Ultrasound-Activated Piezoelectric Polyvinylidene Fluoride-Trifluoroethylene Scaffolds for Tissue Engineering Applications.

Severe peripheral nervous system (PNS) injuries have limited options for therapeutic solutions to regain functional recovery. This can be attributed in part to the lack of regeneration pathways promoted by recapitulating chemical, physical, and electrical cues to direct nerve guidance. To address this, we examined ultrasonic stimulation of a piezoelectric polyvinylidene fluoride-triflouroethylene (PVDF-TrFE) scaffold as a potentially clinically relevant therapy for PNS regeneration. Owing to the piezoelectric modality of PVDF-TrFE, we hypothesize that ultrasound stimulation will activate the scaffold to electrically stimulate cells in response to the mechanical deformation mediated by sound waves. Biocompatible PVDF-TrFE scaffolds were fabricated to be used as an ultrasound-activated, piezoelectric biomaterial to enhance cellular activity for PNS applications. NIH-3T3 fibroblasts were cultured on PVDF-TrFE nanofibers and stimulated with low-, medium-, or high-powered ultrasound. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays were performed on fibroblasts to measure the metabolic activity of the cells following stimulation. MTT assays showed that ultrasound-stimulated fibroblasts on PVDF-TrFE scaffolds had increased metabolic activity as power was increased, whereas on plain polystyrene, an opposite trend was observed where cells had a decreased metabolic activity with ascending levels of ultrasound power. Ultrasound-stimulated PVDF-TrFE nanofibers hold exciting potential as a therapy for PNS injuries by promoting increased metabolic activity and proliferation. The ability to noninvasively stimulate implantable piezoelectric nanofibers to promote mechanical and electrical stimulation for nerve repair offers a promising benefit to severe trauma patients.

Characterization of Virulent T4-Like Acinetobacter baumannii Bacteriophages DLP1 and DLP2.

The world is currently facing a global health crisis due to the rapid increase in antimicrobial-resistant bacterial infections. One of the most concerning pathogens is Acinetobacter baumannii, which is listed as a Priority 1 pathogen by the World Health Organization. This Gram-negative bacterium has many intrinsic antibiotic resistance mechanisms and the ability to quickly acquire new resistance determinants from its environment. A limited number of effective antibiotics against this pathogen complicates the treatment of A. baumannii infections. A potential treatment option that is rapidly gaining interest is "phage therapy", or the clinical application of bacteriophages to selectively kill bacteria. The myoviruses DLP1 and DLP2 (vB_AbaM-DLP_1 and vB_AbaM-DLP_2, respectively) were isolated from sewage samples using a capsule minus variant of A. baumannii strain AB5075. Host range analysis of these phages against 107 A. baumannii strains shows a limited host range, infecting 15 and 21 for phages DLP1 and DLP2, respectively. Phage DLP1 has a large burst size of 239 PFU/cell, a latency period of 20 min, and virulence index of 0.93. In contrast, DLP2 has a smaller burst size of 24 PFU/cell, a latency period of 20 min, and virulence index of 0.86. Both phages show potential for use as therapeutics to combat A. baumannii infections.

Innervation of an Ultrasound-Mediated PVDF-TrFE Scaffold for Skin-Tissue Engineering.

