Biomedical subjects

D W Grainger

Publications and source records attributed to D W Grainger.

At least 19 recordsLinked to original sources

PubMed2005

Photopolymerized hydrogel carriers for live vaccine ballistic delivery.

Photopolymerized poly(ethylene glycol) (PEG)-crosslinked hydrogels were assessed for their ability to serve as a payload vehicle to deliver a viable bacterial vaccine (Brucella abortus strain RB51 (RB51) to bison in Yellowstone National Park) ballistically using thermoplastic degradable Biobullets. PEG modified with degradable glycolide or lactide oligomers capped with photopolymerizable methacrylate groups served to crosslink the hydrogel vaccine carrier inside commercial hydroxypropylcellulose Biobullets. Release of 1 microm diameter model fluorescent particles from hydrogels followed known degradation trends for glycolide- and lactide-modified PEG hydrogels. All particles were released from PEG-co-glycolide hydrogels after approximately 10 days and PEG-co-lactide hydrogels after approximately 45 days following gel degradation. Minimal particle release was observed from pure PEG dimethacrylate hydrogels over 40 days. P. aeruginosa (strain PAO1) and RB51 live vaccines exhibit excellent viability following exposure to photopolymerization encapsulation within these gel matrices. Hydrogels photopolymerized into the payload chamber of Biobullets exhibit similar ballistic properties to commercially available Biobullets and penetrate and remain intact when fired intramuscularly into live elk for release of their gel payload in the host.

Animals↗

PubMed2005

Immunologic responses of bison to vaccination with Brucella abortus strain RB51: comparison of parenteral to ballistic delivery via compressed pellets or photopolymerized hydrogels.

This study compared responses of bison calves to 10(10)CFU of Brucella abortus strain RB51 (SRB51) delivered by parenteral or ballistic methods. Two types of biobullet payloads were evaluated; compacted SRB51 pellets or SRB51 encapsulated in photopolymerized poly(ethylene glycol) hydrogels. Bison were vaccinated with saline, parenteral SRB51 alone, or in combination with Spirovac, or ballistically with compressed SRB51 or hydrogel biobullets. Bison parenterally vaccinated with SRB51 had greater (P<0.05) immunologic responses when compared to control bison. Co-administration of Spirovac as an adjuvant did not influence immunologic responses. As compared to compressed SRB51 biobullets, ballistic vaccination with hydrogel biobullets increased cellular immune responses at some sampling times. Our data suggest that hydrogel formulations of SRB51 may be a superior alternative to compressed SRB51 tablets for ballistic vaccination of bison. Although preliminary, data suggests that immunologic responses of bison to SRB51 hydrogel bullets are similar to responses after parenteral vaccination with SRB51.

Adjuvants, Immunologic↗

PubMed2004

Self-assembled organic monolayers terminated in perfluoroalkyl pentafluoro-lambda(6)-sulfanyl (-SF5) chemistry on gold.

Recently synthesized (Winter, R.; Nixon, P. G.; Gard, G. L.; Radford, D. H.; Holcomb, N. R.; Grainger, D. W. J. Fluorine Chem. 2001, 107, 23-30) SF5-terminated perfluoroalkyl thiols (SF5(CF2)nCH2CH2SH, where n = 2, 4, and 6) and a symmetric SF5-terminated dialkyl disulfide ([SF5-CH=CH-(CH2)8-S-]2) were assembled as thin films chemisorbed onto gold surfaces. The adsorbed monolayer films of these SF5-containing molecules on polycrystalline gold were compared using ellipsometry, contact angle, X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and infrared spectroscopy (FTIR) surface analytical methods. The resulting SF5-dialkyl disulfide monolayer film shows moderate angle dependence in depth-dependent XPS analysis, suggesting a preferentially oriented film. The SF5-terminated perfluoroalkyl thiols exhibit angular-dependent XPS compositional variance depending on perfluoroalkyl chain length, consistent with improved film assembly (increasingly hydrophobic, fewer defects, and more vertical chain orientation increasing film thickness) with increasing chain length. Tof-SIMS measurements indicate that both full parent ions for these film-forming molecules and the unique SF5 terminal group are readily detectable from the thin films without substantial contamination from other adsorbates.

