Chemical and Physical Variability in Structural Isomers of an l/d α-Sheet Peptide Designed To Inhibit Amyloidogenesis.

There has been much interest in synthetic peptides as inhibitors of aggregation associated with amyloid diseases. Of particular interest are compounds that target the cytotoxic soluble oligomers preceding the formation of mature, nontoxic fibrils. This study explores physical and chemical differences between two de novo-designed peptides that share an identical primary structure but differ in backbone chirality at six key positions. We show that the presence of alternating l/d-amino acid motifs dramatically increases aqueous solubility, enforces α-sheet secondary structure, and inhibits aggregation of the ß-amyloid peptide implicated in Alzheimer's disease, in addition to neutralizing its cytotoxicity. In contrast, the all-l-amino acid isomer does not form α-sheet structure and is insoluble and inactive.

Scaffold-mediated delivery for non-viral mRNA vaccines.

mRNA is increasingly being recognized as a promising alternative to pDNA in gene vaccinations. Only recently, owing to the needs of cancer immunotherapies, has the biomaterials/gene delivery community begun to develop new biomaterial strategies for immunomodulation. Here, we report a novel way to use implantable porous scaffolds as a local gene delivery depot to enhance mRNA vaccine immunization in vitro, and in vivo when compared with conventional bolus injections. We first evaluated transfection efficiencies of single-stranded mRNA condensed and charge neutralized with two lipids (Lipofectamine Messenger MAXTM LM-MM and StemfectTM SF) and two cationic polymers (in vivo-jetPEI™, Poly (ß-amino ester)) as gene carriers. As SF demonstrated highest in vitro transfection and cell viability, it was selected for subsequent porous polymer scaffold-loading trials. Enhanced in vitro transfection of SF:mRNA nanoparticle-loaded poly (2-hydroxyethyl methacrylate) (pHEMA) scaffolds was also observed with a DC2.4 cell line. Improved sustained local release and local transgene expression were also demonstrated with SF:mRNA nanoparticle-loaded pHEMA scaffolds in vivo compared with bolus injections. Our results suggest that mRNA polyplex-loaded scaffolds may be a superior alternative to either repeated bolus immunizations or ex vivo transfection cell immunotherapies.

Precision-porous templated scaffolds of varying pore size drive dendritic cell activation.

Scaffold based systems have shown significant potential in modulating immune responses in vivo. While there has been much attention on macrophage interactions with tissue engineered scaffolds for tissue regeneration, fewer studies have looked at the effects of scaffold design on the response of immune cells-that is, dendritic cells (DCs). Here, we present the effects of varying pore size of poly (2-hydroxyethyl methacrylate) (pHEMA) and poly(dimethylsiloxane) (PDMS, silicone) scaffolds on the maturation and in vivo enrichment of DCs. We employ a precision templating method to make 3-D porous polymer scaffolds with uniformly defined and adjustable architecture. Hydrophilic pHEMA and hydrophobic PDMS scaffolds were fabricated in three pore sizes (20, 40, 90 µm) to quantify scaffold pore size effects on DCs activation/maturation in vitro and in vivo. In vitro results showed that both pHEMA and PDMS scaffolds could promote maturation in the DC cell line, JAWSII, that resembled lipopolysaccharide (LPS)-activated/matured DCs (mDCs). Scaffolds with smaller pore sizes correlate with higher DC maturation, regardless of the polymer used. In vivo, when implanted subcutaneously in C57BL/6J mice, scaffolds with smaller pore sizes also demonstrated more DCs recruitment and more sustained activation. Without the use of DC chemo-attractants or chemical adjuvants, our results suggested that DC maturation and scaffold infiltration profile can be modulated by simply altering the pore size of the scaffolds.

Multispectral Optical Tweezers for Biochemical Fingerprinting of CD9-Positive Exosome Subpopulations.

