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Biomedical subjects

K Harper

Publications and source records attributed to K Harper.

At least 19 recordsLinked to original sources
PubMed2004

Rapid screening method for detection of bacteria in platelet concentrates.

Public awareness has long focused on the risks of the transmission of viral agents through blood product transfusion. This risk, however, pales in comparison to the less publicized danger associated with the transfusion of blood products contaminated with bacteria, in particular, platelet concentrates. Up to 1,000 cases of clinical sepsis after the transfusion of platelet concentrates are reported annually in the United States. The condition is characterized by acute reaction symptoms and the rapid onset of septicemia and carries a 20 to 40% mortality rate. The urgent need for a method for the routine screening of platelet concentrates to improve patient safety has long been recognized. We describe the development of a rapid and highly sensitive method for screening for bacteria in platelet concentrates for transfusion. No culture period is required; and the entire procedure, from the time of sampling to the time that the final result is obtained, takes less than 90 min. The method involves three basic stages: the selective removal of platelets by filtration following activation with a monoclonal antibody, DNA-specific fluorescent labeling of bacteria, and concentration of the bacteria on a membrane surface for enumeration by solid-phase cytometry. The method offers a universal means of detection of live, nondividing, or dead gram-negative and gram-positive bacteria in complex cellular blood products. The sensitivity is higher than those of the culture-based methods available at present, with a detection limit of 10 to 10(2) CFU/ml, depending upon the bacterial strain.

Bacteria↗
PubMed2003

Effect of raloxifene on bone mineral density and biochemical markers of bone turnover in Japanese postmenopausal women with osteoporosis: results from a randomized placebo-controlled trial.

The safety and efficacy of raloxifene, a selective estrogen receptor modulator (SERM), has been studied extensively in large, global clinical trials. However, the effect of raloxifene on bone mineral density (BMD) and on biochemical markers of bone turnover in Japanese postmenopausal women with osteoporosis has not been rigorously evaluated. This study was designed to assess the safety and efficacy of raloxifene in Japanese postmenopausal women with osteoporosis following 1 year of therapy. Participants in this multicenter trial were randomly assigned to receive placebo, raloxifene 60 mg/day (RLX60), or raloxifene 120 mg/day (RLX120). Lumbar spine BMD was measured at baseline, 24, 40, and 52 weeks, and biochemical markers of bone turnover were assessed at baseline, 12, 24, and 52 weeks. Serum lipids were assessed at baseline, 12, 24, 40, and 52 weeks, and breast examinations and transvaginal ultrasound of the endometrium were performed at enrollment and 52 weeks. Compared with baseline, women taking RLX60 had significant increases in lumbar spine (L2-L4) BMD at 24 weeks (+3.3%, p<0.001) through 52 weeks (+3.5%, p<0.001) of therapy, and similar results were observed in the RLX120 group. Markers of bone turnover and total cholesterol and LDL-C were significantly reduced, and no significant treatment-group difference was observed for patients reporting at least one adverse event following randomization. In addition, there were no reported venous thromboembolic events (VTE) in any treatment group. The results of this study demonstrate that raloxifene is associated with early increases in lumbar spine BMD, has favorable effects on biochemical markers of bone turnover and lipid profile, and is well tolerated in postmenopausal Japanese women.

Aged↗
PubMed2001

Effect of raloxifene on serum triglycerides in postmenopausal women: influence of predisposing factors for hypertriglyceridemia.

