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Biomedical subjects

Stephen J Wagner

Publications and source records attributed to Stephen J Wagner.

12 recordsLinked to original sources
PubMed2006

Thiazole orange, a DNA-binding photosensitizer with flexible structure, can inactivate pathogens in red blood cell suspensions while maintaining red cell storage properties.

BACKGROUND: Development of a robust pathogen reduction system for red cells (RBCs) utilizing photosensitive dyes has been constrained by hemolysis, usually mediated by reactive oxygen species emanating from dye free in solution as well as dye bound to the RBC membrane. The RBC binding properties of thiazole orange (TO), a flexible nucleic acid intercalating cyanine dye that predominantly acts as a photosensitizer only when bound, were assessed along with its virucidal, bactericidal, and light-induced hemolytic activities. STUDY DESIGN AND METHODS: Leukodepleted 20% hematocrit RBCs suspended in Erythrosol (RAS-2) were oxygenated, inoculated with test organisms, incubated with TO, and illuminated. Control and treated samples were analyzed by appropriate assay. Identically prepared, but uncontaminated samples were phototreated, concentrated to 45% hematocrit, and assayed for potassium leakage, hemolysis, and ATP during storage. RESULTS: Approximately 21 percent TO bound to RBCs. Phototreatment inactivated from 5.4 to 7.1 log(10) of 5 tested viruses and from 2.3 to greater than 7.0 log(10) of 8 tested bacteria. Phototreated RBCs exhibited only slightly increased hemolysis, moderately elevated potassium efflux, and similar levels of ATP compared to controls. CONCLUSION: TO can photoinactivate several model viruses and pathogens in RBCs under conditions that produce limited hemolysis without the addition of quenchers or competitive inhibitors.

Anti-Bacterial Agents↗
PubMed2005

Chalcogenoxanthylium photosensitizers for the photodynamic purging of blood-borne viral and bacterial pathogens.

Thio- and selenoxanthylium dyes were prepared by the addition of 2-lithiothiophene, 4-N,N-dimethylaminophenylmagnesium bromide, and 1-naphthylmagnesium bromide to the appropriate 2,7-bis-N,N-dimethylaminochalcogenoxanthen-9-one, followed by dehydration and ion exchange to the chloride salts. The corresponding chalcogenoxanthylium dyes were evaluated as photosensitizers for the inactivation of intracellular and extracellular virus in red blood cell suspensions and for the inactivation of selected strains of gram (+) and gram (-) bacteria in red blood cell suspensions. Selected combinations of photosensitizer and light gave >6 log10 inactivation of intracellular and extracellular virus, and >4 log10 inactivation of extracellular bacteria with varying levels of hemolyis, following a 42-day storage of red blood cell suspensions. Photocleavage experiments with plasmid DNA and the chalcogenoxanthylium dyes suggested the genomic material contained in the virus and in the bacteria as one possible target for the photodynamic action of some of these dyes.

Anti-Bacterial Agents↗
PubMed2005

Toward closed-system culture of blood origin endothelial cells.

BACKGROUND: Blood outgrowth endothelial cells (BOECs) are thought to arise from very rare progenitors that are present in the mononuclear fraction of marrow or peripheral blood. Recently, BOECs have been expanded from progenitors present in buffy coat into confluent monolayers on fibronectin- or collagen-coated polystyrene surfaces. A method for sterile closed-system culture of these cells has not been described, however. Here, efforts are described toward developing closed-system culture of BOECs derived from progenitors present in a mononuclear apheresis unit by use of a cord blood filter, a sterile connection device, and a fibronectin-coated polycarbonate cassette. STUDY DESIGN AND METHODS: Strongly adherent cells from a mononuclear apheresis unit were eluted from a cord blood filter and resuspended in EGM-2 with 10 percent serum. Approximately 2 x 10(8) eluted cells were introduced into human fibronectin-coated polycarbonate cassettes. Medium was introduced and removed from cassettes with a sterile connection device and changed every 2 days. After expansion, cells were either cryopreserved or characterized by fluorescence-activated cell sorting analysis and ability to take up Dil-Ac-LDL. RESULTS: After 2 to 3 weeks of culture, 3 to 28 colonies with cobblestone morphology were observed in cassettes and passed to new cassettes within 3 to 4 weeks. By approximately 5 weeks of culture, 2 x 10(6) cells were typically obtained. BOECs uniformly took up Dil-Ac-LDL and were CD31+, CD105+, CD146+, CD45-, and CD14-. A population of BOECs was HLA-ABC+ or CD34+. CONCLUSION: BOEC progenitors can be isolated from mononuclear apheresis units with cord blood filters, expanded with fibronectin-coated polycarbonate cassettes, and cryopreserved.

