributed to D L Boyle.

16 recordsLinked to original sources

PubMed1998

What proportion of common diagnostic tests appear redundant?

PURPOSE: To identify ancillary tests for which there are criteria defining the earliest interval at which a repeat test might be indicated, to determine how often each test is repeated earlier than these intervals and, if repeated, provides useful information. SUBJECTS AND METHODS: We performed a retrospective cohort study of 6,007 adults discharged from a large teaching hospital during a 3-month period in 1991. We measured the proportion of commonly performed diagnostic tests that were redundant, and their associated charges. RESULTS: Of the 6,007 patients discharged, 5,289 (88%) had at least one of 12 target tests performed. Overall, 78,798 of the target tests were performed during the study period, of which 22,237 (28%) were repeated earlier than test-specific predefined intervals. This percentage varied substantially by test (range, 2% to 62%). To assess how many early repeats were justified, we performed chart reviews in a random sample stratified by test. For two tests, nearly all the initial results in the sample were abnormal, and all repeats were considered justified. Of early repeats following a normal initial result for the remaining 10 tests, chart review found no clinical indication for 92%, and a weighted mean of 40% appeared redundant. Overall, 8.6% of these 10 tests appeared redundant; if these were not performed, the annual charge reductions would be $930,000 at our hospital, although the impact on costs would be much smaller. CONCLUSIONS: For some tests, an important proportion are repeated too early to provide useful clinical information. Most such tests might be eliminated using computerized reminder systems.

Adult↗

PubMed1998

Regulation of synoviocyte proliferation, apoptosis, and invasion by the p53 tumor suppressor gene.

Recent studies show that 1) the p53 tumor suppressor protein is overexpressed by rheumatoid arthritis (RA) synovium and fibroblast-like synoviocytes (FLS) and 2) somatic mutations previously identified in human tumors are present in RA synovium and FLS. We have hypothesized that abnormalities in p53 can contribute to chronic destructive RA synovitis. To understand the functional consequences of p53 abnormalities in FLS, RA and normal FLS expressing wild-type p53 were transduced with a retroviral vector encoding the human papilloma virus 18 E6 gene, which inactivates endogenous p53 protein. Three RA and one normal FLS lines were infected with recombinant retrovirus encoding the neomycin resistance gene (neo) or E6+neo. FLS proliferation, apoptosis, and invasion was studied in E6, neo, and uninfected parental strains (PS). The growth rate for E6 was significantly increased with a sixfold increase in cell number after 7 days compared with a twofold to threefold increase in neo and PS. When FLS were treated with cytokines, proliferative response of E6, neo, and PS to interleukin-1 and transforming growth factor-beta were similar. However, response to platelet-derived growth factor was significantly greater in E6 FLS compared with neo or PS. Apoptosis was studied by incubating FLS with sodium nitroprusside as a source of nitric oxide or hydrogen peroxide for 8 hours and examining DNA fragmentation and E6 cells were significantly less susceptible to cell death. In addition, E6 FLS were more invasive into cartilage extracts than neo or PS using an in vitro cell invasion assay. These data suggest that p53 is a critical regulator of FLS proliferation, apoptosis, and invasiveness. Abnormalities of p53 function might contribute to synovial lining expansion and joint destruction in RA.

Apoptosis↗

PubMed1998

Direct synovial gene transfer with retroviral vectors in rat adjuvant arthritis.

OBJECTIVE: To evaluate the feasibility of direct in vivo gene transfer in an animal model of arthritis using a retroviral vector. METHODS: The timing and dose of retroviral vector was examined using very high titer retroviral vector (> or = 10(9) CFU) in rat adjuvant arthritis. Retroviral vector expressing beta-galactosidase (beta-gal) or vehicle alone was injected into the right ankle of rats with adjuvant arthritis. Ankles were injected either on Day 7 (pre-arthritis), Day 10 (early arthritis), Day 15 (accelerating arthritis), or Day 28 (chronic arthritis) after adjuvant immunization. Joints were harvested 3 days later and extracts were assayed for beta-gal activity. RESULTS: Synovial beta-gal expression was minimal in the Day 7 group and elevated in the Day 10, Day 15, and Day 28 groups. Gene transfer with retroviral vector did not exacerbate the local inflammatory response. Minimal or no beta-gal expression was observed in the contralateral uninjected paw or in the spleen, lung, liver, and kidneys. Frozen sections of retroviral vector injected joints were stained with X-gal and revealed transduced cells in the lining and superficial sublining layers. To determine the longevity of gene expression, ankle joints were injected with vector on Day 15 post-adjuvant, harvested, and assayed for beta-gal activity for up to 49 days after injection. Expression of the enzyme peaked from Day 3 to 7 and was still readily detected up to 49 days after retrovirus infection. CONCLUSION: This is the first report of successful direct in vivo gene transfer in the rat adjuvant arthritis model using a retroviral vector. Appropriate timing of administration and very high titer retroviral vector preparations are key determinants of adequate gene transduction.