BACKGROUND: Estrogen increases serum triglyceride (TG) levels and induces hypertriglyceridemia in susceptible women. The effect of raloxifene (RLX), a selective estrogen-receptor modulator, on serum TG has not been studied in detail. OBJECTIVE: The purpose of this study was to examine the effect of RLX on serum TG levels in postmenopausal women with and without osteoporosis, including those with predisposing factors for hypertriglyceridemia. METHODS: Fasting serum TG levels were assessed over 36 months in 2738 osteoporotic postmenopausal women (mean age, 67 years) assigned to placebo or RLX (60 or 120 mg/d) in an osteoporosis treatment trial and over 24 months in 1318 postmenopausal women without osteoporosis (mean age, 54 years) assigned to placebo or RLX (60 or 150 mg/d) in 3 osteoporosis prevention trials. RESULTS: In the osteoporosis treatment trial, the median serum TG concentration decreased in all groups, but significantly more in the placebo group (placebo, -3.4%; RLX 60 mg/d, -1.4%; RLX 120 mg/d, -1.3%; P = 0.002). In the osteoporosis prevention trials, the percentage change in median serum TG concentration was not significantly different among treatments (P = 0.22). Among women with varying degrees of hypertriglyceridemia at baseline (>2.82, >3.39, and >4.51 mmol/L), the median serum TG level at the end of the study decreased from baseline in all groups, with no significant differences among treatments (P > or = 0.13). The effect of RLX on serum TG level was not influenced by age, smoking status, use of alcohol, or presence of diabetes (P > or = 0.10 for all interactions). Among women in the highest tertile of body mass index (>26.4 kg/m2), RLX increased serum TG levels significantly compared with placebo (placebo, -3%; RLX 60 mg/d, 6%: RLX 120 mg/d, 4%; P < 0.05); the absolute increase from baseline with RLX in this subgroup was 0.05 mmol/L (4.4 mg/dL). CONCLUSIONS: RLX did not increase serum TG in postmenopausal women overall or among women with elevated TG levels or evidence of diabetes at baseline. TG levels increased slightly but statistically significantly in women in the upper tertile of body mass index who were treated with RLX.

Adult↗
PubMed2000

Turbo STIR magnetic resonance imaging as a whole-body screening tool for metastases in patients with breast carcinoma: preliminary clinical experience.

This study was undertaken to assess the utility of whole-body turbo short tau inversion recovery (STIR) magnetic resonance imaging (MRI) to detect metastases to liver, brain, and bone as a single examination in women with breast cancer. Seventeen patients with biopsy-proven breast cancer and suspected metastatic disease attending over a 12-month period referred for both conventional imaging and whole-body MRI were included in the study. Three patients were found to be free of metastases at both conventional and MR imaging. Appendicular or axial skeletal metastases were identified in 11 of 17 patients, with correlation between findings at whole-body MRI and scintigraphy in 15 of the 17 patients. Five patients had evidence of hepatic metastases on whole-body MRI, of which metastases were identified in only three patients at CT despite contrast enhancement. Four patients had brain abnormalities (metastases in three patients, meningioma in one patient) detected on both whole-body and dedicated brain MRI. Preliminary clinical experience suggests that turbo STIR whole-body MRI may represent a convenient and cost-effective method of total body screening for metastases in patients with breast carcinoma.

Adult↗
PubMed2000

Growth hormone for in vitro fertilization.

