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Biomedical subjects

Richard A Ward

Publications and source records attributed to Richard A Ward.

At least 19 recordsLinked to original sources
PubMed2007

The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets.

A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared with plasma membrane, there was less actin associated with secretory vesicles (SV, 75%), gelatinase granules (GG, 40%), specific granules (SG, 10%), and azurophil granules (AG, 5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, with flow cytometry, and GG exocytosis was measured as gelatinase release with an ELISA. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated exocytosis of SV, GG, and SG with an ED(50) of 15, 31, and 28 nM, respectively, with maximal response at 10(-7) M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC(50) for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis.

Actins↗
PubMed2006

Dialyzer and machine technologies: application of recent advances to clinical practice.

Although hemodialysis is a mature therapy, a growing population of patients with more complex medical problems and limitations on resources will require technological innovations to improve the safety, reliability and efficiency of the therapy. The past several years have seen design changes to dialyzers that have provided incremental improvements in small solute clearance and more substantial improvements in the clearance of large solutes. New functions have been added to dialysis machines that help ensure reliable delivery of the dialysis prescription and enable full advantage to be taken of improvements in dialyzer clearance of large molecules. In addition, feedback control systems have been developed that may help reduce the untoward side effects which many patients experience during hemodialysis. Whether or not a particular innovation enters routine clinical use will depend on demonstrating that it improves clinical outcomes, its cost, and, in some cases, on a more enlightened approach by regulatory authorities.

Dialysis Solutions↗
PubMed2006

Proteomic analysis of human neutrophils.

Proteomics is the study of the set of proteins, or proteome, expressed by a cell under specific conditions. Proteomics methodology consists of protein extraction, protein separation, and protein identification. Currently, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser-desorption ionization time of flight mass spectrometry are the most widespread methods for proteomic studies. The recent introduction of precast immobilized pH gradient gel strips, precast gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and well-designed electrophoresis equipment has made 2DE a highly reproducible and relatively simple method for protein separation. Inherent limitations of the procedure, however, require approaches in sample preparation that may be cell- or tissue-dependent. This chapter describes a methodology for proteomic analysis of human neutrophils and discusses its applications.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2005

Structure-based virtual screening for low molecular weight chemical starting points for dipeptidyl peptidase IV inhibitors.

Structure-based virtual screening was performed against the target dipeptidyl peptidase IV (DPP-IV) to identify good chemical starting points for medicinal chemistry. A database of available compounds was filtered by calculated physical properties and undesired chemistry. This database was matched against two in-house designed DPP-IV pharmacophores, and the hits from these pharmacophore searches were docked into a DPP-IV crystal structure. Compounds were then selected for testing and 51 active compounds were identified from a list of 4000 compounds tested. These had activities ranging from 30% to 82% when tested at a concentration of 30 microM in an enzyme inhibition assay.

Algorithms↗
PubMed2005

Proteomic analysis of human neutrophil granules.

Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining the proteome of neutrophil granule subsets provides a basis for understanding the role of exocytosis in neutrophil biology. Additionally, the described methods may be applied to mobilizable compartments of other secretory cells.

Actins↗
PubMed2005

Protein-leaking membranes for hemodialysis: a new class of membranes in search of an application?

A new class of membranes that leak protein has been developed for hemodialysis. These membranes provide greater clearances of low molecular weight proteins and small protein-bound solutes than do conventional high-flux dialysis membranes but at the cost of some albumin loss into the dialysate. Protein-leaking membranes have been used in a small number of clinical trials. The results of these trials suggest that protein-leaking membranes improve anemia correction, decrease plasma total homocysteine concentrations, and reduce plasma concentrations of glycosylated and oxidized proteins. However, it is not clear yet that routine use of protein-leaking membranes is warranted. Specific uremic toxins that are removed by protein-leaking membranes but not conventional high-flux membranes have not been identified. It is also unclear whether protein-leaking membranes offer benefits beyond those obtained with conventional high-flux membranes used in convective therapies, such as hemofiltration and hemodiafiltration. Finally, the amount of albumin loss that can be tolerated by hemodialysis patients in a long-term therapy has yet to be determined. Protein-leaking membranes offer a new approach to improving outcomes in hemodialysis, but whether their benefits will outweigh their disadvantages will require more basic and clinical research.

Albumins↗
PubMed2005

Myeloid-related protein-14 is a p38 MAPK substrate in human neutrophils.

The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [(32)P]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr(113). MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [(32)P]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.

Amino Acid Sequence↗
PubMed2005

Dialysis water as a determinant of the adequacy of dialysis.

Hemodialysis patients are exposed to large volumes of water in the form of dialysate. Contaminants from the dialysate may cross the dialyzer membrane into the blood and have the potential to compromise the adequacy of dialysis. Several chemicals found commonly in drinking water have long been known to be toxic to hemodialysis patients. More recently, it has become apparent that even low levels of bacterial products in dialysate may adversely impact dialysis adequacy through their ability to stimulate an inflammatory response. Minimum levels of water and dialysate quality have been recommended to protect patients from chemical and microbiologic contaminants. Complying with these recommendations requires an appropriately designed water purification and distribution system, combined with a surveillance program designed to maintain dialysate quality.

