Biomedical subjects

David W Grainger

Publications and source records attributed to David W Grainger.

15 recordsLinked to original sources

PubMed2006

Surface coverage and structure of mixed DNA/alkylthiol monolayers on gold: characterization by XPS, NEXAFS, and fluorescence intensity measurements.

Self-assembly of thiol-terminated single-stranded DNA (HS-ssDNA) on gold has served as an important model system for DNA immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled DNA monolayers containing a short alkylthiol surface diluent [11-mercapto-1-undecanol (MCU)] on gold supports. These mixed DNA monolayers were studied with X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and fluorescence intensity measurements. XPS results on sequentially adsorbed DNA/MCU monolayers on gold indicated that adsorbed MCU molecules first incorporate into the HS-ssDNA monolayer and, upon longer MCU exposures, displace adsorbed HS-ssDNA molecules from the surface. Thus, HS-ssDNA surface coverage steadily decreased with MCU exposure time. Polarization-dependent NEXAFS and fluorescence results both show changes in signals consistent with changes in DNA orientation after only 30 min of MCU exposure. NEXAFS polarization dependence (followed by monitoring the N 1s --> pi* transition) of the mixed DNA monolayers indicated that the DNA nucleotide base ring structures are oriented more parallel to the gold surface compared to DNA bases in pure HS-ssDNA monolayers. This indicates that HS-ssDNA oligomers reorient toward a more-upright position upon MCU incorporation. Fluorescence intensity results using end-labeled DNA probes on gold show little observable fluorescence on pure HS-ssDNA monolayers, likely due to substrate quenching effects between the fluorophore and the gold. MCU diluent incorporation into HS-ssDNA monolayers initially increases DNA fluorescence signal by densifying the chemisorbed monolayer, prompting an upright orientation of the DNA, and moving the terminal fluorophore away from the substrate. Immobilized DNA probe density and DNA target hybridization in these mixed DNA monolayers, as well as effects of MCU diluent on DNA hybridization in complex milieu (i.e., serum) were characterized by surface plasmon resonance (SPR) and (32)P-radiometric assays and reported in a related study (Gong, P.; Lee, C.-Y.; Gamble, L. J.; Castner, D. G.; Grainger, D. W. Anal. Chem. 2006, 78, 3326-3334.).

Alkanes↗

PubMed2006

Hybridization behavior of mixed DNA/alkylthiol monolayers on gold: characterization by surface plasmon resonance and 32P radiometric assay.

Nucleic acid assay from a complex biological milieu is attractive but currently difficult and far from routine. In this study, DNA hybridization from serum dilutions into mixed DNA/mercaptoundecanol (MCU) adlayers on gold was monitored by surface plasmon resonance (SPR). Immobilized DNA probe and hybridized target densities on these surfaces were quantified using 32P-radiometric assays as a function of MCU diluent exposure. SPR surface capture results correlated with radiometric analysis for hybridization performance, demonstrating a maximum DNA hybridization on DNA/MCU mixed adlayers. The maximum target surface capture produced by MCU addition to the DNA probe layer correlates with structural and conformational data on identical mixed DNA/MCU adlayers on gold derived from XPS, NEXAFS, and fluorescence intensity measurements reported in a related study (Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316-3325.). MCU addition into the DNA adlayer on gold also improved surface resistance to both nonspecific DNA and serum protein adsorption. Target DNA hybridization from serum dilutions was monitored with SPR on the optimally mixed DNA/MCU adlayers. Both hybridization kinetics and efficiency were strongly affected by nonspecific protein adsorption from a complex milieu even at a minimal serum concentration (e.g., 1%). No target hybridization was detected in SPR assays from serum concentrations above 30%, indicating nonspecific protein adsorption interference of DNA capture and hybridization from complex milieu. Removal of nonsignal proteins from nucleic acid targets prior to assay represents a significant issue for direct sample-to-assay nucleic acid diagnostics from food, blood, tissue, PCR mixtures, and many other biologically complex sample formats.

Alkanes↗

PubMed2006

Multi-technique comparison of immobilized and hybridized oligonucleotide surface density on commercial amine-reactive microarray slides.