Extracellular vesicles (EVs), including exosomes, are circulating nanoscale particles heavily implicated in cell signaling and can be isolated in vast numbers from human biofluids. Study of their molecular profiling and materials properties is currently underway for purposes of describing a variety of biological functions and diseases. However, the large, and as yet largely unquantified, variety of EV subpopulations differing in composition, size, and likely function necessitates characterization schemes capable of measuring single vesicles. Here we describe the first application of multispectral optical tweezers (MS-OTs) to single vesicles for molecular fingerprinting of EV subpopulations. This versatile imaging platform allows for sensitive measurement of Raman chemical composition (e.g., variation in protein, lipid, cholesterol, nucleic acids), coupled with discrimination by fluorescence markers. For exosomes isolated by ultracentrifugation, we use MS-OTs to interrogate the CD9-positive subpopulations via antibody fluorescence labeling and Raman spectra measurement. We report that the CD9-positive exosome subset exhibits reduced component concentration per vesicle and reduced chemical heterogeneity compared to the total purified EV population. We observed that specific vesicle subpopulations are present across exosomes isolated from cell culture supernatant of several clonal varieties of mesenchymal stromal cells and also from plasma and ascites isolated from human ovarian cancer patients.

Porous Precision-Templated 40 µm Pore Scaffolds Promote Healing through Synergy in Macrophage Receptor with Collagenous Structure and Toll-Like Receptor Signaling.

Porous precision-templated scaffolds (PTS) with uniform, interconnected, 40 µm pores have shown favorable healing outcomes and a reduced foreign body reaction (FBR). Macrophage receptor with collagenous structure (MARCO) and toll-like receptors (TLRs) have been identified as key surface receptors in the initial inflammatory phase of wound healing. However, the role of MARCO and TLRs in modulating monocyte and macrophage phenotypes within PTS remains uncharacterized. In this study, we demonstrate a synergetic relationship between MARCO and TLR signaling in cells inhabiting PTS, where induction with TLR3 or TLR4 agonists to 40 µm scaffold-resident cells upregulates the transcription of MARCO. Upon deletion of MARCO, the prohealing phenotype within 40 µm PTS polarizes to a proinflammatory and profibrotic phenotype. Analysis of downstream TLR signaling shows that MARCO is required to attenuate nuclear factor kappa B (NF-κB) inflammation in 40 µm PTS by regulating the transcription of inhibitory NFKB inhibitor alpha (NFKBIA) and interleukin-1 receptor-associated kinase 3 (IRAK-M), primarily through a MyD88-dependent signaling pathway. Investigation of implant outcome in the absence of MARCO demonstrates an increase in collagen deposition within the scaffold and the development of tissue fibrosis. Overall, these results further our understanding of the molecular mechanisms underlying MARCO and TLR signaling within PTS. Impact statement Monocyte and macrophage phenotypes in the foreign body reaction (FBR) are essential for the development of a proinflammatory, prohealing, or profibrotic response to implanted biomaterials. Identification of key surface receptors and signaling mechanisms that give rise to these phenotypes remain to be elucidated. In this study, we report a synergistic relationship between macrophage receptor with collagenous structure (MARCO) and toll-like receptor (TLR) signaling in scaffold-resident cells inhabiting porous precision-templated 40 µm pore scaffolds through a MyD88-dependent pathway that promotes healing. These findings advance our understanding of the FBR and provide further evidence that suggests MARCO, TLRs, and fibrosis may be interconnected.

Chemokine programming dendritic cell antigen response: part II - programming antigen presentation to T lymphocytes by partially maintaining immature dendritic cell phenotype.