In this work, electrospun polyvinylidene-trifluoroethylene (PVDF-TrFE) was utilized for its biocompatibility, mechanics, and piezoelectric properties to promote Schwann cell (SC) elongation and sensory neuron (SN) extension. PVDF-TrFE electrospun scaffolds were characterized over a variety of electrospinning parameters (1, 2, and 3 h aligned and unaligned electrospun fibers) to determine ideal thickness, porosity, and tensile strength for use as an engineered skin tissue. PVDF-TrFE was electrically activated through mechanical deformation using low-intensity pulsed ultrasound (LIPUS) waves as a non-invasive means to trigger piezoelectric properties of the scaffold and deliver electric potential to cells. Using this therapeutic modality, neurite integration in tissue-engineered skin substitutes (TESSs) was quantified including neurite alignment, elongation, and vertical perforation into PVDF-TrFE scaffolds. Results show LIPUS stimulation promoted cell alignment on aligned scaffolds. Further, stimulation significantly increased SC elongation and SN extension separately and in coculture on aligned scaffolds but significantly decreased elongation and extension on unaligned scaffolds. This was also seen in cell perforation depth analysis into scaffolds which indicated LIPUS enhanced perforation of SCs, SNs, and cocultures on scaffolds. Taken together, this work demonstrates the immense potential for non-invasive electric stimulation of an in vitro tissue-engineered-skin model.

Bacteriophage Isolation, Purification, and Characterization Techniques Against Ubiquitous Opportunistic Pathogens.

Healthcare-associated infection with "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) is a global health crisis due to their extensive intrinsic antibiotic resistance and the ability to quickly acquire resistance determinants. Alternative treatment options are required to combat this crisis, and one possibility is the use of bacteriophages, or viruses that strictly infect the pathogenic bacteria. Currently, there is a renaissance in research and development into the use of phages to target multi-, extensively, and pan-resistant bacterial infections in humans, known as phage therapy. Using A. baumannii as an example, this article describes the isolation and purification of bacteriophages from sewage and soil samples, as well as general methods used in phage research such as precipitation of phages using polyethylene glycol, host range analysis, single-cell burst size determination, DNA extraction, and restriction fragment length polymorphism analysis. © 2022 National Research Council Canada. Current Protocols © 2022 Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Industry. Basic Protocol 1: Isolation of bacteriophages against A. baumannii from sewage samples Alternate Protocol 1: Isolation of bacteriophages against A. baumannii from soil samples Support Protocol 1: Titering a bacteriophage stock Basic Protocol 2: Purification of phage to an axenic working stock Support Protocol 2: Liquid propagation of bacteriophage Basic Protocol 3: Host range analysis using the spot plate method Basic Protocol 4: Single burst size analysis Alternate Protocol 2: One-step growth curve Basic Protocol 5: Precipitation of bacteriophage using PEG 6000 Basic Protocol 6: DNA extraction from dsDNA bacteriophages Basic Protocol 7: Restriction fragment length polymorphism analysis of novel phage genomes.

The impact of physical, biochemical, and electrical signaling on Schwann cell plasticity.

Peripheral nervous system (PNS) injuries are an ongoing health care concern. While autografts and allografts are regarded as the current clinical standard for traumatic injury, there are inherent limitations that suggest alternative remedies should be considered for therapeutic purposes. In recent years, nerve guidance conduits (NGCs) have become increasingly popular as surgical repair devices, with a multitude of various natural and synthetic biomaterials offering potential to enhance the design of conduits or supplant existing technologies entirely. From a cellular perspective, it has become increasingly evident that Schwann cells (SCs), the primary glia of the PNS, are a predominant factor mediating nerve regeneration. Thus, the development of severe nerve trauma therapies requires a deep understanding of how SCs interact with their environment, and how SC microenvironmental cues may be engineered to enhance regeneration. Here we review the most recent advancements in biomaterials development and cell stimulation strategies, with a specific focus on how the microenvironment influences the behavior of SCs and can potentially lead to functional repair. We focus on microenvironmental cues that modulate SC morphology, proliferation, migration, and differentiation to alternative phenotypes. Promotion of regenerative phenotypic responses in SCs and other non-neuronal cells that can augment the regenerative capacity of multiple biomaterials is considered along with innovations and technologies for traumatic injury.

Mechanical stimulation of a bioactive, functionalized PVDF-TrFE scaffold provides electrical signaling for nerve repair applications.