Animals↗

PubMed1997

Adenosine inhibits neutrophil degranulation in activated human whole blood: involvement of adenosine A2 and A3 receptors.

Adenosine, acting via A2 receptors, is a potent inhibitor of neutrophil oxidative burst, but its effects and mechanisms of action on neutrophil degranulation have been less well characterized. We, therefore, investigated the effects of adenosine and its receptor-specific analogues on neutrophil degranulation in stimulated human whole blood. Adenosine dose-dependently inhibited the LPS- and TNF-alpha-induced release of the azurophilic granule proteins bactericidal/permeability-increasing protein, elastase, and defensins to approximately the same extent, with a maximum inhibition of 70 to 80% and an IC50 ranging from 14 to 24 microM. The inhibitory effects of adenosine were partially blocked by the A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine, the A1/A2 antagonist 8(p-sulfophenyl)theophyline, and the A1/A3 antagonist xanthine amine congener, but not by the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine. The highly selective A3 agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide and the nonselective agonist 2-chloroadenosine reduced degranulation more potently than the A1 agonist N6-cyclopentyladenosine. The inhibitory effects of N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide and 2-chloroadenosine were strongly reversed by xanthine amine congener, but were not affected by 8(p-sulfophenyl)theophyline. In addition, the adenosine kinase inhibitor GP515 attenuated degranulation via an adenosine-mediated mechanism. These data indicate that adenosine acts via A2 as well as A3 receptors to inhibit neutrophil degranulation and add to the anti-inflammatory potential of adenosine and adenosine-regulating agents in neutrophil-mediated tissue injury.

Adenosine↗

PubMed1997

Posttranscriptional regulation of collagenase-1 gene expression in synoviocytes by adenosine receptor stimulation.

OBJECTIVE: To characterize the transcriptional and posttranscriptional regulation of collagenase-1 by adenosine receptor stimulation in interleukin-1 (IL-1)-stimulated fibroblast-like synoviocytes (FLS). METHODS: FLS were stimulated with IL-1 and either the nonselective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) or the adenylate cyclase activator forskolin. Electrophoretic mobility shift assays were performed to determine AP-1 and cAMP-responsive element binding protein (CREB) activation. Transcriptional activation was determined by transfecting HS68 dermal fibroblasts with a collagenase-chloramphenicol acetyltransferase construct. Finally, collagenase messenger RNA (mRNA) half-life was determined by activating cells in the presence of IL-1, IL-1 + NECA, or IL-1 + forskolin and culturing cells in the presence of actinomycin D. RESULTS: NECA and forskolin had no effect on AP-1 activation, c-jun or c-fos gene expression, or CREB phosphorylation. IL-1 markedly increased collagenase promoter activity, and neither NECA nor forskolin blocked this action. Studies of mRNA half-life showed that both NECA and forskolin decreased the half-life of collagenase mRNA in IL-1-stimulated FLS and HS68 cells. CONCLUSION: The findings of this study demonstrate that NECA and forskolin decrease collagenase gene expression in FLS and dermal fibroblasts due to enhanced mRNA degradation.

Adenosine↗

PubMed1997

Confocal microscopy of cataracts from animal model systems: relevance to human nuclear cataract.

A recent study demonstrated that cytosolic lipid membrane structures, independent of the plasma membrane, preferentially occurred in human cataractous lenses. Animal model systems of cataractogenesis (selenite treated rats: galactose fed rats; buthionine-sulfoxime treated mice; Emory mice) were screened for possible relevant structures using the lipid membrane probe DiI and confocal microscopy. Well delineated plasma membranes of lens fiber cells with independent cytosolic staining structures were only observed in the selenite model system. These cytosolic structures were not observed in aged matched control lenses or within the transparent cortical regions of selenite treated animals with intense nuclear opacification. These results suggested that the morphological changes in DiI staining structures seen in the nucleus of the human cataractous lens were best approximated by those seen in the selenite model system.

Animals↗

PubMed1997

Confocal microscopy of human lens membranes in aged normal and nuclear cataracts.

