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Biomedical subjects

M Webb

Publications and source records attributed to M Webb.

At least 19 recordsLinked to original sources
PubMed2006

Fresh PBSC harvests, but not BM, show temperature-related loss of CD34 viability during storage and transport.

BACKGROUND: The optimum conditions for storage and transport of freshly harvested HPC in the liquid state are uncertain. It is not specified in commonly applied standards for stem cell transplantation. We used a viable CD34 assay to determine the optimum temperature for maintaining progenitor cell viability in freshly harvested BM and PBSC. Our aim was to identify standardized conditions for storage and transport of marrow or peripheral blood products that would optimize CD34 recovery, leading to better transplant outcomes. METHODS: Samples were aseptically removed from 46 fresh HPC harvests (34 PBSC and 12 BM) and stored at refrigerated temperature (2-8 degrees C), room temperature (18-24 degrees C) and 37 degrees C for up to 72 h. Samples were analyzed for viable CD34+ cells/microL at 0, 24, 48 and 72 h. RESULTS: The mean viable CD34+ yield prior to storage was 7.7 x 10(6)/kg (range 0.7-30.3). The mean loss of viable CD34+ cells in HPC products at refrigerated temperature was 9.4%, 19.4% and 28% at 24, 48 and 72 h, respectively. In contrast, the mean loss of viable CD34+ cells at room temperature was 21.9%, 30.7% and 43.3% at 24, 48 and 72 h, respectively. No viable CD34+ cells remained after storage at 37 degrees C for 24 h. Only PBSC products and not BM showed temperature-related loss of CD34 viability. Greater loss of viable CD34+ cells was observed for allogeneic PBSC compared with autologous PBSC. DISCUSSION: These results demonstrate that the optimum temperature for maintaining the viability of CD34+ cells, during overnight storage and transport of freshly harvested HPC, is 2-8 degrees C. These findings will allow the development of standard guidelines for HPC storage and transport.

Adult↗
PubMed2005

Recovery of viable CD34+ cells from cryopreserved hemopoietic progenitor cell products.

The number of CD34+ cells infused into patients at the time of autologous or allogeneic transplantation is a clinically important variable, but the viability of these cells has not been extensively documented. In this study, we analyzed the recovery of viable CD34+ cells before and after cryopreservation on 79 autologous stem cell products, using a novel flow cytometry assay without red cell lysis. For 70 PBSC harvest samples, the mean viable CD34+ cell count was 5.98 x 10(6)/kg (range 0.3-23 x 10(6)/kg) before freezing and 5.4 x 10(6)/kg (range 0.2-23 x 10(6)/kg) after thawing. The median recovery was 93% (range 48-107%), with 90% recovery for NHL (range 48-100%, n=34), 83% for multiple myeloma (range 56-106%, n=11), 92.3% for acute leukemia (range 71-100% n=7) and 94.5% for nonhematological malignancies (range 50-107% n=18). Similarly, for autologous bone marrows (n=9) the median recovery of viable CD34+ cells was 90% (range 68-100%). The recovery of viable CD34+ cells for adult (n=51) and pediatric (n=28) stem cell collections was 91 and 94%, respectively. Further examination of the correlation between the kinetics of hematological recovery and the number of viable progenitor cells infused, particularly at the lower end of the accepted dose range, may be warranted.

Antigens, CD34↗
PubMed2002

Different Mg to Fe ratios in the mixed metal MgFe hydroxy-carbonate compounds and the effect on phosphate binding compared with established phosphate binders.

Due to the side effects of the current oral phosphate binders, there is a need for effective alternatives. A number of mixed metal hydroxy-carbonate compounds (MMHCs) based on Mg and Fe have recently been established as effective phosphate binders. We have now carried out further studies on the MMHCs with different ratios of Mg(2+):Fe(3+) in different forms to assess for phosphate binding efficacy and ionic release in aqueous solution and food slurries. The compounds that provide the most promise are those with Mg(2+):Fe(3+) ratios of 2:1 and 4:1 in the unaged/dry form. Their phosphate binding efficacy was compared with a wide range of established phosphate binders, such as aluminum hydroxide [Al(OH)(3)], calcium carbonate (CaCO(3)), calcium acetate (CaAc(2)), magnesium hydroxide [Mg(OH)(2)], and lanthanum carbonate [La(2)(CO(3))(3)] in various food slurries. The results showed that the MgFe compounds were much more effective (on a weight for weight basis) than the established binders, and their properties were relatively pH independent. Calcium compounds (CaCO(3) and CaAc(2)) were ineffective under the experimental conditions. Mg(OH)(2) was effective at low pH but not at pHs greater than 5.0, and also released two- to threefold more magnesium than the MgFe compounds. Al(OH)(3) showed some degree of efficacy, but the binding capacity was, at best, less than 50% of the MMHCs. La(2)(CO(3))(3) required at least a 10-fold increase in weight to give comparable binding to the MMHCs. In conclusion, MgFe hydroxy-carbonate compounds are effective phosphate binders and may provide a better alternative to both existing and emerging binders for combating hyperphosphataemia.