To establish a quantitative, corroborative understanding of observed correlations between immobilized probe DNA density on microarray surfaces and target hybridization efficiency in biological samples, we have characterized amine-terminated, single-stranded DNA probes attached to amine-reactive commercial microarray slides and complementary DNA target hybridization using fluorescence imaging, X-ray photoelectron spectroscopy (XPS) and 32P-radiometric assays. Importantly, we have reproduced DNA probe microarray immobilization densities in macroscopic spotted dimensions using high ionic strength, high-concentration DNA probe solutions to permit direct XPS surface analysis of DNA surface chemistry with good reliability and reproducibility. Target capture hybridization efficiency with complementary DNA exhibited an optimum value at intermediate DNA probe immobilization densities. The macroscopic array model provides a new platform for the study of DNA surface chemistry using highly sensitive, quantitative surface analytical techniques (e.g., XPS, ToF-SIMS). Sensitive 32P-DNA radiometric density measurements were calibrated with more routine XPS DNA signals, facilitating future routine DNA density determinations without the use of a hazardous radioactive assay. The objective is to provide new insight into different surface chemistry influences on immobilized DNA probe environments that affect target capture efficiency from solution to improve microarray assay performance.

Amines↗

PubMed2006

Comparison of hydroxylated print additives on antibody microarray performance.

Various hydroxylated additives were added to antibody print buffers at different concentrations to stabilize printed antibodies during normal array spot desiccation on commercial polymer-coated microarray slides. Polyvinyl alcohol addition to print buffers produced the most regular spot morphologies, homogeneous intra-spot antibody distribution, uniform fluorescence intensity, and improved analyte capture activity, maintained up to 1 month at 4 degrees C for capturing model analytes, anti-human IL-1beta, IL-4, and TNFalpha, on these microarraying slides.

Antibodies↗

PubMed2005

Drug/device combinations for local drug therapies and infection prophylaxis.

Combination devices-those comprising drug releasing components together with functional prosthetic implants-represent a versatile, emerging clinical technology promising to provide functional improvements to implant devices in several classes. Landmark antimicrobial catheters and the drug-eluting stent have heralded the entrance, and significantly, routes to FDA approval, for these devices into clinical practice. This review describes recent strategies creating implantable combination devices. Most prominent are new combination devices representing current orthopedic and cardiovascular implants with new added capabilities from on-board or directly associated drug delivery systems are now under development. Wound coverings and implantable sensors will also benefit from this combination enhancement. Infection mitigation, a common problem with implantable devices, is a current primary focus. On-going progress in cell-based therapeutics, progenitor cell exploitation, growth factor delivery and advanced formulation strategies will provide a more general and versatile basis for advanced combination device strategies. These seek to improve tissue-device integration and functional tissue regeneration. Future combination devices might best be completely re-designed de novo to deliver multiple bioactive agents over several spatial and temporal scales to enhance prosthetic device function, instead of the current 'add-on' approach to existing implant device designs never originally intending to function in tandem with drug delivery systems.

Animals↗

PubMed2005

DNA and protein microarray printing on silicon nitride waveguide surfaces.

Sputtered silicon nitride optical waveguide surfaces were silanized and modified with a hetero-bifunctional crosslinker to facilitate thiol-reactive immobilization of contact-printed DNA probe oligonucleotides, streptavidin and murine anti-human interleukin-1 beta capture agents in microarray formats. X-ray photoelectron spectroscopy (XPS) was used to characterize each reaction sequence on the native silicon oxynitride surface. Thiol-terminated DNA probe oligonucleotides exhibited substantially higher surface printing immobilization and target hybridization efficiencies than non-thiolated DNA probe oligonucleotides: strong fluorescence signals from target DNA hybridization supported successful DNA oligonucleotide probe microarray fabrication and specific capture bioactivity. Analogously printed arrays of thiolated streptavidin and non-thiolated streptavidin did not exhibit noticeable differences in either surface immobilization or analyte capture assay signals. Non-thiolated anti-human interleukin-1 beta printed on modified silicon nitride surfaces reactive to thiol chemistry exhibited comparable performance for capturing human interleukin-1 beta analyte to commercial amine-reactive microarraying polymer surfaces in sandwich immunoassays, indicating substantial non-specific antibody-surface capture responsible for analyte capture signal.

Biosensing Techniques↗

PubMed2004

Controlled-release and local delivery of therapeutic antibodies.