PURPOSE: To visualize the structure and determine the continuity of lipid membranes in lens fiber cells (LFCs) from human aged normal and cataractous lenses. METHODS: Thick sections from human nuclear cataracts and aged normal lenses were stained with the lipophilic probe DiI, and then analyzed by confocal microscopy. Staining patterns of membranes were observed in individual optical sections or three-dimensional projections of z-series taken in longitudinal section and cross-section of LFCs from different regions within the lens nucleus. RESULTS: DiI bound to and delineated the plasma membrane of LFCs from all regions of the lens nucleus. Three-dimensional projections of z-series from aged normal and cataractous lenses suggested that some of the stained lipid membranes were not continuous with LFC plasma membrane of cataractous lenses. CONCLUSIONS: The results obtained using these methods demonstrated that lipid membranes, discontinuous with the plasma membrane of LFCs, were indicative of a novel process occurring predominately in cataractous human lenses.

Aged↗

PubMed1996

cDNA cloning and characterization of A3i, an alternatively spliced rat A3 adenosine receptor variant.

A cDNA encoding variant form of the A3 adenosine (Ado) receptor was isolated from rat by reverse transcription of brain mRNA followed by PCR. The full-length receptor (A3i) cDNA encodes 337 amino acids and shares complete sequence identity with the rat A3 Ado receptor, except for the presence of a seventeen amino acid insert located in the second intracellular domain. In contrast to the rat A3 receptor, stable expression of A3i in CHO cells resulted in poor coupling to Gi proteins. Analysis of receptor transcripts by RT-PCR suggests that the A3 Ado receptor mRNAs are products of alternative splicing. Sequence analysis of A3 genomic DNA identified a 1.7 kb intron that is likely alternatively spliced to produce the A3 and A3i receptors.

Adenosine↗

PubMed1996

Inhibition of synoviocyte collagenase gene expression by adenosine receptor stimulation.

OBJECTIVE: To characterize the regulation of matrix metalloproteinases (MMPs) by adenosine. METHODS: Cultured fibroblast-like synoviocytes (FLS) were stimulated with interleukin-1 (IL-1) in the presence or absence of adenosine receptor agonists. Immunoreactive MMPs were measured using specific enzyme-linked immunosorbent assays, and gene expression was assessed by Northern blot analysis. RESULTS: The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) decreased collagenase production by IL-1-stimulated synoviocytes from 196 +/- 28 ng/ml (mean +/- SEM) to 66 +/- 9 ng/ml (P < 0.001). There was minimal effect on stromelysin production (decrease from 107 +/- 16 ng/ml to 97 +/- 15 ng/ml). Selective adenosine receptor agonists implicated the A2b adenosine receptor in this activity, and reverse transcriptase-polymerase chain reaction studies confirmed that FLS express this receptor. Northern blot analysis demonstrated that the mechanism of action was pre-translational since NECA decreased collagenase, but not stromelysin or tissue inhibitor of metalloproteinases 1 (TIMP-1), messenger RNA levels. Cyclic AMP levels were increased by NECA, and a direct adenylate cyclase activator (forskolin) also suppressed collagenase gene expression. These data suggest that cAMP mediates the inhibitory effect of NECA on collagenase production. CONCLUSION: Stimulation of the A2b receptor on FLS decreases collagenase gene expression, with little or no effect on stromelysin and TIMP-1. The combination of antiinflammatory and MMP-regulating properties of adenosine or adenosine-regulating agents suggest that treatment based on this approach might be useful in rheumatoid arthritis.

Adenosine↗

PubMed1996

EM immunolocalization of alpha-crystallins: association with the plasma membrane from normal and cataractous human lenses.

PURPOSE: To integrate past biochemical findings with past morphological observations of area insoluble material isolated from cataract and aged normal lenses, by determining the spatial distribution of alpha-crystallins associated with the plasma membrane (PM) of nuclear cataractous and age matched normal human lenses. METHODS: Lenses were homogenized, pelleted and washed several times in 0.05M Tris-Cl (pH 7.2) containing 100mM KCl, 1 mM MgCl2 and 2mM beta-mercaptoethanol, followed by several washes in 8M urea. Urea insoluble pellets (UIP) were labeled before fixation and embedding with rabbit serum raised against alpha-crystallins, followed by goat anti-rabbit IgG conjugated to 5nm gold. Approximately 300 gold particles associated with the PM were counted, for each lens, on several electron microscopy (EM) micrographs. The number of gold particles/um of PM, number of individual vs clusters of gold particles were determined. RESULTS: Micrographs from both normal and cataractous human lenses clearly demonstrated the association of alpha-crystallins with the PM. Also apparent was the abundant labeling of the PM for cataractous lenses as compared to normal lenses. Quantification of the gold labeling revealed that not only was there an increase in the amount of labeling/um of PM in cataract lenses, but there was also an increased percentage of gold in clusters. These clusters were not only more numerous in cataractous lenses, but also contained a greater number of gold/cluster. CONCLUSIONS: These findings provide morphological evidence that the PM in nuclear cataract lenses is associated with large aggregates of alpha-crystallin.