Binding Sites↗
PubMed2001

Cost of hospitalization for and risk factors associated with vancomycin-resistant Enterococcus faecium infection and colonization.

The increase in costs of hospitalization for patients with drug-resistant infection may be associated with drug resistance itself or with the severity of the underlying illness that predisposes patients to acquire the drug-resistant infection. To address this issue, risk factors and cost of hospitalization were compared for patients infected or colonized with vancomycin-susceptible Enterococcus faecium (VSEF) or vancomycin-resistant E. faecium (VREF) in a large tertiary-care hospital in New York City. From January 1995 through December 1996, 157 patients with VSEF and 262 patients with VREF were identified. CMI (case-mix index) was assigned to each patient as a measure of severity of illness, with a CMI of 1 considered to represent illness of average severity. For all patients who were assigned a CMI of <3, the cost per day of hospitalization for patients with VREF was significantly greater than that for patients with VSEF. However, for patients with a CMI of >3, there was no difference between cost of hospitalization for patients with VREF and that for patients with VSEF. These observations indicate that, although vancomycin resistance is associated with an increased cost of hospitalization for less severely ill patients with VREF, patients with severe underlying illness, regardless of vancomycin resistance, incur similar hospitalization costs.

Adolescent↗
PubMed2001

Structural requirements for cooperative binding of HMG1 to DNA minicircles.

DNA minicircles, where the length of DNA is below the persistence length, are highly effective, preferred, ligands for HMG-box proteins. The proteins bind to them "structure-specifically" with affinities in the nanomolar range, presumably to an exposed widened minor groove. To understand better the basis of this preference, we have studied the binding of HMG1 (which has two tandem HMG boxes linked by a basic extension to a long acidic tail) and Drosophila HMG-D (one HMG box linked by a basic region to a short and less acidic tail), and their HMG-box domains, to 88 bp and 75 bp DNA minicircles. In some cases we see cooperative binding of two molecules to the circles. The requirements for strong cooperativity are two HMG boxes and the basic extension; the latter also appears to stabilize and constrain the complex, preventing binding of further protein molecules. HMG-D, with a single HMG box, does not bind cooperatively. In the case of HMG1, the acidic tail is not required for cooperativity and does not affect binding significantly, in contrast to a much greater effect with linear DNA, or even four-way junctions (another distorted DNA substrate). Such effects could be relevant in the hierarchy of binding of HMG-box proteins to DNA distortions in vivo, where both single-box and two-box proteins might co-exist, with or without basic extensions and acidic tails.

Allosteric Site↗
PubMed2001

Identification of the apical membrane-targeting signal of the multidrug resistance-associated protein 2 (MRP2/MOAT).

The human canalicular multispecific organic anion transporter (cMOAT), known as the multidrug resistance-associated protein 2 (MRP2), is normally expressed in the liver and to a lesser extent in the kidney proximal tubules. In these tissues MRP2 specifically localizes to the apical membrane. The construction of MRP2 fused to the green fluorescent protein, and subsequent site-directed mutagenesis enabled the identification of a targeting signal in MRP2 that is responsible for its apical localization in polarized cells. The specific apical localization of MRP2 is due to a C-terminal tail that is not present in the basolaterally targeted MRP1. Deletion of three amino acids from the C-terminal of MRP2 (DeltaMRP2) causes the protein to be localized predominantly in the basolateral membrane in polarized Madin-Darby canine kidney cells. Interestingly, MRP2 expressed in a mouse leukemia cell line (L1210 cells) predominantly accumulates intracellularly with minimal cell membrane localization. In contrast, DeltaMRP2 was shown to predominantly localize in the cell membrane in L1210 cells. Increased transport of 2,4-dinitrophenyl glutathione from L1210 cells expressing DeltaMRP2 showed that the re-targeted protein retains its normal function.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2001

Flotation of metal-loaded clay anion exchangers. Part I: the case of chromates.