Human and humanised antibodies are now poised to become a major new class of protein-based therapeutic agents. A significant fraction of new drugs in clinical testing (approximately 20% in 2002) are antibody classes. Monoclonal antibodies (mAbs) with high affinities against newly discovered disease targets, both cellularly and extracellularly, are now clinically proven to elicit high bioactivities against numerous diseases, including tumours, infections, asthma, inflammation, arthritis and osteoporosis. Clinical humanised antibody delivery is typically intravenous, with large multiple doses (grams) required for systemic volumes of distribution. Due to the relatively high costs of both this drug type, and its common mode of administration, alternatives are sought where doses might be reduced and the bioavailability and efficacy enhanced. Local, controlled-release methods that deliver antibodies locally to site of disease, offer new possibilities with these potential advantages. However, protein drugs frequently exhibit formulation challenges when packaged in delivery vehicles, and as globular proteins, antibodies are no exception. Several examples of mAb controlled-release and local delivery strategies against several disease targets are reviewed. Importantly, several antibody delivery methods work in tandem with existing clinically-accepted therapeutics, sometimes exhibiting potentiating or synergistic effects in animal models with small molecule, systemically administered drugs.

Aerosols↗

PubMed2004

Morphology and growth of murine cell lines on model biomaterials.

All biomaterial implants are assaulted by the host "foreign body" immune response. Understanding the complex, dynamic relationship between cells, biomaterials and milieu is an important first step towards controlling this reaction. Material surface chemistry dictates protein adsorption, and thus subsequent cell interactions. The cell-implant is a microenvironment involving 1) proteins that coat the surface and 2) cells that interact with these proteins. Macrophages and fibroblasts are two cell types that interact with proteins on biomaterials surfaces and play different related, but equally important, roles in biomaterials rejection and implant failure. Growth characteristics of four murine cell lines on model biomaterials surfaces were examined. Murine monocyte-macrophages (RAW 264.7 and J774A.1), murine macrophage (IC-21) and murine fibroblast (NIH 3T3) cell lines were tested to determine whether differences exist in adhesion, proliferation, differentiation, spreading, and fusion (macrophage lineages only) on these surfaces. Differences were observed in the ability of cells to adhere to and subsequently proliferate on polymer surfaces. (Monocyte-) macrophages grew well on all surfaces tested and growth rates were measured on three representative polymer biomaterials surfaces: tissue culture polystyrene (TCPS), polystyrene, and Teflon-AF. J774A.1 cultures grown on TCPS and treated with exogenous cytokines IL-4 and GM-CSF were observed to contain multinucleate cells with unusual morphologies. Thus, (monocyte-) macrophage cell lines were found to effectively attach to and interrogate each surface presented, with evidence of extensive spreading on Teflon-AF surfaces, particularly in the IC-21 cultures. The J774A.1 line was able to proliferate and/or differentiate to more specialized cell types (multinucleate/dendritic-like cells) in the presence of soluble chemokine cues.

3T3 Cells↗

PubMed2003

Correlating fibronectin adsorption with endothelial cell adhesion and signaling on polymer substrates.

Fibronectin (Fn) adsorption was studied on different commercial polymer surface chemistries, including tissue culture polystyrene (TCPS), bacteriologic polystyrene (BPS), fluoropolymer Teflon AF, and poly-L-lactide (PLLA). Antibody probes detected the availability of Fn's cell binding domain on adsorbed Fn in the competitive presence and absence of bovine serum albumin (BSA). Domain availability was highest for Fn adsorbed on TCPS, especially in the presence of either serum albumin or dilute serum. Attachment and growth efficiencies for human umbilical venous endothelial cells (HUVECs) cultured on surfaces preadsorbed with Fn in serum and serum-free media correlated with antibody cell-binding domain availability: TCPS > BPS, Teflon AF > PLLA. Intracellular signaling from the GTPase, RhoA, was highest (RhoA:RhoGDI inhibitor ratio) in cells cultured on the Teflon AF surfaces, indicating that despite lower attached cell numbers on Teflon AF compared to TCPS, cell signaling remained activated after 24 h of growth. Up-regulated cellular Fn mRNA messages, assessed using RT-PCR techniques, supported HUVECs' producing the endogenous extracellular matrix (ECM) protein Fn in order to attach and survive on the suboptimal Teflon AF culture surfaces.

Adsorption↗

PubMed2003

Assessment of fibronectin conformation adsorbed to polytetrafluoroethylene surfaces from serum protein mixtures and correlation to support of cell attachment in culture.