In a companion article to this study,(1) the successful programming of a JAWSII dendritic cell (DC) line's antigen uptake and processing was demonstrated based on pre-treatment of DCs with a specific 'cocktail' of select chemokines. Chemokine pre-treatment modulated cytokine production before and after DC maturation [by lipopolysaccharide (LPS)]. After DC maturation, it induced an antigen uptake and processing capacity at levels 36% and 82% higher than in immature DCs, respectively. Such programming proffers a potential new approach to enhance vaccine efficiency. Unfortunately, simply enhancing antigen uptake does not guarantee the desired activation and proliferation of lymphocytes, e.g. CD4(+) T cells. In this study, phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs were examined in long-term co-culture with antigen-specific CD4(+) T cells to quantify how chemokine pre-treatment may impact the adaptive immune response. When a model antigen, ovalbumin (OVA), was added after intentional LPS maturation of chemokine-treated DCs, OVA-biased CD4(+) T-cell proliferation was initiated from ~ 100% more undivided naive T cells as compared to DCs treated only with LPS. Secretion of the cytokines interferon-γ, interleukin-1ß, interleukin-2 and interleukin-10 in the CD4(+) T cell : DC co-culture (with or without chemokine pre-treatment) were essentially the same. Chemokine programming of DCs with a 7 : 3 ratio of CCL3 : CCL19 followed by LPS treatment maintained partial immature phenotypes of DCs, as indicated by surface marker (CD80 and CD86) expression over time. Results here and in our companion paper suggest that chemokine programming of DCs may provide a novel immunotherapy strategy to obviate the natural endocytosis limit of DC antigen uptake, thus potentially increasing DC-based vaccine efficiency.

Chemokine programming dendritic cell antigen response: part I - select chemokine programming of antigen uptake even after maturation.

Here, we report on the successful programming of dendritic cells (DCs) using selectively applied mixtures of chemokines as a novel protocol for engineering vaccine efficiency. Antigen internalization by DCs is a pivotal step in antigen uptake/presentation for bridging innate and adaptive immunity and in exogenous gene delivery used in vaccine strategies. Contrary to most approaches to improve vaccine efficiency, active enhancement of antigen internalization by DCs as a vaccine strategy has been less studied because DCs naturally down-regulate antigen internalization upon maturation. Whereas chemokines are mainly known as signal proteins that induce leucocyte chemotaxis, very little research has been carried out to identify any additional effects of chemokines on DCs following maturation. Here, immature DCs are pre-treated with select chemokines before intentional maturation using lipopolysaccharide (LPS). When pre-treated with a mixture of CCL3 and CCL19 in a 7 : 3 ratio, then matured with LPS, chemokine pre-treated DCs exhibited 36% higher antigen uptake capacity than immature DCs and 27% higher antigen-processing capacity than immature DCs treated only with LPS. Further, CCL3 : CCL19 (7 : 3) pre-treatment of DCs modulated MHC molecule expression and secretion of various cytokines of DCs. Collectively, DC programming was feasible using a specific chemokine combination and these results provide a novel strategy for enhancing DC-based vaccine efficiency. In Part II, we report on the phenotype changes and antigen presentation capacity of chemokine pre-treated murine bone marrow-derived DCs examined in long-term co-culture with antigen-specific CD4(+) T cells.

Non-invasive in situ monitoring and quantification of TOL plasmid segregational loss within Pseudomonas putida biofilms.

Methods for the detection of plasmid loss in natural environments have typically relied on replica plating, selective markers and PCR. However, these traditional methods have the limitations of low sensitivity, underestimation of specific cell populations, and lack of insightful data for non-homogeneous environments. We have developed a non-invasive microscopic analytical method to quantify local plasmid segregational loss from a bacterial population within a developing biofilm. The probability of plasmid segregational loss in planktonic and biofilm cultures of Pseudomonas putida carrying the TOL plasmid (pWWO::gfpmut3b) was determined directly in situ, in the absence of any applied selection pressure. Compared to suspended liquid culture, we report that the biofilm mode of growth enhances plasmid segregational loss. Results based on a biofilm-averaged analysis reveal that the probability of plasmid loss in biofilm cultures (0.016 ± 0.004) was significantly greater than that determined in planktonic cultures (0.0052 ± 0.0011). Non-invasive assessments showed that probabilities of plasmid segregational loss at different locations in a biofilm increased dramatically from 0.1% at the substratum surface to 8% at outside layers of biofilm. Results suggest that higher nutrient concentrations and subsequentially higher growth rates resulted in higher probability of plasmid segregational loss at the outer layers of the biofilm.