Dialysis Solutions↗
PubMed2004

Methylglyoxal: a stimulus to neutrophil oxygen radical production in chronic renal failure?

BACKGROUND: Chronic renal failure is characterized by oxidant stress, resulting in part from increased reactive oxygen species production by neutrophils. Plasma concentrations of methylglyoxal are increased in uraemia. Methylglyoxal activates p38 mitogen-activated protein kinase (MAPK) in endothelial cells. Activation of p38 MAPK in neutrophils enhances reactive oxygen species production through exocytosis of intracellular storage granules. We tested the hypothesis that methylglyoxal enhances reactive oxygen species production by activating p38 MAPK in neutrophils. METHODS: Neutrophils were exposed to methylglyoxal in vitro. Activation of p38 MAPK was determined by immunoblot analysis. Exocytosis was determined by measuring plasma membrane expression of CD35 and CD66b, specific markers of secretory vesicles and specific granules, respectively. Reactive oxygen species production was determined by measuring H(2)O(2) and O(-)(2) production. RESULTS: Methylglyoxal activated p38 MAPK and caused dose-dependent increases in CD35 and CD66b expression, which were blocked by the methylglyoxal scavenger, aminoguanidine, or the p38 MAPK inhibitor, SB203580. Methylglyoxal caused dose-dependent increases in basal and Staphylococcus aureus-stimulated H(2)O(2) production and basal and formyl-methionyl-leucyl-phenylalanine-stimulated O(-)(2) production. Enhancement of reactive oxygen species production was blocked by aminoguanidine and SB203580. CONCLUSIONS: Methylglyoxal enhances reactive oxygen species production in neutrophils through a process involving p38 MAPK-dependent exocytosis of intracellular storage granules. These findings, together with the observation that methylglyoxal concentrations are increased in renal failure, suggest a possible role for methylglyoxal as a uraemic toxin that contributes to the oxidant stress associated with renal failure.

Exocytosis↗
PubMed2003

Effects of high-flux hemodialysis on oxidant stress.

BACKGROUND: Neutrophil oxygen radical production is increased in end-stage renal disease (ESRD) patients and it is further enhanced during dialysis with low-flux cellulosic membranes. This increased oxygen radical production may contribute to the protein and lipid oxidation observed in ESRD patients. We tested the hypothesis that high-flux hemodialysis does not increase oxygen radical production and that it is not associated with protein oxidation. METHODS: Neutrophil oxygen radical production was measured during dialysis with high-flux dialyzers containing polysulfone and cellulose triacetate membranes. Free sulfhydryl and carbonyl groups and advanced oxidation protein products were measured to assess plasma protein oxidation. RESULTS: Pre-dialysis, neutrophil oxygen radical production was significantly greater than normal and increased significantly as blood passed through the dialyzer in the first 30 minutes of dialysis. Post-dialysis, however, neutrophil oxygen radical production had decreased and was not different from normal. Pre-dialysis, significant plasma protein oxidation was evident from reduced free sulfhydryl groups, increased carbonyl groups, and increased advanced oxidation protein products. Post-dialysis, plasma protein free sulfhydryl groups had increased to normal levels, while plasma protein carbonyl groups increased slightly, and advanced oxidation protein products remained unchanged. CONCLUSIONS: The results of this study show that neutrophil oxygen radical production normalizes during high-flux dialysis, despite a transient increase early in dialysis. This decrease in oxygen radical production is associated with an improvement in some, but not all, measures of protein oxidation.

Blood Proteins↗
PubMed2003

Impact of bleach cleaning on the performance of dialyzers with polysulfone membranes processed for reuse using peracetic Acid.

Dialyzer performance may change with reuse depending on whether or not the dialyzer is cleaned with bleach. Bleach is usually used in conjunction with formaldehyde as the germicide. Because few data are available for dialyzers cleaned with bleach and disinfected with peracetic acid, we examined dialyzer performance in a cross-over study of dialyzers containing polysulfone membranes reprocessed using bleach and peracetic acid or peracetic acid alone. Each dialyzer was used for a total of 16 treatments, or until it failed standard criteria for continued use. Urea, beta2-microglobulin, and albumin removal were determined during the first, second, seventh, and 16th use of each dialyzer. Urea removal did not differ between the two reprocessing methods and did not change with reuse. Overall, beta2-microglobulin removal remained unchanged in dialyzers reprocessed with peracetic acid alone, but tended to increase after the seventh use in dialyzers reprocessed with bleach and peracetic acid. Approximately 60% of beta2-microglobulin removal resulted from trapping of beta2-microglobulin at the dialyzer membrane. Albumin loss into the dialysate was clinically insignificant throughout the study with both reprocessing methods. These data show that the clearance of both small and large molecules by dialyzers containing polysulfone membranes is well maintained by reprocessing with peracetic acid and that additional cleaning with bleach has limited impact on performance.

Adult↗
PubMed2003

Oxidant stress in hemodialysis patients: what are the determining factors?