BACKGROUND: Ovulation induction protocols for in vitro fertilization (IVF) are constantly under review and revision in an attempt to decrease gonadotropin requirements while improving follicular recruitment and pregnancy rate. Most studies of the effect of Growth Hormone on ovulation induction have investigated normally ovulating infertile women. Attention has begun to turn to its effect on women who fail to produce an optimal number of follicles in response to ovulation induction. OBJECTIVES: To assess the effectiveness of growth hormone adjuvant therapy for women undergoing ovulation induction prior to IVF in two patient groups: a) those with no previous history of poor response and b) those with a history of poor response. SEARCH STRATEGY: This review has drawn on the search strategy developed for the Subfertility Group as a whole. Relevant trials were identified in the Group's Specialised Register of Controlled Trials. See Review Group details for more information. SELECTION CRITERIA: All RCTs were included if they addressed the research question posed and provided outcome data for intervention and control subjects. Outcomes of interest included pregnancy, oocyte/embryo number and peak estradiol level. DATA COLLECTION AND ANALYSIS DATA EXTRACTION: A diverse search strategy was employed, including hand-search of 43 core journals from 1966 to the present, bibliographies of relevant trials, MEDLINE database, abstracts from North American and European meetings and contact with authors of relevant papers. Relevant data were extracted independently by two reviewers using the standardized data extraction sheet. Validity was assessed in terms of method of randomization, completeness of follow-up, presence or absence of crossover and co-intervention. DATA SYNTHESIS: 2x2 tables were generated for all relevant outcomes. Odds ratios were generated using the Peto modified Mantel-Haenszel technique. Statistical heterogeneity was assessed using x2. MAIN RESULTS: In women with no previous history of poor stimulation, the common odds ratio for pregnancy per cycle was 0.97 (95% CI 0.34-2. 76) Total dose of gonadotropin and number of oocytes retrieved were similar between treated and placebo groups. In previous poor responders, the common odds ratio for pregnancy per cycle instituted was 2.55 (95% CI 0.64-10.12). Again, no statistically significant difference was noted in gonadotropin dosage or oocyte number obtained, when all studies were considered. REVIEWER'S CONCLUSIONS: Data from 6 small trials suggest that in women with no previous poor response, GH augmentation does not improve the rate of pregnancy. In poor responders, a trend towards improved outcome with GH treatment deserves further study.

Female↗
PubMed2000

Celecoxib does not significantly alter the pharmacokinetics or hypoprothrombinemic effect of warfarin in healthy subjects.

The objective of this study was to determine the effects of celecoxib, an anti-inflammatory/analgesic agent that primarily inhibits COX-2 and not COX-1 at therapeutic doses, on the steady-state pharmacokinetic profile and hypoprothrombinemic effect of racemic warfarin in healthy volunteers. Twenty-four healthy adult volunteers on maintenance doses of racemic warfarin (2-5 mg daily), stabilized to prothrombin times (PT) 1.2 to 1.7 times pretreatment PT values for 3 consecutive days, were randomized to receive concomitant celecoxib (200 mg bid) or placebo for 7 days in an open-label, multiple-dose, randomized, placebo-controlled, parallel-group study of warfarin pharmacokinetics and PT. Steady-state exposure of S- and R-warfarin (area under the curve [AUC]) and maximum plasma concentration (Cmax) in subjects receiving celecoxib were within 2% to 8% of the warfarin AUC and Cmax in subjects receiving placebo during the concomitant treatment period. In addition, PT values were not significantly different in subjects receiving warfarin and celecoxib concomitantly compared with subjects receiving warfarin and placebo. In conclusion, concomitant administration of celecoxib has no significant effect on PT or steady-state pharmacokinetics of S- or R-warfarin in healthy volunteers.

Adult↗
PubMed1999

pSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins.

The vector pSKAP/S was constructed to enable overexpression of single-chain variable fragment antibody (scFv)-alkaline phosphatase fusion proteins. In pSKAP/S, the scFv were genetically fused to the mutated Escherichia coli PhoA/S gene that encodes an alkaline phosphatase with increased specific activity. The restriction sites incorporated into pSKAP/S allowed the scFv genes to be easily transferred from pUC119-derived phagemid vectors that are used frequently in phage display antibody library technology. Strong transcriptional control of expression was achieved using the tetracycline promoter, and induction of different individual clones with anhydrotetracycline resulted in secretion of most of the scFv-alkaline phosphatase fusion proteins into the culture medium. Although some of the clones secreted fusion proteins that were retained in the periplasm, these proteins could be isolated with a simple extraction procedure. Increased amounts of a scFv-alkaline phosphatase fusion protein were obtained when expressed in the pSKAP/S vector compared with expression in a vector incorporating the lac promoter. Testing for binding of the scFv-alkaline phosphatase fusion proteins to antigen was possible in an ELISA without the need for additional enzyme-conjugated antibodies. The pSKAP/S vector was successfully used to obtain scFv fragments from a preparation of phage-antibody clones after subcloning and expression of individual clones as scFv-alkaline phosphatase fusions, whereas fewer clones (and clones with different properties) were obtained from the same phage-antibody preparations when expressed as soluble scFv fragments. Therefore, the pSKAP/S vector was shown to be useful in extending the range of scFv obtained from phage display libraries.