Aged↗

PubMed1996

Apoptosis in rheumatoid arthritis: p53 overexpression in rheumatoid arthritis synovium.

DNA damage induces p53 tumor suppressor gene expression and protein production, which in turn facilitates DNA repair or apoptosis. Wild-type p53 protein has a short half-life, so it is rarely detected in non-neoplastic tissue. Because DNA fragmentation is abundant in the intimal lining in rheumatoid arthritis (RA) synovial tissue (ST) using in situ end-labeling (Firestein GS, Yeo M, Zvaifler NJ: Apoptosis in rheumatoid arthritis synovium. J Clin Invest 1995, 96:1631-1638), we assessed ST p53 expression. Immunohistochemical analysis of fixed RA synovium using antibody PAb 1801 showed prominent p53 staining in the cytoplasm and nuclei of intimal lining cells. Noninflammatory and osteoarthritis (OA) ST had significantly less p53 in the lining. These data were confirmed by Western blot analysis of ST extracts, with abundant p53 found in RA compared with OA. p53 expression in cultured fibroblast-like synoviocytes (FLS) was then examined. Flow cytometry on permeabilized cells showed that RA FLS constitutively express p53 protein. Western blots showed that RA FLS expressed significantly more p53 than either OA FLS or dermal fibroblasts. Immunohistochemistry of FLS cultured in chamber slides localized the p53 to the cytoplasm of most resting FLS, with nuclear staining in only 10.7 +/- 2.4%. Exposure to hydrogen peroxide for increased nuclear staining to 70.7 +/- 12.8% after 8 hours (P = 0.003). These data indicate that p53 is overexpressed in RA ST in the intimal lining, which is the primary site of DNA damage, and is constitutively expressed by FLS.

Aged↗

PubMed1995

Relationship between medication errors and adverse drug events.

OBJECTIVE: To evaluate the frequency of medication errors using a multidisciplinary approach, to classify these errors by type, and to determine how often medication errors are associated with adverse drug events (ADEs) and potential ADEs. DESIGN: Medication errors were detected using self-report by pharmacists, nurse review of all patient charts, and review of all medication sheets. Incidents that were thought to represent ADEs or potential ADEs were identified through spontaneous reporting from nursing or pharmacy personnel, solicited reporting from nurses, and daily chart review by the study nurse. Incidents were subsequently classified by two independent reviewers as ADEs or potential ADEs. SETTING: Three medical units at an urban tertiary care hospital. PATIENTS: A cohort of 379 consecutive admissions during a 51-day period (1,704 patient-days). INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Over the study period, 10,070 medication orders were written, and 530 medications errors were identified (5.3 errors/100 orders), for a mean of 0.3 medication errors per patient-day, or 1.4 per admission. Of the medication errors, 53% involved at least one missing dose of a medication; 15% involved other dose errors, 8% frequency errors, and 5% route errors. During the same period, 25 ADEs and 35 potential ADEs were found. Of the 25 ADEs, five (20%) were associated with medication errors; all were judged preventable. Thus, five of 530 medication errors (0.9%) resulted in ADEs. Physician computer order entry could have prevented 84% of non-missing dose medication errors, 86% of potential ADEs, and 60% of preventable ADEs. CONCLUSIONS: Medication errors are common, although relatively few result in ADEs. However, those that do are preventable, many through physician computer order entry.

Boston↗

PubMed1994

Mechanisms of methotrexate action in rheumatoid arthritis. Selective decrease in synovial collagenase gene expression.

