Characterization of Inflammatory and Fibrotic Encapsulation Responses of Implanted Materials with Bacterial Infection.

Medical device infections are costly, while preclinical assessment of antimicrobial properties for new materials is time intensive and imperfect at capturing the interrelated aspects of infection response and wound resolution. Herein, we developed an in vivo model for quantification of inflammatory and biocompatibility responses in the presence of a sustained implant-associated infection. The antimicrobial effectiveness of commercially available polymer materials was compared to that of thermoplastic polyurethane (TPU) materials modified with putative antimicrobial strategies as example test materials. Materials were incubated with bioluminescent Escherichia coli prior to implantation in a dorsal subcutaneous pocket in rats with an additional intraluminal bolus of bacteria. Infection kinetics were monitored with bioluminescence, and inflammatory infiltrate and fibrous capsule thickness were determined from stained histological sections. Our model resulted in a persistent infection, sensitive to antimicrobial effects, as the materials modified with a putative antimicrobial surface were able to significantly reduce the level of infection in animals at day 4 postimplantation with efficacy similar to that of commercially available antimicrobial drug-eluting polymers (positive controls). At day 30 postimplantation, the antimicrobial surface modified TPU tubing was found to promote complete elimination of intraluminal bacteria in the absence of antibiotics. Differences were also measurable in acute inflammation, as Wright-Giemsa staining demonstrated reduced inflammatory cell infiltration at day 4 postimplantation for antimicrobial TPU materials. Additionally, antimicrobial materials exhibited reduced fibrous capsule thickness coinciding with infection resolution, as compared to unmodified TPU controls. The developed model can be utilized for testing antimicrobial polymers in the context of a prolonged infection while also revealing concurrent differences in the infiltrating immune cell profiles and fibrous capsule thickness, thus improving the relevance of preclinical medical device material testing.

An ex vivo model of medical device-mediated bacterial skin translocation.

The skin is a barrier and part of the immune system that protects us from harmful bacteria. Because indwelling medical devices break this barrier, they greatly increase the risk of infection by microbial pathogens. To study how these infections can be prevented through improved clinical practices and medical device technology, it is important to have preclinical models that replicate the early stages of microbial contamination, ingress, and colonization leading up to infection. At present, there are no preclinical ex vivo models specifically developed to simulate conditions for indwelling medical devices. Translocation of pathogens from outside the body across broken skin to normally sterile internal compartments is a rate-limiting step in infectious pathogenesis. In this work, we report a sensitive and reproducible ex vivo porcine skin-catheter model to test how long antimicrobial interventions can delay translocation. Skin preparation was first optimized to minimize tissue damage. The presence of skin dramatically decreased bacterial migration time across the polyurethane catheter interface from > 96 h to 12 h. Using visual colony detection, fluorescence, a luminescent in vitro imaging system, and confocal microscopy, the model was used to quantify time-dependent differences in translocation for eluting and non-eluting antimicrobial catheters. The results show the importance of including tissue in preclinical biofilm models and help to explain current gaps between in vitro testing and clinical outcomes for antimicrobial devices.

Role of calcium in the conformational dynamics of factor XIII activation examined by hydrogen-deuterium exchange coupled with MALDI-TOF MS.

Factor XIII catalyzes formation of γ-glutamyl-ε-lysyl crosslinks within fibrin clots. FXIII A(2) can be activated proteolytically with thrombin and low mM Ca(2+) or nonproteolytically with high monovalent/divalent cations along with low mM Ca(2+). Physiologically, FXIII A(2) is poised to respond to transient influxes of Ca(2+) in a Na(+) containing environment. A successful strategy to monitor FXIII conformational events is hydrogen-deuterium exchange (HDX) coupled with mass spectrometry. FXIII A(2) was examined in the presence of different cations (Ca(2+), Mg(2+), Ba(2+), Cu(2+), Na(+), TMAC(+), and EDA(2+)) ranging from 1 to 2mM, physiological Ca(2+) concentration, to 50-500mM for nonproteolytic activation. Increases in FXIII solvent exposure could already be observed at 1mM Ca(2+) for the dimer interface, the catalytic site, and glutamine substrate regions. By contrast, solvent protection was observed at the secondary cleavage site. These events occurred even though 1mM Ca(2+) is insufficient for FXIII activation. The metals 1mM Mg(2+), 1mM Ba(2+), and 1mM Cu(2+) each led to conformational changes, many in the same FXIII regions as Ca(2+). FXIII could also be activated nonproteolytically with 500mM tetramethylammonium chloride (TMAC(+)) and 500mM ethylenediamine (EDA(2+)), both with 2mM Ca(2+). These different HDX studies help reveal the first FXIII segments that respond to physiological Ca(2+) levels.