Alkaline Phosphatase↗
PubMed1999

Properties of a panel of single chain variable fragments against Potato leafroll virus obtained from two phage display libraries.

Twelve single chain variable fragment (scFv) antibodies that bind to particles of Potato leafroll virus (PLRV) were obtained from two naive phage display libraries. Phages were selected against PLRV particles or dissociated PLRV particles immobilised onto tubes. Individual PLRV-binding scFv were identified by ELISA, after their expression either fused to the surface of phage particles, or as soluble scFv (scFv-c-myc), or as scFv-alkaline phosphatase fusion proteins (scFv-AP), obtained by subcloning into pSKAP/S. These procedures resulted in the isolation of scFv with different properties. For example, some of the scFv reacted strongly with virus particles but not with dissociated capsid protein, which suggests that they had reacted with discontinuous epitopes. Others reacted with dissociated capsid proteins and SDS-denatured protein, which suggests that they had reacted with continuous epitopes. ScFv were also subcloned into pC(L) for expression as fusion proteins with human kappa constant region (scFv-C(L)). Expression of these constructs in Escherichia coli yielded 0.2-1 mg protein per litre of bacterial culture. The different scFv fusion proteins were evaluated in ELISA to detect PLRV in leaf extracts of Physalis floridana. Absorbance values obtained with the fusion proteins were greater than those obtained with the scFv-c-myc, and were similar to those obtained in assays done using monoclonal or polyclonal antibodies.

Alkaline Phosphatase↗
PubMed1998

Serological characterization of the 3'-proximal encoded proteins of beet yellows closterovirus.

The 3'-proximal open reading frames (ORFs) of beet yellows closterovirus, California isolate (BYV-CA), were sequenced and the expression of the corresponding proteins analyzed. The nucleotide sequence of ORF 5 (coding for p24) was most conserved compared with ORF 7 (coding for p20) and ORF 8 (coding for p21) among the isolates analyzed. Polyclonal antisera were produced to GST fusion proteins of p24, p20, and p21. Accumulation of p24, CP, p20 and p21 was studied in infected Tetragonia expansa plants and Chenopodium quinoa protoplasts. All four proteins were expressed in all tissues (old leaves, young leaves and stems), and most abundantly in young leaves. The subcellular localization of each protein in different tissues showed that compared with p24, CP and p21, p20 accumulated less in transfected protoplasts. Immunogold labeling in sugarbeet with p24 and CP antisera demonstrated co-localization of p24 and CP in vascular petiole tissues. In infectivity neutralization tests, antisera against p24 and CP greatly reduced transmission of BYV by viruliferous aphids compared with viruliferous aphids fed on preimmune serum or antiserum to p21.

Amino Acid Sequence↗
PubMed1997

A scFv-alkaline phosphatase fusion protein which detects potato leafroll luteovirus in plant extracts by ELISA.

A single chain Fv antibody fragment (scFv) was obtained from a synthetic phage-antibody library after four rounds of selection against purified preparations of potato leafroll luteovirus (PLRV). Nucleotide sequence analysis showed that the scFv belongs to the human V(H)3 family. DNA encoding the scFv was sub-cloned into pDAP2 such that a scFv-alkaline phosphatase fusion protein was produced by transformed bacteria following induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The fusion protein was obtained at concentrations of 10 mg/l of Escherichia coli culture medium and these fusion protein preparations were used directly in ELISA to detect PLRV in sap extracts from infected plants. Our work is the first report of the selection of a scFv specific for a luteovirus from a synthetic phage-display library and the production of a fusion protein with alkaline phosphatase for the detection of PLRV in infected plants. The results demonstrate the potential of scFv and enzyme-scFv fusion proteins in routine testing for plant virus infection.