Antigens, CD↗
PubMed2005

Monocyte enrichment of mononuclear apheresis preparations with a multistep back-flush procedure on a cord blood filter.

INTRODUCTION: Monocytes or mononuclear cells have been investigated for the treatment of chronic wounds and spinal cord injuries, as well as serve as a source for dendritic or endothelial cell culture. Because these cells may have clinical benefit yet no rapid and inexpensive closed system for monocyte purification is commercially available, a method was investigated to enrich monocytes from mononuclear apheresis units using a cord blood filter. METHODS: A 4-step method for monocyte enrichment was developed which involved 1) filtering a mononuclear apheresis unit through a cord blood filter, 2) chasing with medium to remove non-adherent residual cells and plasma, 3) back-flushing under low shear conditions to remove loosely adherent lymphocytes, and 4) back-flushing under high shear conditions to collect a fraction enriched in monocytes. Apheresis units and enriched monocyte preparations were characterized by cell count and differential, filter-isolated preparations were cryopreserved, and thawed preparations were assayed for viability, and phagocytosis. Enriched monocyte preparations were also assayed for inflammatory cytokines secretion and secretion of prostaglandin E2 during short-term culture. RESULTS: Monocytes were viable, capable of phagocytosis, and enriched using the multistep filter elution technique to represent 42 +/- 13-percent of white cells in the final preparation. Fifty-three-percent of monocytes were recovered in the final preparation, while total cell counts of red cells, platelets, neutrophils and lymphocytes were reduced to 3.0, 3.0, 4.5 and 16-percent, respectively, from levels present in mononuclear apheresis units. Filter enriched monocyte preparations secreted IL-8, IL-6, MCP-1, and MIP-1alpha, during short term culture. CONCLUSION: The use of a multi-step back flush procedure with a cord blood filter resulted in rapid enrichment of viable and functional monocytes from mononuclear apheresis units with significant reduction of contaminating platelets and red cells.

Blood Component Removal↗
PubMed2005

Use of a flexible thiopyrylium photosensitizer and competitive inhibitor for pathogen reduction of viruses and bacteria with retention of red cell storage properties.

BACKGROUND: Progress in developing photochemical methods for pathogen reduction of red blood cells (RBCs) has been hampered by hemolysis. A flexible, nucleic acid-intercalating thiopyrylium (TP) dye that is only photochemically active in the bound state and a competitive inhibitor of RBC membrane binding, dipyridamole (DP), was used to reduce photoinduced hemolysis stemming from free- and membrane-bound dye. STUDY DESIGN AND METHODS: Oxygenated leukodepleted 20% hct RBC suspensions were deliberately inoculated with virus or bacteria, incubated with 200 micromol per L DP and less than or equal to 100 micromol per L TP, illuminated with 1.1 J/cm(2) of red light, and titered. RBC suspensions containing 200 micromol per L DP and 160 micromol per L TP were identically phototreated, concentrated to 45% hct, and assayed for RBC storage properties. RESULTS: In RBC suspensions containing DP, TP photoinactivated vesicular stomatitis virus, pseudorabies virus, duck hepatitis B virus, bovine virus diarrhea virus, extracellular human immunodeficiency virus (HIV) to the limit of detection and 6.2 log intracellular HIV. More than 5 log inactivation of 6 bacterial species was demonstrated. DP prevented approximately 30% of TP binding to RBCs. Phototreated RBCs that were subsequently stored for 42 days exhibited acceptable levels of hemolysis, morphology scores, extracellular pH, ATP, glucose utilization rates, and lactate production. Treated samples exhibited substantially increased potassium efflux compared to controls. CONCLUSION: Use of TP photosensitizer and DP enables significant levels of pathogen reduction while retaining most, but not all RBC properties during 42 day storage.

Aniline Compounds↗
PubMed2004

Evaluation of the sensitivity of red blood cell markers to detect photodynamic membrane damage.

BACKGROUND: Photodynamic dyes have been used to investigate pathogen reduction methods in RBC suspensions, but treatment with sensitizers and light can produce unwanted membrane damage during routine blood bank storage. This study compares the relative sensitivity of three assays for detecting RBC membrane damage. STUDY DESIGN AND METHODS: RBCs were treated with dimethylmethylene blue and red light-emitting diode light under four conditions differing in photodynamic stringency and subsequently stored under refrigerated conditions for up to 42 days. Hemolysis, potassium release, and the apoptosis marker annexin V binding were assayed immediately after phototreatment and following storage. RESULTS: In terms of increasing sensitivity for detecting photodynamic RBC membrane damage was enhanced ion leakage >> hemolysis > annexin V binding. CONCLUSION: Although very stringent photodynamic treatment conditions generate annexin V-positive RBCs, the assay will not detect more subtle membrane damage that is readily detected by other well-established methods. In addition, many RBCs destined for photo-induced hemolysis later in storage are annexin V-negative throughout the storage period, suggesting that the two measures are not directly related.