Non-invasive determination of conjugative transfer of plasmids bearing antibiotic-resistance genes in biofilm-bound bacteria: effects of substrate loading and antibiotic selection.

Biofilms cause much of all human microbial infections. Attempts to eradicate biofilm-based infections rely on disinfectants and antibiotics. Unfortunately, biofilm bacteria are significantly less responsive to antibiotic stressors than their planktonic counterparts. Sublethal doses of antibiotics can actually enhance biofilm formation. Here, we have developed a non-invasive microscopic image analyses to quantify plasmid conjugation within a developing biofilm. Corroborating destructive samples were analyzed by a cultivation-independent flow cytometry analysis and a selective plate count method to cultivate transconjugants. Increases in substrate loading altered biofilm 3-D architecture and subsequently affected the frequency of plasmid conjugation (decreases at least two times) in the absence of any antibiotic selective pressure. More importantly, donor populations in biofilms exposed to a sublethal dose of kanamycin exhibited enhanced transfer efficiency of plasmids containing the kanamycin resistance gene, up to tenfold. However, when stressed with a different antibiotic, imipenem, transfer of plasmids containing the kan(R+) gene was not enhanced. These preliminary results suggest biofilm bacteria "sense" antibiotics to which they are resistant, which enhances the spread of that resistance. Confocal scanning microscopy coupled with our non-invasive image analysis was able to estimate plasmid conjugative transfer efficiency either averaged over the entire biofilm landscape or locally with individual biofilm clusters.

Giant extracellular matrix binding protein expression in Staphylococcus epidermidis is regulated by biofilm formation and osmotic pressure.

Staphylococcus epidermidis is an opportunistic bacterium that thrives as a commensal cutaneous organism and as a vascular pathogen. The S. epidermidis extracellular matrix binding protein (Embp) has been reported to be a virulence factor involved in colonization of medical device implants and subsequent biofilm formation. Here, we characterize the expression patterns of Embp in planktonic and biofilm cultures, as well as under high osmotic stresses that typify the commensal environment of the skin. Embp expression without osmotic stress was similar for planktonic and adherent cultures. Addition of osmotic stress via NaCl caused slight increases in embp expression in planktonic cultures. However, in adherent cultures a 100-fold increase in embp expression with NaCl versus controls occurred and coincided with altered biofilm morphology. Results suggest that the central role of Embp lies in commensal skin colonization, stabilizing the cell wall against osmotic stresses, rather than as a virulence factor promoting adhesion.

Designed α-sheet peptides disrupt uropathogenic E. coli biofilms rendering bacteria susceptible to antibiotics and immune cells.

Uropathogenic Escherichia coli account for the largest proportion of nosocomial infections in the United States. Nosocomial infections are a major source of increased costs and treatment complications. Many infections are biofilm associated, rendering antibiotic treatments ineffective or cause additional complications (e.g., microbiome depletion). This work presents a potentially complementary non-antibiotic strategy to fight nosocomial infections by inhibiting the formation of amyloid fibrils, a proteinaceous structural reinforcement known as curli in E. coli biofilms. Despite extensive characterization of the fibrils themselves and their associated secretion system, mechanistic details of curli assembly in vivo remain unclear. We hypothesized that, like other amyloid fibrils, curli polymerization involves a unique secondary structure termed "α-sheet". Biophysical studies herein confirmed the presence of α-sheet structure in prefibrillar species of CsgA, the major component of curli, as it aggregated. Binding of synthetic α-sheet peptides to the soluble α-sheet prefibrillar species inhibited CsgA aggregation in vitro and suppressed amyloid fibril formation in biofilms. Application of synthetic α-sheet peptides also enhanced antibiotic susceptibility and dispersed biofilm-resident bacteria for improved uptake by phagocytic cells. The ability of synthetic α-sheet peptides to reduce biofilm formation, improve antibiotic susceptibility, and enhance clearance by macrophages has broad implications for combating biofilm-associated infections.