Synthetic hydrotalcite-like layered materials are known for their ability to remove anions, like the chromates. These sorbents usually exist in powder form, thereby exhibiting high surface area and rapid kinetics for adsorption, but presenting appreciable problems in the subsequent solid/liquid separation process. Almost complete removals were obtained in this paper, from batchwork dispersed-air flotation in presence of a flocculant. Due to the experienced difficulty of flotation of thermally activated (at 500 degrees C) hydrotalcite metal-loaded particles, the application of various surfactants was studied. Continuous-flow laboratory runs certified also the effectiveness of this combined process of sorptive flotation, a promising innovative treatment technology.

Adsorption↗
PubMed2001

Pharmacological differences between the human and rat vanilloid receptor 1 (VR1).

Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells. Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC(50) values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90+/-0.20 microM; human=1.90+/-0.30 microM: CHO cells: rat=0.20+/-0.06 microM; human=0.19+/-0.08 microM). In CHO cell lines co-expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC(50) values for capsaicin-induced responses were similar in both cell lines (rat=0.35+/-0.06 microM, human=0.53+/-0.03 microM). The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC(50) pH 5.49+/-0.04 and pH 5.78+/-0.09, respectively). The threshold for heat activation was identical (42 degrees C) for rat and human VR1. PPAHV was an agonist at rat VR1 (EC(50) between 3 and 10 microM) but was virtually inactive at the human VR1 (EC(50)>10 microM). Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1. Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more effective at inhibiting the noxious heat response of human than of rat VR1.

Amino Acid Sequence↗
PubMed2001

Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spread.

Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, mu1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.

Adaptor Protein Complex 1↗
PubMed2001

Community based universal neonatal hearing screening by health visitors using otoacoustic emissions.

OBJECTIVES: To carry out a pilot study to test the feasibility of health visitors (HVs) performing neonatal otoacoustic emissions (OAE) hearing screening in the community using Echoport ILO288 and to evaluate its acceptability to parents and HVs. DESIGN: Prospective cohort study. SETTING: Local health centres and babies' homes in urban and rural settings in West Gloucestershire. PARTICIPANTS: Twelve HVs, 683 babies, and their parents. MAIN OUTCOME MEASURES: Coverage rate, age at testing, referral rate for formal audiology testing, and parental anxiety scores. RESULTS: Of the 683 babies registered with the study HVs, 99% (675) were tested, with a median age at first test of 18 days. Parental consent for the study was refused for six of the eight not tested. Taking a unilateral pass as a screening pass (for comparison with other studies), 4% (27/675) failed the first OAE test, and 1.9% (13/675) failed a second OAE test performed by the HV within a further two weeks and were referred for formal audiology testing. One baby (0.15%) was found to have a moderate sensorineural hearing loss on brain stem auditory evoked responses, giving a false positive rate of 1.7% (12/675). Some 18% (120/675) were tested at home, of which 80% (96/120) were combined with another planned reason for HV contact. In all, 82% (555/675) of tests were carried out in health centre clinics, of which 47% (260/555) were combined purpose visits. Mean parental anxiety scores (possible range 0-5) were 0.86, 2.27, and 3.45 before the first test, first retest, and audiology test respectively. The median time taken for one HV to complete testing was 12.2 minutes (range 3-65), compared with the 15 minutes currently allocated for two HVs to perform distraction testing. Based on the results of questionnaires, the test was very well received by parents and HVs alike. CONCLUSION: HVs are able to perform OAE testing in the neonatal period at home and in local health centre clinics. They achieve high population coverage rates and low false positive rates. Universal neonatal hearing screening by HVs using OAE testing is feasible, well received, and could be less demanding of HV time than the current distraction testing. This model of universal neonatal hearing screening should be considered by the National Screening Committee.

Attitude to Health↗
PubMed2001

The development and in-vitro evaluation of novel mixed metal hydroxy-carbonate compounds as phosphate binders.