Traumatic nerve injuries have limited success in achieving full functional recovery, with current clinical solutions often including implementation of nerve grafts or the use of nerve conduits to guide damaged axons across injury gaps. In search of alternative, and complimentary solutions, piezoelectric biomaterials demonstrate immense potential for tissue engineering applications. Piezoelectric poly(vinylidene fluoride-triflouroethylene) (PVFD-TrFE) scaffolds can be harnessed to non-invasively stimulate and direct function of key peripheral nervous system (PNS) cells in regeneration strategies. In this study, electrospun PVDF-TrFE was characterized, fabricated into a 3D scaffold, and finally rendered bioactive with the incorporation of a cell-secreted, decellularized extracellular matrix (dECM). PVDF-TrFE scaffolds were characterized extensively for piezoelectric capacity, mechanical properties, and cell-material interactions with fibroblasts and Schwann cells. Through functionalization of PVDF-TrFE scaffolds with a native, cell-assembled dECM, the ability to promote cell adhesion and enhanced viability was also demonstrated. Additionally, incorporation of bioactive functionalization improved the assembly of key regenerative ECM proteins and regenerative growth factors. PVDF-TrFE scaffolds were then fabricated into a conduit design that retained key physical, chemical, and piezoelectric properties necessary for PNS repair. This work shows great promise for multi-cue, electrospun biomaterials for regeneration of the PNS in traumatic injury.

Universal antibody targeting the highly conserved fusion peptide provides cross-protection in mice.

Influenza is a major public health concern causing millions of hospitalizations every year. The current vaccines need annual updating based on prediction of likely strains in the upcoming season. However, mismatches between vaccines and the actual circulating viruses can occur, reducing vaccine effectiveness significantly because of the remarkably high rate of mutation in the viral glycoprotein, hemagglutinin (HA). Clearly, it would be of great interest to determine the potential role of universally conserved epitopes in inducing protective immunity. Here, an antibody against the 14-aa fusion peptide sequence at the N-terminus of the HA2 subunit (Uni-1) was investigated for its ability to elicit antibody-dependent cellular cytotoxicity (ADCC) in vitro and cross-protection against lethal infection in animals. Uni-1, known to neutralize influenza type A (IAV) in vitro, was found to induce strong ADCC against diverse influenza viruses, including human and avian IAVs and both lineages of type B (IBV). The ADCC effects against human IAVs by Uni-1 was comparable to ADCC induced by well-characterized antibodies, F10 and FI6V3. Importantly, mice treated with Uni-1 were protected against lethal challenge of IAV and IBV. These results revealed the versatile effector functions of this universal antibody against markedly diverse strains of both IAV and IBV.

The fusion peptide is the only universally conserved epitope in both IAV and IBVMono-specific universal antibody induces strong ADCC against human and avian IAVMono-specific universal antibody induces strong ADCC against IBV from both genetic lineages of IBVThe antibody has bi-functional effector functions against several influenza viruses.

The use of intra-articular platelet rich plasma for the symptomatic management of osteoarthritis of the knee: a pilot study.

BACKGROUND: Osteoarthritis of the knee is a chronic inflammatory condition resulting in significant patient disability, with intra-articular platelet rich plasma (PRP) injections having shown potential to improve symptomatic outcomes. This retrospective cohort pilot study aimed to observe whether PRP injections were beneficial in the symptomatic management of knee osteoarthritis in an Australian population, based on patient reported outcomes. An additional aim was to observe for an association between the number of injections and patient characteristics, such as body mass index, age, sex and radiologically determined severity of the disease. METHODS: The cohort was drawn from those who attended Ballarat Orthopaedic and Sports Medicine for PRP injections and who had completed the appropriate pre- and post-injection Western Ontario and McMaster Universities Arthritis Index (WOMAC) questionnaire. WOMAC scores were analysed to observe for any difference following a course of PRP injections. RESULTS: The data suggest that the use of PRP improved patient reported WOMAC scores. Additionally, it was shown that two injections had a greater effect than one injection, with a third injection providing no further benefit. Finally, there was an association with lower WOMAC scores post PRP therapy amongst male participants compared to female participants. CONCLUSION: These results suggest two PRP injections are optimal for the symptomatic management of knee osteoarthritis, identifying a need for further prospective research in this Australian population.