Novel HALO® image analysis to determine cell phenotype in porous precision-templated scaffolds.

Image analysis platforms have gained increasing popularity in the last decade for the ability to automate and conduct high-throughput, multiplex, and quantitative analyses of a broad range of pathological tissues. However, imaging tissues with unique morphology or tissues containing implanted biomaterial scaffolds remain a challenge. Using HALO®, an image analysis platform specialized in quantitative tissue analysis, we have developed a novel method to determine multiple cell phenotypes in porous precision-templated scaffolds (PTS). PTS with uniform spherical pores between 30 and 40 µm in diameter have previously exhibited a specific immunomodulation of macrophages toward a pro-healing phenotype and an overall diminished foreign body response (FBR) compared to PTS with larger or smaller pore sizes. However, signaling pathways orchestrating this pro-healing in 40 µm PTS remain unclear. Here, we use HALO® to phenotype PTS resident cells and found a decrease in pro-inflammatory CD86 and an increase in pro-healing CD206 expression in 40 µm PTS compared to 100 µm PTS. To understand the mechanisms that drive these outcomes, we investigated the role of myeloid-differentiation-primary-response gene 88 (MyD88) in regulating the pro-healing phenomenon observed only in 40 µm PTS. When subcutaneously implanted in MyD88KO mice, 40 µm PTS reduced the expression of CD206, and the scaffold resident cells displayed an average larger nuclear size compared to 40 µm PTS implanted in mice expressing MyD88. Overall, this study demonstrates a novel image analysis method for phenotyping cells within PTS and identifies MyD88 as a critical mediator in the pore-size-dependent regenerative healing and host immune response to PTS.

Engineering biomaterials to integrate and heal: the biocompatibility paradigm shifts.

This article focuses on one of the major failure routes of implanted medical devices, the foreign body reaction (FBR)--that is, the phagocytic attack and encapsulation by the body of the so-called "biocompatible" biomaterials comprising the devices. We then review strategies currently under development that might lead to biomaterial constructs that will harmoniously heal and integrate into the body. We discuss in detail emerging strategies to inhibit the FBR by engineering biomaterials that elicit more biologically pertinent responses.

Antimicrobial effects of nanofiber poly(caprolactone) tissue scaffolds releasing rifampicin.

This study quantified the antibiotic release kinetics and subsequent bactericidal efficacy of rifampicin (RIF) against Gram-positive and Gram-negative bacteria under in vitro static conditions. Antibiotic-loaded scaffolds were fabricated by electrospinning poly(caprolactone) (PCL) with 10% or 20% (w/w) RIF. Scaffold fiber diameter and RIF loading were characterized, and RIF release kinetics were measured. RIF-releasing and RIF-free scaffolds were inoculated with Pseudomonas aeruginosa and Staphylococcus epidermidis, and the suspended concentration live and dead bacteria were determined by fluorescent microscopy. Adherent bacteria and biofilm formation were examined using scanning electron microscopy. Mean fiber diameters were 557 ± 399 nm for RIF-free, 402 ± 225 nm for 10% RIF, and 665 ± 402 nm for 20% RIF scaffolds. RIF release kinetics exhibited a short-burst release during the first hour, followed by a 7 h, zero-order release during which both RIF scaffolds released ~50% of their initial RIF mass loading. P. aeruginosa and S. epidermidis suspended cell populations proliferated in accordance with logarithmic growth models when exposed to control scaffolds; however both RIF-containing scaffolds completely inhibited bacterial growth in suspension and, subsequently, prevented biofilm formation within the scaffolds through the first 6 h.

Monocytes contribute to a pro-healing response in 40 µm diameter uniform-pore, precision-templated scaffolds.

