Charles J. Czuprynski

Surfaces modified with nanometer-thick silver-impregnated polymeric films that kill bacteria but support growth of mammalian cells.

Silver is widely used as a biocidal agent in ointments and wound dressings. However, it has also been associated with tissue toxicity and impaired healing. In vitro characterization has also revealed that typical loadings of silver employed in ointments and dressings (approximately 100 microg/cm(2)) lead to cytotoxicity. In this paper, we report the results of an initial study that sought to determine if localization of carefully controlled loadings of silver nanoparticles within molecularly thin films immobilized on surfaces can lead to antimicrobial activity without inducing cytotoxicity. Polymeric thin films of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were prepared by layer-by-layer deposition and loaded with approximately 0.4 microg/cm(2) to approximately 23.6 microg/cm(2) of silver nanoparticles. Bacterial killing efficiencies of the silver-loaded films were investigated against Staphylococcus epidermidis, a gram-positive bacterium, and it was determined that as little as approximately 0.4 microg/cm(2) of silver in the polymeric films caused a reduction of 6log(10)CFU/mL (99.9999%) bacteria in suspensions incubated in contact with the films (water-borne assays). Significantly, whereas the antibacterial films containing high loadings of silver were found to be toxic to a murine fibroblast cell line (NIH-3T3), the polymeric films containing approximately 0.4 microg/cm(2) of silver were not toxic and allowed attachment, and growth of the mammalian cells. Thus, the results of this study go beyond prior reports by identifying silver-impregnated, polymeric thin films that are compatible with in vitro mammalian cell culture yet exhibit antibacterial activity. These results support the hypothesis that localization of carefully controlled loadings of silver nanoparticles within molecularly thin polymeric films can lead to antimicrobial activity without cytotoxicity. More broadly, this strategy of modifying surfaces with minimal loadings of bioactive molecules indicates the basis of approaches that may permit management of microbial burden in wound beds without impairment of wound healing.

Priming and Stimulation of Bovine Neutrophils by Recombinant Human interleukin‐1 Alpha and Tumor Necrosis Factor Alpha

We have examined the in vitro effects of two recombinant human monokines, interleukin‐1 alpha (rHulL‐1α) and tumor necrosis factor alpha (rHuTNFα), on bovine neutrophil functions. Both rHulL‐1α (10 to 1,000 ng/ml) and rHuTNFα (5 to 50 ng/ml) directly stimulated the oxidative burst of bovine neutrophils as measured by Luminol‐dependent chemiluminescence, superoxide anion generation, and hydrogen peroxide production. In addition, both rHulL‐1α (1 to 1,000 ng/ml) and rHuTNFα (0.5 to 50 ng/ml) primed bovine neutrophils for an enhanced oxidative burst to subsequent stimulation with opsonized zymosan. Neutrophils pretreated with either monokine exhibited an earlier, as well as stronger, zymosan‐stimulated Luminol‐dependent chemiluminescence response, as compared to untreated neutrophils. Exposure of bovine neutrophils to combinations of suboptimal doses of rHulL‐1α (10 and 100 ng/ml) and rHuTNFα (0.5 and 5 ng/ml) resulted in a synergistic stimulation of Luminol‐dependent chemiluminescence, whereas, no synergism was observed when using optimal doses of each monokine. Pre‐incubation of bovine neutrophils with an optimal concentration of recombinant bovine interferon gamma (100 U/ml), and either rHulL‐1α or rHuTNFα further augmented the maximal oxidative response of neutrophils stimulated with opsonized zymosan. Bovine neutrophils released both primary and secondary granules in response to rHulL‐1α and rHuTNFα, and also exhibited enhanced adherence in the presence of either monokine.

Use of Hoechst 33342 Staining To Detect Apoptotic Changes in Bovine Mononuclear Phagocytes Infected with Mycobacterium avium subsp. paratuberculosis

ABSTRACT Mycobacterium avium subsp. paratuberculosisis an intracellular pathogen of macrophages that causes a chronic enteritis (Johnes disease) in ruminants. The purpose of this study was to determine whether M. avium subsp.paratuberculosis infection causes apoptosis in bovine monocytes. Using Hoechst 33342 staining, we observed increased numbers of apoptotic monocytes within 6 h of infection withM. avium subsp. paratuberculosis, and these numbers increased further at 24 and 48 h. This effect appeared to require viable bacilli, because monocytes infected with heat-killed M. avium subsp. paratuberculosisdid not exhibit a significant increase in apoptosis. Preincubation of monocytes with bovine growth hormone prior to infection with M. avium subsp.paratuberculosis did not significantly alter the number of apoptotic cells.