A Slam-dependent hemophore contributes to heme acquisition in the bacterial pathogen Acinetobacter baumannii.

Nutrient acquisition systems are often crucial for pathogen growth and survival during infection, and represent attractive therapeutic targets. Here, we study the protein machinery required for heme uptake in the opportunistic pathogen Acinetobacter baumannii. We show that the hemO locus, which includes a gene encoding the heme-degrading enzyme, is required for high-affinity heme acquisition from hemoglobin and serum albumin. The hemO locus includes a gene coding for a heme scavenger (HphA), which is secreted by a Slam protein. Furthermore, heme uptake is dependent on a TonB-dependent receptor (HphR), which is important for survival and/or dissemination into the vasculature in a mouse model of pulmonary infection. Our results indicate that A. baumannii uses a two-component receptor system for the acquisition of heme from host heme reservoirs.

Cell Shape and Matrix Stiffness Impact Schwann Cell Plasticity via YAP/TAZ and Rho GTPases.

Schwann cells (SCs) are a highly plastic cell type capable of undergoing phenotypic changes following injury or disease. SCs are able to upregulate genes associated with nerve regeneration and ultimately achieve functional recovery. During the regeneration process, the extracellular matrix (ECM) and cell morphology play a cooperative, critical role in regulating SCs, and therefore highly impact nerve regeneration outcomes. However, the roles of the ECM and mechanotransduction relating to SC phenotype are largely unknown. Here, we describe the role that matrix stiffness and cell morphology play in SC phenotype specification via known mechanotransducers YAP/TAZ and RhoA. Using engineered microenvironments to precisely control ECM stiffness, cell shape, and cell spreading, we show that ECM stiffness and SC spreading downregulated SC regenerative associated proteins by the activation of RhoA and YAP/TAZ. Additionally, cell elongation promoted a distinct SC regenerative capacity by the upregulation of Rac1/MKK7/JNK, both necessary for the ECM and morphology changes found during nerve regeneration. These results confirm the role of ECM signaling in peripheral nerve regeneration as well as provide insight to the design of future biomaterials and cellular therapies for peripheral nerve regeneration.

Micropatterning Decellularized ECM as a Bioactive Surface to Guide Cell Alignment, Proliferation, and Migration.

Bioactive surfaces and materials have displayed great potential in a variety of tissue engineering applications but often struggle to completely emulate complex bodily systems. The extracellular matrix (ECM) is a crucial, bioactive component in all tissues and has recently been identified as a potential solution to be utilized in combination with biomaterials. In tissue engineering, the ECM can be utilized in a variety of applications by employing the biochemical and biomechanical cues that are crucial to regenerative processes. However, viable solutions for maintaining the dimensionality, spatial orientation, and protein composition of a naturally cell-secreted ECM remain challenging in tissue engineering. Therefore, this work used soft lithography to create micropatterned polydimethylsiloxane (PDMS) substrates of a three-dimensional nature to control cell adhesion and alignment. Cells aligned on the micropatterned PDMS, secreted and assembled an ECM, and were decellularized to produce an aligned matrix biomaterial. The cells seeded onto the decellularized, patterned ECM showed a high degree of alignment and migration along the patterns compared to controls. This work begins to lay the groundwork for elucidating the immense potential of a natural, cell-secreted ECM for directing cell function and offers further guidance for the incorporation of natural, bioactive components for emerging tissue engineering technologies.

Fucoidan-Doxorubicin Nanoparticles Targeting P-Selectin for Effective Breast Cancer Therapy.