Surfaces of polytetrafluoroethylene (PTFE) were exposed to buffered aqueous solutions containing radio-labeled human fibronectin ([125I]Fn), Fn/bovine serum albumin (BSA) binary mixtures of various ratios or whole human plasma dilutions for 1 h. Total adsorbed Fn and albumin adsorption following rinsing was quantified on this surface. 125I-labeled monoclonal antibodies against either the tenth type-III Fn repeat unit (containing the cell-binding RGDS integrin recognition motif) or the Fn amino-terminal domain were used to probe the accessibility of each of these respective Fn regions post-adsorption. Human umbilical vein endothelial cells (HUVECs) were cultured on PTFE surfaces pre-exposed to each of these protein adsorption conditions and compared to identical conditions on tissue culture polystyrene (TCPS). Fn adsorption to PTFE is dependent upon the concentration of albumin co-adsorbing from solution: albumin out-competes Fn for PTFE surface sites even at non-physiological Fn/HSA ratios 10-100-fold biased in Fn. Antibodies against Fn do not readily recognize Fn adsorbed on PTFE as the HSA co-adsorption concentration in either binary mixtures or in plasma increases, indicating albumin masking of adsorbed Fn. At Fn/HSA ratios rich in Fn (1:1, 1:100), albumin co-adsorption actually improves anti-Fn antibody recognition of adsorbed Fn. HUVEC attachment efficiency to PTFE after protein adsorption correlates with amounts of Fn adsorbed and levels of anti-Fn antibody recognition of Fn on PTFE, linking cell attachment to integrin recognition of both adsorbed Fn density and Fn adsorbed conformation on PTFE surfaces.

Adsorption↗

PubMed2002

Effect of neutral lipids on coexisting phases in monolayers of pulmonary surfactant.

We previously established that compression of monolayers containing the lipids in pulmonary surfactant, with or without the surfactant proteins, initially leads to phase separation. On further compression, however, phase coexistence terminates at a critical point that requires the presence of cholesterol. The studies reported here address the changes in the phospholipid phase diagram produced by cholesterol. We used the two systems of the lipids from calf surfactant with and without the surfactant proteins. For both systems, we began with the postulate that cholesterol had no effect on the composition of other constituents in the two phases, and then used the known behavior of interfacial tension at a critical point to test the two extreme cases in which the cholesterol partitions exclusively into condensed domains or into the surrounding film. Measurements of surface potential along with the fraction of the nonfluorescent area and the radius of the domains, both obtained by fluorescence microscopy, for films with and without cholesterol allowed calculation of the interfacial tension between the two phases. Only the model that assumes the presence of cholesterol within the domains accurately predicts a decreasing line tension during film compression toward the critical point. That model, however, also predicts an unlikely decrease during compression of the dipole moment density for the condensed phase. Our results are best explained in terms of cholesterol partitioning predominantly into the condensed domains, with a resulting partial redistribution of the phospholipids between the two phases.

Animals↗

PubMed2002

Fabrication of poly(alpha-hydroxy acid) foam scaffolds using multiple solvent systems.

The present studies describe the fabrication and characterization of highly porous and interconnected poly(alpha-hydroxy acid) foam scaffolds produced using a phase separation multisolvent system, followed by a sublimation process. Fabrication parameters, including solvent composition, polymer concentration, freezing temperature, polymer type, and polymer molecular weight, were optimized to produce the desired foam microstructure. Analyses of selected samples with scanning electron microscopic images and mercury intrusion porosimetry indicated polymer foams with pore size ranges of 100-350 microm, a porosity >90%, and an interconnecting open-pore foam structure. Scaffold degradation profiles varied according to the type and molecular weight of the polymers. Cytocompatibility assays demonstrated that the preferred foam structures were nontoxic and osteoprecursor cells seeded into the scaffolds exhibited the ability to attach, propagate, and differentiate into a calcified structure.

Absorbable Implants↗

PubMed2002

Prophylactic treatment of gram-positive and gram-negative abdominal implant infections using locally delivered polyclonal antibodies.