Fluorocarbons↗

PubMed2004

Analysis of regenerated amine-reactive polymer microarray slides.

Reactive polymer-coated microarray substrates based on N-hydroxyl succinimide (NHS) chemistry lose their bio-immobilization reactivity to probe nucleophiles over time, both in use and in storage, due to their intrinsic hydrolytic instability. Poor DNA and protein probe immobilization efficiency is often observed with routine microarray printing conditions, with accompanying reliability and stability issues for assay. We report a one-step reaction to regenerate NHS-reactive chemistry in situ on these microarray polymer surfaces with simple, straightforward reaction chemistry. Surfaces regenerated with this method perform equal to or better than freshly prepared slides in print-immobilization of oligonucleotide probes functionalized with primary amine reactive groups. DNA probe specific and non-specific surface binding as well as target hybridization were analyzed using both fresh and regenerated slides. Commercial microarray substrates appear to retain DNA probes with both substantial covalent immobilization and some non-specific adsorption to the surface.

Amines↗

PubMed2004

Design and synthesis of a new polymer drug delivery conjugate.

This work describes the design and synthesis of a novel polymer conjugate to facilitate intracellular release of attached cargo. Water soluble poly[N(2-hydroxypropyl) methacrylamide] with derivatizable pendant side chains was used as the polymer carrier. The membrane active Tat peptide was masked with poly(ethylene glycol) to inhibit non-specific interactions with exterior cell membranes, and then covalently linked to poly[N-(2-hydroxypropyl) methacrylamide]. A novel heterobifunctional crosslinker was synthesized containing both maleimide and aldehyde functionality to allow attachment of peptides to the polymer backbone through a pH sensitive bond. Analysis of the proposed conjugate by gel permeation chromatography, nuclear absorbance spectroscopy, and absorbance measurements indicate that the desired compound has been synthesized.

Acrylamides↗

PubMed2004

Toward immobilized antibody microarray optimization: print buffer and storage condition comparisons on performance.

Performance issues in protein arrays differ substantially from DNA microarrays because of the different intrinsic construction and stability of proteins versus DNA, and the impact on biological recognition phenomena required for assay from a surface-immobilized state. Maintenance of the capture bioactivity and performance of antibodies on microarray substrates for relatively long periods (under storage or shipping) requires carefully designed and optimized print buffer and storage conditions. We have focused on selection of several additives added to antibody print buffers with different concentrations, and storage of printed antibody slides under different temperatures from 1 day to 4 weeks. Activities of these slides were compared in capture assay formats in order to compare the effects of print buffer and storage conditions of antibody microarrays. Commercial polymer-coated Optarray slides exhibited better performance over Codelink slides in terms of activity of anti-human TNF alpha and IL-1 beta. IL-1 beta antibodies immobilized on both commercial surfaces showed much higher activity than TNF alpha antibodies, regardless of source or vendor. Addition of 1% (v/v) PEO to the print buffer increased the printed analyte capture activity of IL-1 beta on Optarray slides compared to standard print buffer without any additives. This helps to maintain antibody immobilized analyte capture activity for 4 weeks when stored at -70 degrees C.

Adsorption↗

PubMed2003

Design strategies to improve soluble macromolecular delivery constructs.

Macromolecular therapeutics provide numerous benefits for the delivery of cytotoxic or poorly soluble drugs in vivo. However, these constructs often encounter barriers for drug delivery on both the systemic and subcellular level. Many soluble polymer carriers have been designed to surmount specific physiological barriers individually, but less work has been dedicated to designing an all-encompassing construct that addresses multiple therapeutic barriers at once. Incorporation of multiple agents already individually known to increase effectiveness into one carrier could further improve current drug delivery technology. Recent developments in subcellular delivery of therapeutic agents in soluble macromolecular carriers are discussed in the context of the future possibility for the design of an all-encompassing soluble multi-functional drug delivery vehicle.