Oxidant stress contributes to morbidity in hemodialysis patients. Three possible causes of oxidant stress have been suggested: the uremic state, the dialyzer membrane, and bacterial contaminants from the dialysate. Oxidant stress occurs in uremia before dialysis therapy is initiated, as evidenced by increased production of reactive oxygen species, increased levels of oxidized plasma proteins and lipids, and decreased antioxidant defenses. It has been proposed that increased production of reactive oxygen species during hemodialysis is also an important contributor to oxidant stress. Hemodialysis is associated with a transient increase in production of reactive oxygen species, particularly with cellulose membranes. In addition, surveys have shown widespread contamination of dialysate by endotoxin, which may cross membranes and prime production of reactive oxygen species by phagocytic cells. Recent studies, however, show a decrease in protein oxidation from pre- to post-dialysis and a normalization of neutrophil reactive oxygen species production. Taken together, these data suggest that uremia, per se, is the most important cause of oxidant stress in hemodialysis patients. Dialysate quality may also contribute to oxidant stress, but evidence that the dialyzer membrane plays a role is weak.

Cardiovascular Diseases↗
PubMed2000

A comparison of on-line hemodiafiltration and high-flux hemodialysis: a prospective clinical study.

Some of the morbidity associated with chronic hemodialysis is thought to result from retention of large molecular weight solutes that are poorly removed by diffusion in conventional hemodialysis. Hemodiafiltration combines convective and diffusive solute removal in a single therapy. The hypothesis that hemodiafiltration provides better solute removal than high-flux hemodialysis was tested in a prospective, randomized clinical trial. Patients were randomized to either on-line postdilution hemodiafiltration or high-flux hemodialysis. The groups did not differ in body size, treatment time, blood flow rate, or net fluid removal. The filtration volume in hemodiafiltration was 21 +/-1 L. Therapy prescriptions were unchanged for a 12-mo study period. Removal of both small (urea and creatinine) and large (ss(2)-microglobulin and complement factor D) solutes was significantly greater for hemodiafiltration than for high-flux hemodialysis. The increased urea and creatinine removal did not result in lower pretreatment serum concentrations in the hemodiafiltration group. Pretreatment plasma beta(2)-microglobulin concentrations decreased with time (P< 0.001); however, the decrease was similar for both therapies (P = 0.317). Pretreatment plasma complement factor D concentrations also decreased with time (P<0.001), and the decrease was significantly greater with hemodiafiltration than with high-flux hemodialysis (P = 0.010). The conclusion is that on-line hemodiafiltration provides superior solute removal to high-flux hemodialysis over a wide molecular weight range. The improved removal may not result in lower pretreatment plasma concentrations, however, possibly because of limitations in mass transfer rates within the body.

Anemia↗
PubMedDate not supplied

Water treatment for hemodialysis: ensuring patient safety.

Patient safety has become an important focus of the Institute of Medicine and the medical community. Although hemodialysis is a routine therapy, it is nonetheless a complex procedure where errors can occur. In particular, errors related to water quality can lead to patient injury and to increased medical costs. Using the Institute of Medicine report on errors in medicine as a basis, this article discusses previously published incidents of patient injury related to water quality in terms of the types of errors that occurred. Epidemiologic techniques provide a framework to identify, correct, and possibly avert these types of errors in the future. While the ultimate responsibility for ensuring water quality rests with the medical director of the hemodialysis unit, patient safety should be a concern of all members of the nephrology community.

Humans↗
PubMedDate not supplied

Ultrapure dialysate.

To prevent pyrogenic reactions during hemodialysis, it is recommended that bacteria and endotoxin in dialysate not exceed 100-200 colony forming units (CFU)/ml and 0.25-2 endotoxin units (EU)/ml, respectively. While these limits are adequate to prevent acute pyrogenic reactions, data are accumulating to suggest they may not prevent stimulation of chronic inflammation in hemodialysis patients. Fragments of endotoxin and other bacterial products capable of stimulating immune cells cross low-flux and high-flux membranes in vitro. In clinical studies, use of ultrapure dialysate (bacteria < 0.1 CFU/ml and endotoxin < 0.03 EU/ml) is associated with lower concentrations of inflammatory markers and acute phase reactants than are observed with dialysate meeting current quality recommendations. Moreover, observational studies suggest a link between clinical outcomes and dialysate purity. Treatment of patients with ultrapure dialysate is reported to improve nutritional status, increase responsiveness to erythropoietin, slow the decline in residual renal function, lessen cardiovascular morbidity, and decrease the incidence of beta(2)-microglobulin amyloidosis. To date, however, none of these studies has shown a cause-and-effect relationship between dialysate purity and outcome. Further, there are no data defining the concentration dependence of outcomes on dialysate purity and the relative importance of dialysate purity as a trigger of inflammation remains unclear. While the technology exists to routinely provide ultrapure dialysate, controlled clinical trials are still needed to answer the question of whether or not introducing ultrapure dialysate into routine clinical practice represents an efficient use of limited resources in terms of decreasing inflammation and improving outcomes in hemodialysis patients.

Bacteria↗