Fucoidan, a type of sulfated polysaccharide known for its anticoagulant, anti-tumor and anti-inflammatory effects, has been reported to have strong affinity towards P-selectin. P-selectin, which plays an important role in metastasis by enhancing the adhesion of cancer cells to endothelium and activated platelets in distant organs, is overexpressed on many cancer types. This study demonstrates the synthesis of a fucoidan-based drug delivery system for minimizing the side effects of doxorubicin (Dox) with the help of active targeting toward P-selectin. Fucoidan-doxorubicin nanoparticles (FU-Dox NPs), developed by direct conjugation of Dox to the fucoidan backbone, showed a well-controlled size distribution and sustained release. The active targeting capability of FU-Dox NPs toward P-selectin resulted in enhanced cellular uptake and cytotoxicity against the MDA-MB-231 cell line with high P-selectin expression compared to the MDA-MB-468 cell line with low P-selectin expression.

Development of a Piezoelectric PVDF-TrFE Fibrous Scaffold to Guide Cell Adhesion, Proliferation, and Alignment.

Severe peripheral nervous system injuries currently hold limited therapeutic solutions. Existing clinical techniques such as autografts, allografts, and newer nerve guidance conduits have shown variable outcomes in functional recovery, adverse immune responses, and in some cases low or minimal availability. This can be attributed in part to the lack of chemical, physical, and electrical cues directing both nerve guidance and regeneration. To address this pressing clinical issue, electrospun nanofibers and microfibers composed of piezoelectric polyvinylidene flouride-triflouroethylene (PVDF-TrFE) have been introduced as an alternative template for tissue engineered biomaterials, specifically as it pertains to their relevance in soft tissue and nerve repair. Here, biocompatible scaffolds of PVDF-TrFE are fabricated and their ability to generate an electrical response to mechanical deformations and produce a suitable regenerative microenvironment is examined. It is determined that 20% (w/v) PVDF-TrFE in (6:4) dimethyl formamide (DMF):acetone solvent maintains a desirable piezoelectric coefficient and the proper physical and electrical characteristics for tissue regeneration. Further, it is concluded that scaffolds of varying thickness promoted the adhesion and alignment of Schwann cells and fibroblasts. This work offers a prelude to further advancements in nanofibrous technology and a promising outlook for alternative, autologous remedies to peripheral nerve damage.

Systemic and heart autonomous effects of sphingosine Δ4 desaturase deficiency in lipotoxic cardiac pathophysiology.

Lipotoxic cardiomyopathy (LCM) is characterized by cardiac steatosis, including the accumulation of fatty acids, triglycerides and ceramides. Model systems have shown the inhibition of ceramide biosynthesis to antagonize obesity and improve insulin sensitivity. Sphingosine Δ4 desaturase (encoded by ifc in Drosophila melanogaster) enzymatically converts dihydroceramide into ceramide. Here, we examine ifc mutants to study the effects of desaturase deficiency on cardiac function in Drosophila Interestingly, ifc mutants exhibited classic hallmarks of LCM: cardiac chamber dilation, contractile defects and loss of fractional shortening. This outcome was phenocopied in global ifc RNAi-mediated knockdown flies. Surprisingly, cardiac-specific ifc knockdown flies exhibited cardiac chamber restriction with no contractile defects, suggesting heart autonomous and systemic roles for ifc activity in cardiac function. Next, we demonstrated that ifc mutants exhibit suppressed Sphingosine kinase 1 (Sk1) expression. Ectopic overexpression of Sk1 was sufficient to prevent cardiac chamber dilation and loss of fractional shortening in ifc mutants. Partial rescue was also observed with cardiac- and fat-body-specific Sk1 overexpression. Finally, we showed that cardiac-specific expression of Drosophila inhibitor of apoptosis (dIAP) also prevented cardiac dysfunction in ifc mutants, suggesting a role for caspase activity in the observed cardiac pathology. Collectively, we show that spatial regulation of sphingosine Δ4 desaturase activity differentially affects cardiac function in heart autonomous and systemic mechanisms through tissue interplay.