Alkaline Phosphatase↗
PubMed1997

Ongoing Y-chromosome instability defines sub-clonal variants in radiation-induced leukaemias in the mouse.

Forty primary leukaemias that arose in vivo as a consequence of 3 Gy X-irradiation of inbred mouse strains were analysed for Y-chromosome aberrations by conventional cytogenetics and fluorescent in situ hybridization (FISH). Compared with control mice which were X-irradiated but which exhibited no overt signs of leukaemia, the loss and gain of Y-chromosomes in leukaemic spleen cells defined subclonal variants in the radiation-induced haemopoietic malignancies that arose in CBA/H, DBA/2 and (C57BL/6 x DBA/2)F1 mice. This Y-chromosome instability was significantly higher than that observed in spleen cells of age-matched (or older) irradiated control mice that had not developed overt leukaemia. The detection of Y-chromosome aberrations is considered in the context of the high numbers of potential gene regulatory sequences in the murine Y-chromosome and the potential for the insertional activation of cellular genes during multi-stage radiation leukaemogenesis.

Acute Disease↗
PubMed1997

Delayed appearance of radiation-induced mutations at the Hprt locus in murine hemopoietic cells.

Earlier work in this laboratory identified high levels of non-clonal chromatid aberrations in the clonal descendants of murine hemopoietic stem cells after many cell divisions postirradiation with densely-ionizing, high linear energy transfer (LET) alpha-particles, but not with sparsely ionizing low LET X-rays. Using the Hprt locus as a marker, we have now demonstrated genomic instability in murine hemopoietic stem cells for greater than 20 cell divisions following both high and low LET irradiation. The increase in Hprt-deficient variants demonstrated following X- as well as alpha-particle and neutron irradiation indicates that there is a difference in the LET-dependence of delayed gene mutations and higher-order cytogenetic effects. Over 90% of the mutations identified arose in the expanding colonies after more than 12 cell divisions postirradiation and therefore cannot be attributed to the initial DNA damage. Such a high frequency of delayed mutations has important implications for mechanistic studies of radiation mutagenesis and for risk estimation.

Alpha Particles↗
PubMed1996

Expression of extracellular matrix molecules in human mesangial cells in response to prolonged hyperglycaemia.

Post-mitotic cultures of human mesangial cells were maintained in media containing 4-30 mM D-glucose for up to 28 days. Changes in mRNA and protein levels for specific macromolecules occurred between 7 and 14 days after initiating hyperglycaemic conditions. Slot blot analysis showed 2-3-fold increases in mRNAs for collagen type I, fibronectin, versican and perlecan, whereas mRNA for decorin was increased by up to 20-fold. Levels of mRNAs for biglycan and syndecan were unaffected by hyperglycaemic culture. Reverse transcriptase PCR (RT-PCR) confirmed that decorin mRNA levels are greatly elevated and also showed increased transcription of the TGF-beta 1 gene in hyperglycaemic cultures. Western analysis and ELISA indicated accumulations of collagen types I and III, laminin and fibronectin in the cell layers and media of hyperglycaemic cultures with increasing time. Type IV collagen did not accumulate in either compartment of hyperglycaemic mesangial cell cultures. Collagen types I, III, and fibronectin did not accumulate in the cell layers of hyperglycaemic human dermal fibroblasts, indicating a cell-specific response in mesangial cultures. Decorin and versican, but not biglycan, were increased in the hyperglycaemic mesangial cell culture media. There were no apparent changes in core proteins for decorin and biglycan in fibroblast media. Transforming growth factor beta 1 (TGF-beta 1) in hyperglycaemic mesangial cell cultures increased 5-fold after 7 days, but decreased thereafter to only approx. 2-fold after 28 days. The changes in TGF-beta 1 mRNA, as detected by RT-PCR, and protein followed one another closely.

Base Sequence↗