Animals↗

PubMed2000

Modulating fibroblast adhesion, spreading, and proliferation using self-assembled monolayer films of alkylthiolates on gold.

Ultrathin, highly organized functionalized alkylthiol monolayers were applied as model substrates for cell growth and protein adsorption studies. The aim of this approach was to improve the understanding of molecular surface determinants required for adhesion-dependent cell growth and proliferation using well-controlled surface chemistry. Carboxyl- and methyl-terminated alkylthiol monolayers on gold were used to monitor Swiss 3T3 fibroblast adhesion, spreading, and growth. Stress fiber and focal contact formation were determined by immunostaining of actin filaments and paxillin. Fibronectin deposition and conformation on these surface chemistries in the presence and absence of competing proteins were also determined. The relative levels of adsorbed fibronectin were assessed using radiolabeled proteins. Exposure of the 10th type III cell integrin binding domain of fibronectin was assessed using a radiolabeled monoclonal antibody. Distinct alkylthiol substrate chemistry-dependent differences were observed in fibroblast adhesion, spreading, and growth. The formation of focal contacts and stress fibers was enhanced on the carboxyl-terminated surface relative to the methyl surface. Relative deposition and conformations of adsorbed fibronectin were shown to be dependent on surface chemistry in both the presence and absence of competing proteins. The results indicated that well-controlled culture surfaces modulate differential cell adhesion, spreading, and growth through modulations of the amounts and conformations of adsorbed extracellular matrix molecules (e.g., fibronectin).

3T3 Cells↗

PubMed2000

A novel spinal implant infection model in rabbits.

STUDY DESIGN: A new spinal implant model was designed to study device-centered infection with methicillin-resistant Staphylococcus aureus in multiple noncontiguous surgical sites in the lumbar spine region of a rabbit. OBJECTIVE: To develop a multiple-site spinal implant device-centered infection model in rabbits. SUMMARY OF BACKGROUND DATA: Results in many recent studies show that postoperative wound infection after spinal implant surgery and the increase in antibiotic-resistant bacteria are a concern. Anti-infection strategies must be tested in relevant animal models that will lead to appropriate clinical studies. METHODS: Eight anesthetized New Zealand White rabbits underwent completely isolated partial laminectomy and subsequent stainless steel Kirschner wire implantation directly into the transverse processes of vertebrae T13, L3, and L6. The middle sites (L3) were used as sterile control sites, and the outer sites (T13, L6) were challenged with different amounts of methicillin-resistant Staphylococcus aureus. Rabbits were killed after 7 days, and biopsies were performed to provide evidence for device-centered infection. Bacterial growth on the implant surfaces and in surrounding tissues and bone was assayed. RESULTS: Overall device-centered infection was established after 7 days in 100% of the sites challenged with 10(3) colony-forming units methicillin-resistant Staphylococcus aureus or higher. No infection was seen in any of the control sites located between infected vertebrae. Multiple blood and liver samples showed that the separate localized infections did not become systemic after 7 days. CONCLUSIONS: This new animal model demonstrates that multiple biomaterial implants can be evaluated in the same animal and provides a technique for investigating postoperative device-centered infection of the spine. Infection was demonstrated in noncontiguous lumbar sites of the spine, whereas adjacent control sites remained sterile. Because there was no cross contamination or systemic spread of the infection, multiple anti-infection strategies or implant materials can now be tested for efficacy in a single animal to combat dramatic and costly postoperative implant infections.

Animals↗

PubMed2000

Polyurethane coatings release bioactive antibodies to reduce bacterial adhesion.