The increasing clinical incidence and host risk of biomaterial-centered infections, as well as the reduced effectiveness of clinically relevant antibiotics to treat such infections, provide compelling reasons to develop new approaches for treating implanted biomaterials in a surgical context. We describe the direct local delivery of polyclonal human antibodies to abdominal surgical implant sites to reduce infection severity and mortality in a lethal murine model of surgical implant-centered peritoneal infection. Surgical implant-centered peritonitis was produced in 180 female CF-1 mice by the direct inoculation of surgical-grade polypropylene mesh disks placed in the peritoneal cavity with lethal doses of either methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa. Mice randomly received a resorbable antibody delivery vehicle at the implant site: either a blank carboxymethylcellulose (CMC) aqueous gel or the same CMC gel containing 10 mg of pooled polyclonal human immunoglobulin G locally on the implant after infection, either alone or in combination with systemic doses of cefazolin or vancomycin antibiotics. Human antibodies were rapidly released (first-order kinetics) from the gel carrier to both peritoneal fluids and serum in both infection scenarios. Inocula required for lethal infection were substantially reduced by surgery and the presence of the implant versus a closed lethal peritonitis model. Survival to 10 days with two different gram-negative P. aeruginosa strains was significantly enhanced (p < 0.01) by the direct application of CMC gel containing antibodies alone to the surgical implant site. Human-equivalent doses of systemic vancomycin provided a significantly improved benefit (p < 0.01) against lethal, implant-centered, gram-positive MRSA infection. However, locally delivered polyclonal human antibodies in combination with a range of systemic vancomycin doses against MRSA failed to improve host survival. Successful antibody therapy against gram-negative, implant-centered infections complements the clinically routine use of systemic antibiotics, providing a mechanism of protection independent of antibiotic resistance.

Abdomen↗

PubMed2002

Polyclonal human antibodies reduce bacterial attachment to soft contact lens and corneal cell surfaces.

Bacterial keratitis due to Pseudomonas aeruginosa is a potentially serious complication of extended-wear contact lens use. Adhesion of P. aeruginosa to soft contact lens materials or corneal endothelial cells in the presence of pooled human immunoglobulins and/or neutrophils in artificial tear fluid was studied in vitro as a potential method to treat contact lens-associated infection. Soft hydrophilic contact lens materials equilibrated in sterile saline were soaked in artificial tear fluid for 18 h prior to use. P. aeruginosa IFO 3455 was added to groups of lenses or confluent cultured bovine corneal endothelial cells with varying amounts of human polyclonal immunoglobulin (IgG) and human blood neutrophils or serum albumin as a control. After 2 or 4 h incubation, adherent viable bacteria on lenses were quantified. Fluorescence microscopy was used to assess bacterial adherence to bovine corneal endothelial cells in the presence and absence of IgG and neutrophils. Various concentrations of albumin had no effect on adhesion. Human immunoglobulin solutions (25 mg/ml) reduced P. aeruginosa adhesion by nearly 1 log and 2 logs after 2 and 4 h incubations, respectively. Neutrophils in combination with 25 mg/ml IgG reduced bacterial adhesion approximately 1 log over reduction in adhesion by neutrophils alone. Diluted human IgG (10 mg/ml) did not significantly decrease bacterial adhesion after 2 or 4 h, but did reduce adhesion in combination with human neutrophils at both time points. Similar reductions in amounts of fluorescently labeled bacteria adhered to cultured monolayers of corneal endothelial cells under these conditions were qualitatively observed.

Albumins↗

PubMed2002

Locally delivered polyclonal antibodies potentiate intravenous antibiotic efficacy against gram-negative infections.

PURPOSE: Comparison of the anti-microbial efficacy of locally delivered antibodies in tandem with conventional systemic administration of ceftazidime antibiotic therapy in two lethal gram-negative animal infection models. METHODS: Previously published lethal E. coli-induced closed peritonitis and Klebsiella-induced burn wound infections were generated in outbred female CF-1 mice cohorts. Pooled human polyclonal antibodies were injected locally into sites of infection in these mice simultaneously with intravenous infusions of the broad-spectrum antibiotic, ceftazidime. Mouse survival was compared in sham control cohorts vs. both ceftazidime-alone or antibody-alone systemically infused cohorts as well as local antibody-systemic ceftazidime combination therapy cohorts. Microbial burdens in blood and tissue samples (by agar plating), as well as interleukin-6 cytokine levels (using ELISA) correlated with sepsis, were monitored in sacrificed animals as a function of antimicrobial treatment regimen. RESULTS: Local delivery of human polyclonal antibodies to infection sites was shown to produce synergistic therapeutic efficacy in combination with systemic antibiotic administration in these lethal wound infection models in mice. Enhanced benefits of the unique combination therapy included host survival, bacterial burden both locally and systemically, and IL-6 levels in host serum. CONCLUSIONS: Commercial pooled human antibodies contain a broad spectrum of antimicrobial activity against gram-negative pathogens. Prevention of systemization of infection correlates with host survival in these models. Local control of infection using doses of local, high-titer polyclonal antibodies can enhance traditional approaches to curb systemic spread of infection using intravenous antibiotics. Antibodies provide antimicrobial efficacy independent of known pathogen resistance mechanisms.

Animals↗