A/J Mice Are Susceptible and C57BL/6 Mice Are Resistant to Listeria monocytogenes Infection by Intragastric Inoculation

ABSTRACT Previous studies demonstrated that the innate resistance of mice to Listeria monocytogenes infection by intravenous or intraperitoneal inoculation is regulated principally by the Hc locus on mouse chromosome 2. The A/J and C57BL/6 mouse strains were identified as prototype L. monocytogenes-susceptible and -resistant strains, respectively. In the present study, we compared the relative susceptibilities of A/J and C57BL/6 mice to intragastric (i.g.) inoculation with L. monocytogenes. The results of our study indicate that A/J mice are significantly more susceptible than C57BL/6 mice to an i.g. challenge with L. monocytogenes. This was reflected in the estimated 50% lethal doses for the two strains (106 and 108 CFU for A/J and C57BL/6 mice, respectively) and a more rapid and severe dissemination of the infection to the spleen and liver in A/J mice than in C57BL/6 mice. Histopathological examination of tissues from the infected mice confirmed the greater severity of disease in A/J mice. Clearance of a primary infection enhanced the resistance of both A/J and C57BL/6 mice to reinfection with L. monocytogenes via the gastrointestinal tract. However, the relative difference in susceptibility between the two strains was evident even after immunization. The A/J mouse holds promise as a model for investigating the pathogenesis of gastrointestinal listeriosis because of its ability to develop systemic infection following challenge with numbers of organisms similar to those recovered from some L. monocytogenes-contaminated food products.

The Aryl Hydrocarbon Receptor Is Required for Optimal Resistance to Listeria monocytogenes Infection in Mice

The aryl hydrocarbon receptor (AhR) is part of a powerful signaling system that is triggered by xenobiotic agents such as polychlorinated hydrocarbons and polycyclic aromatic hydrocarbons. Although activation of the AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin or certain polycyclic aromatic hydrocarbons can lead to immunosuppression, there is also increasing evidence that the AhR regulates certain normal developmental processes. In this study, we asked whether the AhR plays a role in host resistance using murine listeriosis as an experimental system. Our data clearly demonstrate that AhR null C57BL/6J mice (AhR−/−) are more susceptible to listeriosis than AhR heterozygous (AhR+/−) littermates when inoculated i.v. with log-phase Listeria monocytogenes. AhR−/− mice exhibited greater numbers of CFU of L. monocytogenes in the spleen and liver, and greater histopathological changes in the liver than AhR+/− mice. Serum levels of IL-6, MCP-1, IFN-γ, and TNF-α were comparable between L. monocytogenes-infected AhR−/− and AhR+/− mice. Increased levels of IL-12 and IL-10 were observed in L. monocytogenes-infected AhR−/− mice. No significant difference was found between AhR+/− and AhR−/− macrophages ex vivo with regard to their ability to ingest and inhibit intracellular growth of L. monocytogenes. Intracellular cytokine staining of CD4+ and CD8+ splenocytes for IFN-γ and TNF-α revealed comparable T cell-mediated responses in AhR−/− and AhR+/− mice. Previously infected AhR−/− and AhR+/− mice both exhibited enhanced resistance to reinfection with L. monocytogenes. These data provide the first evidence that AhR is required for optimal resistance but is not essential for adaptive immune response to L. monocytogenes infection.

Ability of the Listeria monocytogenes Strain Scott A To Cause Systemic Infection in Mice Infected by the Intragastric Route

ABSTRACT Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37°C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.

Haemophilus somnus Induces Apoptosis in Bovine Endothelial Cells In Vitro

ABSTRACT Haemophilus somnus causes pneumonia, reproductive failure, infectious myocarditis, thrombotic meningoencephalitis, and other diseases in cattle. Although vasculitis is commonly seen as a result of systemic H. somnus infections, the pathogenesis of vascular damage is poorly characterized. In this study, we demonstrated that H. somnus (pathogenic isolates 649, 2336, and 8025 and asymptomatic carrier isolates 127P and 129Pt) induce apoptosis of bovine endothelial cells in a time- and dose-dependent manner, as determined by Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end labeling, DNA fragmentation, and transmission electron microscopy. H. somnus induced endothelial cell apoptosis in as little as 1 h of incubation and did not require extracellular growth of the bacteria. Viable H. somnus organisms induced greater endothelial cell apoptosis than heat-killed organisms. Since viableH. somnus cells release membrane fibrils and blebs, which contain lipooligosaccharide (LOS) and immunoglobulin binding proteins, we examined culture filtrates for their ability to induce endothelial cell apoptosis. Culture filtrates induced similar levels of endothelial cell apoptosis, as did viable H. somnus organisms. Heat inactivation of H. somnus culture filtrates partially reduced the apoptotic effect on endothelial cells, which suggested the presence of both heat-labile and heat-stable factors. We found thatH. somnus LOS, which is heat stable, induced endothelial cell apoptosis in a time- and dose-dependent manner and was inhibited by the addition of polymyxin B. These data demonstrate that H. somnus and its LOS induce endothelial cell apoptosis, which may play a role in producing vasculitis in vivo.