This study describes the formulation of a biomedical grade polyurethane hydrogel coating containing solid dispersed bioactive antibodies cast from an organic solvent onto a model polymer biomaterial substrate. A prepolymer dispersion in anhydrous isopropanol containing a uniformly distributed slurry of 22 microm sieved commercial lyophilized polyclonal pooled human immunoglobulin G (IgG) solids was coated onto polymer substrates by simple immersion. Maximum antibody release was approximately 50 microg/cm(2) from a 15% w/w IgG polymer coating. In vitro antimicrobial studies utilized Escherichia coli to compare performance of bare uncoated tubing, hydrogel-coated tubing with added aqueous phase antibodies, and antibody-dispersed hydrogel-coated tubing. Bacterial adhesion was reduced significantly (p<0.05) in the presence of antibodies with the greatest reduction seen with the antibody releasing coating. The presence of antibody also significantly enhanced the killing of the bacteria in an in vitro opsonophagocytic assay using freshly isolated blood neutrophils over 2 h indicating that antibody bioactivity is maintained. This controlled release polyurethane hydrogel coating imparts infection resistance by exploiting the low adhesive properties of the biomedical grade hydrogel and the intrinsic bioactive role of the antibodies to reduce bacterial adhesion and promote clearance via natural immune mechanisms.

Antibodies, Bacterial↗

PubMed2000

Pooled human immunoglobulins reduce adhesion of Pseudomonas aeruginosa in a parallel plate flow chamber.

The influence of pooled polyclonal immunoglobulin (IgG) interactions with both bacteria and model substrates in altering Pseudomonas aeruginosa surface adhesion is reported. Opsonization of this pathogen by polyclonal human IgG and preadsorption of IgG to glass surfaces both effectively reduce initial deposition rates and surface growth of P. aeruginosa IFO3455 from dilute nutrient broth in a parallel plate flow chamber. Polyclonal IgG depleted of P. aeruginosa-specific antibodies reduces the initial deposition rate or surface growth to levels intermediate between exposed and nonexposed IgG conditions. Bacterial surface properties are changed in the presence of opsonizing IgG. Plateau contact angle analysis via sessile drop technique shows a drop in P. aeruginosa surface hydrophobicity after IgG exposure consistent with a more hydrophilic IgG surface coat. Zeta potential values for opsonized versus nonopsonized bacteria exhibit little change. X-ray photoelectron spectroscopy measurements provide surface compositional evidence for IgG attachment to bacterial surfaces. Surface elemental ratios attributed to IgG protein signals versus those attributed primarily to bacterial polysaccharide surface or lipid membrane change with IgG opsonization. Direct evidence for antibody-modified P. aeruginosa surface properties correlates both with reduction of bacterial adhesion to glass surfaces under flow in nutrient medium reported and previous reports of IgG efficacy against P. aeruginosa motility in vitro and infection in vivo.

Adsorption↗

PubMed2000

Surgical irrigation with pooled human immunoglobulin G to reduce post-operative spinal implant infection.

A multiple-site, nonlethal rabbit surgical model of spinal implant infection was used to assess the efficacy of a spinal wound lavage to reduce post-operative infection from methicillinresistant Staphylococcus aureus (MRSA). Multiple aqueous lavages of isotonic saline were compared to the same procedure using 1wt% pooled human immunoglobulin G (IgG) applied directly to the surgical implant sites. Visually observed clinically relevant signs of infection (e.g. , swelling, erythema, pus) were supported by bacterial enumeration from multiple biopsied tissue and bone sites post-mortem at 7 and 28 days post-challenge. Clinical signs of infection were significantly reduced in IgG-lavaged infected spinal sites. Bacterial enumeration also exhibited statistically significant reductions in soft tissues, bone and on K-wire spinal implants using IgG lavage compared with saline. Complete healing of all surgical wounds was seen after 28 days, although isolated fibrosed abscesses were observed in autopsied sites treated with both IgG and saline lavages. Local use of IgG wound lavage is proposed as supplementary infection prophylaxis against antibiotic resistant implant-centered or surgical wound infection.

Animals↗

PubMed2000

Locally delivered antibodies combined with systemic antibiotics confer synergistic protection against antibiotic-resistant burn wound infection.