Porous precision-templated scaffolds (PTS) with uniformly distributed 40 µm spherical pores have shown a remarkable ability in immunomodulating resident cells for tissue regeneration. While the pore size mediated pro-healing response observed only in 40 µm pore PTS has been attributed to selective macrophage polarization, monocyte recruitment and phenotype have largely been uncharacterized in regulating implant outcome. Here, we employ a double transgenic mouse model for myeloid characterization and a multifaceted phenotyping approach to quantify monocyte dynamics within subcutaneously implanted PTS. Within 40 µm PTS, myeloid cells were found to preferentially infiltrate into the scaffold. Additionally, macrophage receptor with collagenous structure (MARCO), an innate activation marker, was significantly upregulated within 40 µm PTS. When 40 µm PTS were implanted in monocyte-depleted mice, the transcription of MARCO was significantly decreased and an increase in pro-inflammatory inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNFα) were observed. Typical of a foreign body response (FBR), 100 µm PTS significantly upregulated pro-inflammatory iNOS, secreted higher amounts of TNFα, and displayed a pore size dependent morphology compared to 40 µm PTS. Overall, these results identify a pore size dependent modulation of circulating monocytes and implicates MARCO expression as a defining subset of monocytes that appears to be responsible for regulating a pro-healing host response.

Uniform 40-µm-pore diameter precision templated scaffolds promote a pro-healing host response by extracellular vesicle immune communication.

Implanted porous precision templated scaffolds (PTS) with 40-µm spherical pores reduce inflammation and foreign body reaction (FBR) while increasing vascular density upon implantation. Larger or smaller pores, however, promote chronic inflammation and FBR. While macrophage (MØ) recruitment and polarization participates in perpetuating this pore-size-mediated phenomenon, the driving mechanism of this unique pro-healing response is poorly characterized. We hypothesized that the primarily myeloid PTS resident cells release small extracellular vesicles (sEVs) that induce pore-size-dependent pro-healing effects in surrounding T cells. Upon profiling resident immune cells and their sEVs from explanted 40-µm- (pro-healing) and 100-µm-pore diameter (inflammatory) PTS, we found that PTS pore size did not affect PTS resident immune cell population ratios or the proportion of myeloid sEVs generated from explanted PTS. However, quantitative transcriptomic assessment indicated cell and sEV phenotype were pore size dependent. In vitro experiments demonstrated the ability of PTS cell-derived sEVs to stimulate T cells transcriptionally and proliferatively. Specifically, sEVs isolated from cells inhabiting explanted 100 µm PTS significantly upregulated Th1 inflammatory gene expression in immortalized T cells. sEVs isolated from cell inhabiting both 40- and 100-µm PTS upregulated essential Treg transcriptional markers in both primary and immortalized T cells. Finally, we investigated the effects of Treg depletion on explanted PTS resident cells. FoxP3+ cell depletion suggests Tregs play a unique role in balancing T cell subset ratios, thus driving host response in 40-µm PTS. These results indicate that predominantly 40-µm PTS myeloid cell-derived sEVs affect T cells through a distinct, pore-size-mediated modality.

Non-invasive method to quantify local bacterial concentrations in a mixed culture biofilm.

To better engineer and analyze beneficial biofilms as well as to develop strategies to control detrimental biofilms (e.g., biomedical device-based infections), it is critical to quantify bacterial species compositions within biofilms. A non-invasive method is described here that determines local and overall bacterial concentrations within a biofilm, using optical microscopy and digital image analysis techniques. The method is based upon a calibration of cell fluorescence to known cell number concentrations and is verified by direct cell counts of destructive samples of cultivated biofilms. Two GFP mutants, each with unique emission colors were used with both epi-fluorescent microscopy and one-photon confocal microscopy to determine local spatial biofilm cell concentrations in pure and mixed-strain biofilms. Our microbial system comprises Pseudomonas putida containing either green fluorescent protein (GFP) or containing the red fluorescent protein (DsRed). Strains expressing a green or red fluorescent protein were detected by two different microscopy methods: epi-fluorescence and single-photon confocal laser scanning microscopy. Overall biofilm cell concentrations determined directly from destructive samples were in good agreement with non-invasive measurements of adherent cell concentrations calculated from the measured "integrated fluorescent density" minus any background fluorescence. Results show the areal cell concentration (cell number/area) determined from non-destructive direct counts in a pure culture or binary-strain biofilm varied with the biofilm depth. Use of this method to estimate local dynamic plasmid segregational loss and plasmid conjugation transfer kinetics will be reported in a subsequent manuscript.