The currently available phosphate binders are relatively inefficient and suffer from clinical side-effects of increased absorption of calcium and aluminium and the diarrhoea-inducing effects of magnesium. A new class of compounds based on mixed metal hydroxides has been developed and evaluated for their potential as phosphate binders. The mixed metal hydroxides were prepared using a standard procedure for hydrotalcite (Al2Mg6(OH)16.CO3.4H2O) by substituting Fe3+ for Al3+, with Mg2+ or Ca2+ as the divalent metal ion. Phosphate precipitation (binding) was examined at different pH values in aqueous solution and in various food mixtures in comparison with hydrotalcite, Al(OH)3, CaCO3 and Mg(OH)2 on the same weight-to-weight basis. A series of compounds with differing ratios of metal ions (Fe:Mg/Ca 1:2 or 1:3) gave analytically similar ratios to those predicted from the initial amounts added. CTFeCa bound > 90% phosphate in aqueous solution compared with 65% binding with CTFeMg, 85% binding with Mg(OH)2, and less than 30% binding for CaCO3 and Al(OH)3. The mixed metal compounds also bound up to 80% phosphate in various food matrices, which was relatively independent of changes in pH, compared with Mg(OH)2, where binding decreased from 85% at pH 3.0 to 25% at pH 8.0. Al(OH)3 and CaCO3 were relatively ineffective phosphate binders under all the conditions tested. The mixed metal hydroxides compounds show considerable promise as phosphate binders over those currently available and warrant further patient-based in-vivo testing.

Aluminum↗
PubMed2001

The evaluation of novel mixed metal hydroxy-carbonates as phosphate binders: an in-vivo study in the rat.

A number of novel phosphate binders based on mixed metal hydroxide structures incorporating Fe and Ca, or Fe and Mg (classified as CT, Crosfield test compounds), were compared with the established phosphate binders Mg(OH)2, Al(OH)3, CaCO3 and a commercial hydrotalcite (Al- and Mg-based) using a rat model. The changes in urine and soluble faecal phosphate were used to evaluate efficacy of phosphate binding. The binders were mixed into a standard rat maintenance food at a concentration of 1% (w/w). Four rats were used for each binder study group and fed over 7 days. Urine and faeces were collected (in a metabolic cage) over the last 24-h study period and the phosphate content measured. The urinary phosphate was significantly reduced (P < 0.001) with CTFeCa (72+/-44 microm), CTFeMg (13+/-4 microm), CT100 (26+/-11 microm), and Mg(OH)2 (65+/-53 microm), compared with control (766+/-188 microm), Al(OH)3 (1,256+/-279 microm), and CaCO3 (857+/-25 microm). The soluble phosphate content of the faeces was significantly reduced (P < 0.05) by up to 60 % with CTFeCa, CTFeMg and Mg(OH)2, and up to 40% with CT100 and Al(OH)3, compared with 30% in controls and 10% with CaCO3. The new mixed metal hydroxy-carbonate compounds based on FeCa or FeMg are effective phosphate binders in-vivo and warrant further testing in patients.

Animals↗
PubMed2001

Abnormal endothelial function in young African-American females: discordance with blood flow.

In this pilot study, we sought to compare the vasodilatory and hemodynamic properties of the peripheral vasculature in the forearms of young, healthy African-American females to similarly matched white females. We used high-resolution ultrasound of the brachial artery to evaluate 11 African-American females and 8 white females. When normalized to nitrate-induced dilation, endothelium-dependent dilation was reduced in young African American females compared to white females (0.6 in African American females compared to 1.0 in white females). These results indicate the need for a larger study to examine this phenomenon.

Adult↗
PubMed2000

Acute basophilic leukemia presenting with abnormal liver function tests and the absence of blast cells in the peripheral blood.

Acute basophilic leukemia is an uncommon form of acute leukemia, rarely occurring as a de novo disease. We describe a case of de novo acute basophilic leukemia occurring in a 47-year-old man who presented with abnormal liver function tests in the absence of leukemic infiltration of the liver. We postulate that this presentation occurred as a consequence of pathophysiological features of the malignant basophilic blast cells.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2000

Ultra-high throughput screen of two-million-member combinatorial compound collection in a miniaturized, 1536-well assay format.

Results of a complete survey of the more than 2-million-member Pharmacopeia compound collection in a 1536-well microvolume screening assay format are reported. A complete technology platform, enabling the performance of ultra-high throughput screening in a miniaturized 1536-well assay format, has been assembled and utilized. The platform consists of tools for performing microvolume assays, including assay plates, liquid handlers, optical imagers, and data management software. A fluorogenic screening assay for inhibition of a protease enzyme target was designed and developed using this platform. The assay was used to perform a survey screen of the Pharmacopeia compound collection for active inhibitors of the target enzyme. The results from the survey demonstrate the successful implementation of the ultra-high throughout platform for routine screening purposes. Performance of the assay in the miniaturized format is equivalent to that of a standard 96-well assay, showing the same dependence on kinetic parameters and ability to measure enzyme inhibition. The survey screen identified an active class of compounds within the Pharmacopeia compound collection. These results were confirmed using a standard 96-well assay.

Chemistry, Pharmaceutical↗