BACKGROUND: Nosocomially derived gram-negative infections, particularly from antibiotic-resistant pathogens, are a cause of morbidity in patients with severe burn wounds. METHODS: Locally delivered polyclonal antibodies and systemically infused ceftazidime were combined in a lethal murine burn wound model against a virulent Pseudomonas aeruginosa strain that exhibits intermediate resistance to ceftazidime. RESULTS: Survival was synergistically enhanced in cohorts of burned mice treated both locally (subeschar) with pooled polyclonal human immunoglobulin G (1-mg dose) and intravenously with infused ceftazidime (0.44 mg dose). Enhancement of survival correlated with reduced bacterial quantitation in local and systemic tissue observed in separate burned cohorts. Burned, infected mice treated prophylactically with either individual treatment at the same dose or a combination of both treatments administered systemically showed no survival enhancement as compared with the untreated control group. CONCLUSION: Treatment of antibiotic-resistant burn wound infections with antibiotics together with locally delivered immunoglobulins may improve antibiotic protective effects against antibiotic-resistant pathogens.

Animals↗

PubMed1999

Neutral lipids induce critical behavior in interfacial monolayers of pulmonary surfactant.

We have shown previously that lateral compression of pulmonary surfactant monolayers initially induces separation of two phases but that these remix when the films become more dense (1). In the studies reported here, we used fluorescence microscopy to examine the role of the different surfactant constituents in the remixing of the separated phases. Subfractions containing only the purified phospholipids (PPL), the surfactant proteins and phospholipids (SP&PL), and the neutral and phospholipids (N&PL) were obtained by chromatographic separation of the components in extracted calf surfactant (calf lung surfactant extract, CLSE). Compression of the different monolayers produced nonfluorescent domains that emerged for temperatures between 20 and 41 degreesC at similar surface pressures 6-8 mN/m higher than values observed for dipalmitoyl phosphatidylcholine (DPPC), the most prevalent component of pulmonary surfactant. Comparison of the different preparations showed that the neutral lipid increased the total nonfluorescent area at surface pressures up to 25 mN/m but dispersed that total area among a larger number of smaller domains. The surfactant proteins also produced smaller domains, but they had the opposite effect of decreasing the total nonfluorescent area. Only the neutral lipids caused remixing. In images from static monolayers, the domains for N&PL dropped from a maximum of 26 +/- 3% of the interface at 25 mN/m to 4 +/- 2% at 30 mN/m, similar to the previously reported behavior for CLSE. During continuous compression through a narrow range of pressure and molecular area, in N&PL, CLSE, and mixtures of PPL with 10% cholesterol, domains became highly distorted immediately prior to remixing. The characteristic transition in shape and abrupt termination of phase coexistence indicate that the remixing caused by the neutral lipids occurs at or close to a critical point.

Animals↗

PubMed1999

RhoA-induced changes in fibroblasts cultured on organic monolayers.

Substantial previous work indicates that adherent cell morphology in culture is modulated by surface chemistry. Activation of the intracellular small molecular weight GTPase, RhoA, has recently been shown to play an essential role in controlling initiation of key integrin-mediated events in surface adhesion and proliferation. RhoA is interconvertible between an active, membrane-bound form and an inactive, cytosolic RhoGDI-bound form in response to integrin stimulation. This study reports the use of self-assembled functionalized organic alkylthiol monolayers (SAMs) as well-defined cell culture substrates to investigate the relationships between surface chemistry, RhoA activation and subsequent cell morphological and molecular level signal transduction responses in cells attaching to derivatized SAMs. Well-controlled alkylthiol surface chemistries were used to monitor and modulate the activation state of RhoA in attaching cells. Activation states were determined indirectly by fractionating cell lysates into membrane and cytosolic fractions by ultracentrifugation. Western blots were then performed, showing RhoA localization to be surface chemistry-dependent. RhoGDI levels and its intracellular localization were also shown to be surface-chemistry dependent. Cells cultured on -CH3 terminated SAMs, which normally exhibit a low-growth phenotype, were transfected with a constitutively active mutant form of RhoA. Subsequent cell morphological changes were observed on SAM surfaces by fluorescence microscopy. Results support surface chemistry influences on the activation state of RhoA mediated by adsorbed proteins and distinct changes in adherent cell morphology resulting from modulation of this activation state.

3T3 Cells↗

PubMed1999

Efficacy of locally delivered polyclonal immunoglobulin against Pseudomonas aeruginosa infection in a murine burn wound model.