Injectable Biodegradable Chitosan-Alginate 3D Porous Gel Scaffold for mRNA Vaccine Delivery.

mRNA vaccines have proven to be more stable, effective, and specific than protein/peptide-based vaccines in stimulating both humoral and cellular immune response. However, mRNA's fast degradation rate and low-transfection efficiency in vivo impede its potential in vaccination. Recent research in gene delivery has focused on nonviral vaccine carriers and either implantable or injectable delivery systems to improve transgene expression in vivo. Here, an injectable chitosan-alginate gel scaffold for the local delivery of mRNA vaccines is reported. Gel scaffold biodegradation rates and biocompatibility are quantified. Scaffold-mediated mRNA in vivo transgene expression as well as ovalbumin antigen specific cellular and humoral immune responses are evaluated in vivo. Luciferase reporter protein expression resulting from mRNA lipoplex-loaded gel scaffolds is five times higher than systemic injection. Compared to systemic injections of naked mRNA or mRNA:lipoplexes, elevated levels of T cell proliferation and IFN-γ secretion are seen with in vivo scaffold-mediated mRNA lipoplex delivery. Furthermore, a humoral response (ovalbumin antigen specific IgG levels) is observed as early as week 1 for scaffold-mediated mRNA lipoplex delivery, while protein-based immunization did not elicit IgG production until 2 weeks post-injection. Results suggest that injectable scaffold mRNA vaccine delivery maybe a viable alternative to traditional nucleic acid immunization methods.

Surface modification of a perfluorinated ionomer using a glow discharge deposition method to control protein adsorption.

Nafion is the membrane material preferred for in situ glucose sensors. Unfortunately, surface properties of Nafion promote random protein adsorption and eventual foreign body encapsulation thus leading to loss of glucose signal over time. Here we detail surface modifications made by RF plasma deposition to Nafion with the intent to prevent random protein adsorption while providing enough functional sites (hydroxyl groups) to bind a biologically active peptide known to induce cellular adhesion (YRGDS). Nafion surfaces were modified by RF plasma polymerizing five different combinations of (1) tetraethylene glycol dimethyl ether (tetraglyme) and (2) 2-hydroxyethyl methacrylate (HEMA): pure tetraglyme, 2.5% HEMA with 97.5% tetraglyme, 5% HEMA with 95% tetraglyme, 10% HEMA with 90% tetraglyme, and pure HEMA. Resultant surfaces were characterized by XPS (low and high resolution), dynamic contact angle, and atomic force microscopy. Protein adsorption and retention was determined and correlated to surface layer composition. The ability to bind a cell adhesion peptide was also determined and correlated well with surface layer composition.

Medical biofilms.

For more than two decades, Biotechnology and Bioengineering has documented research focused on natural and engineered microbial biofilms within aquatic and subterranean ecosystems, wastewater and waste-gas treatment systems, marine vessels and structures, and industrial bioprocesses. Compared to suspended culture systems, intentionally engineered biofilms are heterogeneous reaction systems that can increase reactor productivity, system stability, and provide inherent cell:product separation. Unwanted biofilms can create enormous increases in fluid frictional resistances, unacceptable reductions in heat transfer efficiency, product contamination, enhanced material deterioration, and accelerated corrosion. Missing from B&B has been an equivalent research dialogue regarding the basic molecular microbiology, immunology, and biotechnological aspects of medical biofilms. Presented here are the current problems related to medical biofilms; current concepts of biofilm formation, persistence, and interactions with the host immune system; and emerging technologies for controlling medical biofilms.