OBJECTIVE: To measure the effect of methotrexate (MTX) treatment in rheumatoid arthritis (RA) on the expression of synovial collagenase, stromelysin, and tissue inhibitor of metalloproteinase 1 (TIMP-1) gene expression in a prospective study. METHODS: Serial percutaneous synovial biopsies (pretreatment and after 3-4 months) were performed on the knees of 8 patients (7 with RA, 1 with seronegative arthritis) who were beginning oral MTX therapy. Synovial gene expression was determined by quantitative in situ hybridization using computer-assisted image analysis. RESULTS: After therapy, patients had decreased joint counts, morning stiffness, and erythrocyte sedimentation rates. Synovial inflammation in the biopsy tissues was slightly decreased after therapy. In situ hybridization on pretreatment and posttreatment frozen sections was performed to quantify synovial messenger RNA (mRNA) levels. Collagenase gene expression significantly decreased after MTX therapy (P = 0.006) even though cell density in the region was unchanged. TIMP-1 and stromelysin mRNA levels were not changed by MTX therapy. To study the mechanism of MTX action in vitro, MTX-treated and control fibroblast-like synoviocytes were stimulated with interleukin-1 beta (IL-1 beta). MTX did not alter collagenase or TIMP-1 mRNA levels after IL-1 exposure. CONCLUSION: MTX therapy decreases collagenase gene expression but not TIMP-1 or stromelysin gene expression in the synovium. This action is probably an indirect effect due to an alteration in the synovial cytokine milieu, rather than a direct effect on gene expression.

Adult↗

PubMed1994

Synovial interleukin-1 receptor antagonist and interleukin-1 balance in rheumatoid arthritis.

OBJECTIVE: To quantify interleukin-1 receptor antagonist (IL-1ra) and IL-1 production and gene expression by rheumatoid arthritis (RA) synovial tissue (ST) cells. METHODS: IL-1 alpha, IL-1 beta, and IL-1ra protein levels were measured by enzyme-linked immunosorbent assay in fresh and cultured ST cells, purified synovial macrophages, and fibroblast-like synoviocytes (FLS). The relative expression of the secreted form of IL-1ra (sIL-1ra) and the alternatively spliced intracellular form (icIL-1ra) was determined by reverse transcription polymerase chain reaction (RT-PCR) techniques. RESULTS: IL-1 alpha, IL-1 beta, and IL-1ra were present in fresh and cultured ST cell samples of synovium from RA and osteoarthritis patients. IL-1ra:IL-1 ratios ranged from 1.2 to 3.6, which is below the 10-100-fold excess of IL-1ra needed to inhibit IL-1 bioactivity. Isolated CD14+ synovial macrophages secreted IL-1ra, but the amount was much less than that of alveolar or in vitro-derived macrophages. Cultured FLS contained intracellular IL-1ra but secreted little IL-1ra into the culture supernatants. RT-PCR showed that icIL-1ra mRNA was more abundant than sIL-1ra mRNA in FLS and unfractionated ST cells. CONCLUSION: IL-1ra production by RA ST cells is deficient relative to total production of IL-1.

Arthritis, Rheumatoid↗

PubMed1994

Impact of computerized physician order entry on physician time.

We examined the effect of computerized physician order entry on housestaff time use patterns, using time motion techniques. For both medical and surgical house officers, writing orders on the computer took about twice as long (p < 0.001), or 44 minutes for medical and 73 minutes for surgical house officers. Medical house officers recovered about half this time because some administrative tasks--e.g. looking for charts--were made easier. Within types of orders, sets of stereotyped orders took much less time with order entry, but one-time orders took longer. We have since developed strategies to make it easier to enter one-time orders.

Hospital Information Systems↗

PubMed1990

The effect of swainsonine on the phagocytosis of rod outer segments by rat RPE.

From studies using inhibitors such as tunicamycin and castanospermine, it has been suggested that plasma membrane glycoproteins may function as receptors in the phagocytosis of rod outer segments (ROS) by the retinal pigment epithelium (RPE). The exact structure of the oligosaccharide side chain of the glycoprotein may not be critical for this process. We have employed another inhibitor, swainsonine, which inhibits mannosidase II, a terminal enzyme in the protein glycosylation pathway, which results in membrane glycoproteins having hybrid-type oligosaccharide chains and fewer complex oligosaccharide chains. We have examined the ability of cultured rat RPE explants to phagocytize fluorescein isothiocyanate (FITC) labelled ROS or latex beads in the presence and absence of swainsonine. A significant (p less than 0.05) reduction in the phagocytosis of FITC-ROS was found between the swainsonine treated (37.7 +/- 4.1%) and untreated (85.4 +/- 2.7%) RPE explants. The nonspecific uptake of latex beads in both swainsonine treated (85.3 +/- 2.4%) and untreated (89.3 +/- 2.0%) RPE explants indicate that the RPE cells retained their ability to phagocytize. Major differences in spectrophotometric analysis of WGA-stained blots were an absence of a peak at 201 kD, a doublet at 86 kD and an overall reduction in all peak absorbances in the swainsonine treatments as compared to the untreated controls. These results suggest that the alterations in RPE glycoprotein formation due to swainsonine alter the ability of RPE to phagocytize ROS.

Alkaloids↗