Mannheimia haemolytica and Its Leukotoxin Cause Neutrophil Extracellular Trap Formation by Bovine Neutrophils

ABSTRACT Mannheimia haemolytica is an important member of the bovine respiratory disease complex, which is characterized by abundant neutrophil infiltration into the alveoli and fibrin deposition. Recently several authors have reported that human neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of trapping and killing pathogens. Here, we demonstrate that the leukotoxin (LKT) of M. haemolytica causes NET formation by bovine neutrophils in a CD18-dependent manner. Using an unacylated, noncytotoxic pro-LKT produced by an ΔlktC mutant of M. haemolytica, we show that binding of unacylated pro-LKT stimulates NET formation despite a lack of cytotoxicity. Inhibition of LKT binding to the CD18 chain of lymphocyte function-associated antigen 1 (LFA-1) on bovine neutrophils reduced NET formation in response to LKT or M. haemolytica cells. Further investigation revealed that NETs formed in response to M. haemolytica are capable of trapping and killing a portion of the bacterial cells. NET formation was confirmed by confocal microscopy and by scanning and transmission electron microscopy. Prior exposure of bovine neutrophils to LKT enhanced subsequent trapping and killing of M. haemolytica cells in bovine NETs. Understanding NET formation in response to M. haemolytica and its LKT provides a new perspective on how neutrophils contribute to the pathogenesis of bovine respiratory disease.

Bacterial lipopolysaccharide induces release of tumor necrosis factor-alpha from bovine peripheral blood monocytes and alveolar macrophages in vitro.

In this study, we demonstrate that freshly adherent bovine monocytes release tumor necrosis factor‐α (TNF‐α) in response to stimulation with bacterial lipopolysaccharide (LPS). TNF‐α was detected using actinomycin D‐treated WEHI‐164 murine fibrosarcoma cells as targets in an 18 hr cytotoxicity assay. Doses of LPS from 20 ng/ml to 20 μg/ml were capable of inducing bovine TNF‐α. The kinetics of TNF‐α release from bovine monocytes demonstrated peak levels of cytotoxic activity at 1–3 hr post‐LPS treatment, with a subsequent decline to background levels by 18 hr post‐LPS treatment. A monoclonal antibody that neutralizes recombinant human TNF‐α (rHuTNF‐α) significantly reduced the cytotoxicity of LPS‐stimulated bovine monocyte culture supematants. Size exclusion high‐performance liquid chromatography (HPLC) analysis of LPS‐stimulated monocyte and alveolar macrophage culture supematants resulted in a molecular weight elution profile similar to that of recombinant human TNF‐α. These elution profiles are consistent with the presence of multimers of TNF‐α. This is believed to be the first report of the in vitro production of bovine TNF‐α.

Bone marrow cytotoxicity of benzo[a]pyrene is dependent on CYP1B1 but is diminished by Ah receptor-mediated induction of CYP1A1 in liver

We have previously used CYP1B1-null mice to demonstrate that dimethylbenz(a)anthracene (DMBA) requires CYP1B1 for bone marrow (BM) toxicity. Benzo(a)pyrene (BP), a much more potent Ah receptor ligand, shows very different responses that nevertheless depend on CYP1B1. Wild-type (AhR(b)) mice treated with DMBA for 48 h exhibit a large loss in BM cellularity and disruption of marrow structure that is not seen for BP treatment. In congenic mice with a low affinity AhR (AhR(d)), DMBA and BP are equally toxic to the BM whereas AhR(d) x CYP1B1-null mice are fully protected. In situ hybridization demonstrates that CYP1B1 mRNA is constitutively expressed in marrow cells and is induced by PAHs according to their AhR affinity (BP>DMBA), including lower levels in AhR(d) mice. Importantly, expression of CYP1A1 mRNA was undetectable in BM. In wild-type mice, BP treatment leads to a fivefold greater induction of hepatic CYP1A1 than that of DMBA treatment. Neither induction occurs in AhR(d) mice. Thus, hepatic metabolism may prevent BP from reaching the BM, where it can be bioactivated by CYP1B1. Flow cytometric analyses of BM cells showed that there were decreases in granulocytes and lymphocytes following DMBA treatment, but not after BP treatment. These data suggest that there is an inverse relationship between liver metabolism and BM toxicity resulting from limitations on the delivery of PAH to CYP1B1 present in BM, where only very low constitutive levels are needed.