Annexin A5↗
PubMed2002

Virus inactivation in blood components by photoactive phenothiazine dyes.

The virucidal properties of photoactive phenothiazine dyes, such as methylene blue, have been known approximately 70 years. The mechanism of virus inactivation involves binding of the dye to nucleic acid, absorption of light, generation of reactive oxygen species, guanine oxidation in the viral genome. The first practical research involving phenothiazine photosensitizers in transfusion medicine was sparked by efforts to prevent transmission of hepatitis from pooled plasma administered during the Korean War. During the last decade, methylene blue was used by some European countries for virus photoinactivation in fresh frozen plasma, whereas other phenothiazine derivatives with greater affinities for nucleic acids and capabilities to inactivate intracellular virus have been investigated for photochemical decontamination of red cell suspensions. The toxicity of methylene blue is well characterized; the drug has been used for many years to treat methemoglobinemia using concentrations orders of magnitude greater than those used for virus photoinactivation, and several filters have been developed to remove the dye from plasma. Future widespread use of phenothiazines for virus photoinactivation in blood components may depend on whether the risk inherent in their use is less than the residual risk from transfusion-transmitted viruses.

Blood↗
PubMed2002

Quinacrine enhances vesicular stomatitis virus inactivation and diminishes hemolysis of dimethylmethylene blue-phototreated red cells.

Several photodynamic methods for virus inactivation in red blood cell (RBC) suspensions have resulted in unwanted hemolysis during extended 1-6 degrees C storage. To explore the possibility that hemolysis may be mediated by a membrane-bound dye, a molecule similar in structure to yet different in light absorption properties from the photosensitizer was used as an inhibitor for RBC membrane binding in virus photoinactivation and photohemolysis studies. The addition of 500 pM quinacrine to oxygenated RBC before treatment with 3.6 microM dimethylmethylene blue (DMMB) and 219 mJ/cm2 red light resulted in an increased extracellular concentration of the sensitizer, increased extracelluar viral inactivation kinetics, and decreased hemolysis during 1-6 degrees C storage without alteration of quinacrine absorption properties. These results collectively suggest that despite its recognized affinity for viral nucleic acid, DMMB also binds to RBC membranes and that the bound dye is, in part, responsible for photoinduced hemolysis.

Blood Preservation↗
PubMedDate not supplied

Sterile isolation and cryopreservation of human adherent monocytes/macrophages from mononuclear cell apheresis preparations.

There is evidence from animal and human study that suggests clinical use of monocytes/macrophages may be of benefit in wound healing, tissue regeneration, and cancer therapy. To facilitate further study, a method was developed for sterile isolation and cryopreservation of adherent monocyte/macrophages from mononuclear cell apheresis units collected from unstimulated normal human donors. Preparations contained approximately 1 x 108 total cells and were comprised of approximately 60% monocytes, 38% lymphocytes, and 2% granulocytes. Cells could be cryopreserved for up to 8 months and subsequently thawed and stored at 1-6 degrees C for up to 4 hours with retention of viability and adequate phagocytic function. These cells can be used in clinical trials to determine their possible therapeutic benefit, e.g., whether administration of exogenously supplied cells improves the healing of chronic wounds or promotes the regeneration of transected nerves.

Cryopreservation↗
PubMedDate not supplied

Use of a red cell band 3-ligand/antioxidant to improve red cell storage properties following virucidal phototeatment with chalcogenoxanthylium photosensitizers.

The efficacy of two chalcogenoxanthylium photosensitizers to reduce pathogens in red cell suspensions while maintaining red cell storage properties was investigated in the presence and absence of the red cell band-3 ligand and antioxidant, dipyridamole (DP). In the presence of DP, both sensitizers, TMR-S and DJD-42, displayed potent virucidal photoactivity (>6 log10) and species-dependent bactericidal activities that ranged from 0.6 to 8.7 log10. Addition of DP to red cell suspensions containing TMR-S or DJD-42 reduced Day 42 photo-induced hemolysis by approximately eight-fold and four-fold, respectively. Red cell binding studies revealed only a small degree of competition between DP and TMR-S, and no competition between DP with DJD-42 for binding to red cell membranes, suggesting that protection of red cells by DP in this system may primarily stem from its antioxidant properties.

Anti-Bacterial Agents↗