Distribution of Brevetoxin to Lipoproteins in human plasma.

To better understand the distribution of brevetoxins in lipoproteins, including their role in tissue delivery and toxin elimination in humans, we examined the interaction of brevetoxin congener PbTx-3 with human lipoproteins. In a scintillation proximity assay (SPA) and microtiter equilibrium dialysis, brevetoxin bound linearly to purified human high density, low density, and very low density lipoproteins (HDL, LDL, and VLDL). Both methods demonstrated higher binding capacity per weight for HDL over the other lipoproteins; approximately 50% higher with SPA and 100% higher with equilibrium dialysis. The preferential binding of brevetoxin to HDL particles is consistent with the higher surface to volume ratio of these particles and the association of the toxin with the surface phospholipid/cholesterol domain of the lipoprotein particle. Lipoprotein components were next separated from a well-characterized human plasma sample to determine the mass distribution of brevetoxin within plasma. Equilibrium dialysis of the fractionated and recombined lipoproteins and plasma proteins determined that brevetoxin distributed predominately (>80%) to lipoproteins associating with each lipoprotein class. These results provide useful information to consider human susceptibility differences, such as those based on dyslipidemia, to the transport and elimination of polyether toxins.

Distribution of brevetoxin (PbTx-3) in mouse plasma: association with high-density lipoproteins.

We investigated the brevetoxin congener PbTx-3 to determine its distribution among carrier proteins, including albumin and blood lipoproteins. Using a radiolabeled brevetoxin tracer (PbTx-3), we found that 39% of the radiolabel remained associated with components in mouse plasma after > 15 kDa cutoff dialysis. Of this portion, only 6.8% was bound to serum albumin. We also examined the binding of brevetoxin to various lipoprotein fractions. Plasma, either spiked with PbTx-3 or from mice treated for 30 min with PbTx-3, was fractionated into different-sized lipoproteins by iodixanol gradient ultracentrifugation. Each fraction was then characterized and quantified by agarose gel electrophoresis and brevetoxin radioimmunoassay, respectively. In both the in vitro and in vivo experiments, the majority of brevetoxin immunoreactivity was restricted to only those gradient fractions that contained high-density lipoproteins (HDLs). Independent confirmation of brevetoxin binding to HDLs was provided by high molecular weight (100 kDa cutoff) dialysis of [3H]PbTx-3 from lipoprotein fractions as well as a scintillation proximity assay using [3H]PbTx-3 and purified human HDLs. This information on the association of brevetoxins with HDLs provides a new foundation for understanding the process by which the toxin is delivered to and removed from tissues and may permit more effective therapeutic measures to treat intoxication from brevetoxins and the related ciguatoxins.

Uptake and elimination of brevetoxin in blood of striped mullet (Mugil cephalus) after aqueous exposure to Karenia brevis.

There is a critical need to simply and reliably monitor brevetoxins routinely in the blood of humans and aquatic animals. We used striped mullet as laboratory test animals to better define the uptake and elimination kinetics of brevetoxin during an aqueous exposure to the brevetoxin-producing dinoflagellate Karenia brevis. Striped mullet were first exposed to sublethal densities of K. brevis (approximately 250,000 cells/L) for 1, 4, 8, 12, and 24 hr. No mortality was observed in the aquaria, and at each time point blood samples were taken and applied to blood collection cards for brevetoxin analysis using radioimmunoassay (RIA). The RIA indicated that blood levels of brevetoxin (PbTx-3) increased to values significantly different from that of the controls at all five time points during exposure (p < 0.05). Striped mullet were then exposed to a K. brevis culture with a known brevetoxin concentration of 0.5 ng/mL. Even after exposures at a low brevetoxin concentration, RIA was able to detect 2.25 +/- 0.62 ng/mL PbTx-3 equivalents in the blood of the mullet at 8 hr of exposure. When exposed to higher brevetoxin concentrations (3.5 and 5.4 ng/mL), blood brevetoxin increased to peak levels at 12 hr and then reached equilibrium after 24 hr in the continued presence of K. brevis. During this time of equilibrium, the mullet maintained brevetoxins with a blood:water coefficient of 2.2. To define the elimination of brevetoxin, striped mullet were next exposed for 8-10 hr and then transferred to fresh seawater containing no K. brevis for up to 116 hr. Blood brevetoxin levels remained elevated and decreased only by 50% 116 hr after transfer. The rate of elimination fit best to a two-phase exponential decay with a biologic half-life of 12 and 266 hr. This study, using RIA in conjunction with blood collection cards, demonstrates an effective means to monitor blood brevetoxin levels in finfish and provides a foundation to characterize biologically relevant levels of brevetoxin in other species impacted by red tide events.