The leading cause of morbidity and mortality in severe burn wound patients is infection. Treatment of burn wound infection is complicated by the emergence of antibiotic resistant organisms. A potential therapeutic alternative to antibiotic drugs is the local administration of polyclonal antibodies, termed passive local immunotherapy (PLI), directly to the burned tissue. A mouse burn wound infection model to simulate full thickness burn wound infection was used to evaluate the efficacy of passive local immunotherapy as a viable prophylactic or therapeutic agent. Pooled human immunoglobulins (IgG), delivered locally to the site of infection, are shown to be more effective at preventing fatal burn wound sepsis than treatment by intravenous infusion of IgG. A single 10 mg dose of human IgG administered locally to the burned, infected tissue site, either 24 hours prior to bacterial challenge, or within 3 hours after bacterial challenge, enhanced animal survival significantly (P < 0.001 and P < 0.05 respectively) compared to control animals. In addition, reduced levels of bacteria were found in local and systemic tissues of IgG-treated mice compared to control mice (P < 0.05). These data support the local use of polyclonal immunoglobulin preparations as an efficacious and cost effective means to prevent and treat burn wound infections.

Animals↗

PubMed1999

Efficacy of locally delivered polyclonal immunoglobulin against Pseudomonas aeruginosa peritonitis in a murine model.

Infectious peritonitis results from bacterial contamination of the abdominal cavity. Conventional antibiotic treatment is complicated both by the emergence of antibiotic-resistant bacteria and by increased patient populations intrinsically at risk for nosocomial infections. To complement antibiotic therapies, the efficacy of direct, locally applied pooled human immunoglobulin G (IgG) was assessed in a murine model (strains CF-1, CD-1, and CFW) of peritonitis caused by intraperitoneal inoculations of 10(6) or 10(7) CFU of Pseudomonas aeruginosa (strains IFO-3455, M-2, and MSRI-7072). Various doses of IgG (0.005 to 10 mg/mouse) administered intraperitoneally simultaneously with local bacterial challenge significantly increased survival in a dose-dependent manner. Local intraperitoneal application of 10 mg of IgG increased animal survival independent of either the P. aeruginosa or the murine strains used. A local dose of 10 mg of IgG administered up to 6 h prophylactically or at the time of bacterial challenge resulted in 100% survival. Therapeutic 10-mg IgG treatment given up to 12 h postinfection also significantly increased survival. Human IgG administered to the mouse peritoneal cavity was rapidly detected systemically in serum. Additionally, administered IgG in peritoneal lavage fluid samples actively opsonized and decreased the bacterial burden via phagocytosis at 2 and 4 h post-bacterial challenge. Tissue microbial quantification studies showed that 1.0 mg of locally applied IgG significantly reduced the bacterial burden in the liver, peritoneal cavity, and blood and correlated with reduced levels of interleukin-6 in serum.

Animals↗

PubMed1996

Lateral phase separation in interfacial films of pulmonary surfactant.

To determine if lateral phase separation occurs in films of pulmonary surfactant, we used epifluorescence microscopy and Brewster angle microscopy (BAM) to study spread films of calf lung surfactant extract (CLSE). Both microscopic methods demonstrated that compression produced domains of liquid-condensed lipids surrounded by a liquid-expanded film. The temperature dependence of the pressure at which domains first emerged for CLSE paralleled the behavior of its most prevalent component, dipalmitoyl phosphatidylcholine (DPPC), although the domains appeared at pressures 8-10 mN/m higher than for DPPC over the range of 20-37 degrees C. The total area occupied by the domains at room temperature increased to a maximum value at 35 mN/m during compression. The area of domains reached 25 +/- 5% of the interface, which corresponds to the predicted area of DPPC in the monolayer. At pressures above 35 mN/m, however, both epifluorescence and BAM showed that the area of the domains decreased dramatically. These studies therefore demonstrate a pressure-dependent gap in the miscibility of surfactant constituents. The monolayers separate into two phases during compression but remain largely miscible at higher and lower surface pressures.

Animals↗