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification.

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

In vitro Production and Immunogenicity of a Clostridium Difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine.

Abstract: The BclA3 glycoprotein is a major component of the exosporangial layer of Clostridium difficile spores and in this study we demonstrate that this glycoprotein is a major spore surface associated antigen. Here, we confirm the role of SgtA glycosyltransferase (SgtA GT) in BclA3 glycosylation and recapitulate this process by expressing and purifying SgtA GT fused to MalE, the maltose binding protein from Escherichia coli. In vitro assays using the recombinant enzyme and BclA3 synthetic peptides demonstrated that SgtA GT was responsible for the addition of ß-O-linked GlcNAc to threonine residues of each synthetic peptide. These peptide sequences were selected from the central, collagen repeat region of the BclA3 protein. Following optimization of SgtA GT activity, we generated sufficient glycopeptide (10 mg) to allow conjugation to KLH (keyhole limpet hemocyanin) protein. Glycosylated and unglycosylated versions of these conjugates were then used as antigens to immunize rabbits and mice. Immune responses to each of the conjugates were examined by Enzyme Linked Immunosorbent Assay ELISA. Additionally, the BclA3 conjugated peptide and glycopeptide were used as antigens in an ELISA assay with serum raised against formalin-killed spores. Only the glycopeptide was recognized by anti-spore polyclonal immune serum demonstrating that the glycan moiety is a predominant spore-associated surface antigen. To determine whether antibodies to these peptides could modify persistence of spores within the gut, animals immunized intranasally with either the KLH-glycopeptide or KLH-peptide conjugate in the presence of cholera toxin, were challenged with R20291 spores. Although specific antibodies were raised to both antigens, immunization did not provide any protection against acute or recurrent disease.

Extracellular matrix cues modulate Schwann cell morphology, proliferation, and protein expression.

Peripheral nerve injuries require a complex set of signals from cells, macrophages, and the extracellular matrix (ECM) to induce regeneration across injury sites and achieve functional recovery. Schwann cells (SCs), the major glial cell in the peripheral nervous system (PNS), are critical to nerve regeneration due to their inherent capacity for altering phenotype postinjury to facilitate wound healing. The ECM plays a vital role in wound healing as well as regulating cell phenotype during tissue repair. To examine the underlying mechanisms between the ECM and SCs, this work sought to determine how specific ECM cues regulate the phenotype of SCs. To address this, SCs were cultured on polydimethylsiloxane substrates of a variable Young's modulus coated with ECM proteins. Cells were analyzed for spreading area, proliferation, cell and nuclear shape, and c-Jun expression. It was found that substrates with a stiffness of 8.67 kPa coated with laminin promoted the highest expression of c-Jun, a marker signifying a "regenerative" SC. Microcontact printed, cell adhesive areas were then utilized to precisely control the geometry and spreading of SCs and by controlling spreading area and cellular elongation; expression of c-Jun was either promoted or downregulated. These results begin to address the significant interplay between ECM cues and phenotype of SCs, while offering a potential means to enhance PNS regeneration through cellular therapies.

Potential Mechanisms of Mucin-Enhanced Acinetobacter baumannii Virulence in the Mouse Model of Intraperitoneal Infection.

Porcine mucin has been commonly used to enhance the infectivity of bacterial pathogens, including Acinetobacter baumannii, in animal models, but the mechanisms for enhancement by mucin remain relatively unknown. In this study, using the mouse model of intraperitoneal (i.p.) mucin-enhanced A. baumannii infection, we characterized the kinetics of bacterial replication and dissemination and the host innate immune responses, as well as their potential contribution to mucin-enhanced bacterial virulence. We found that mucin, either admixed with or separately injected with the challenge bacterial inoculum, was able to enhance the tissue and blood burdens of A. baumannii strains of different virulence. Intraperitoneal injection of A. baumannii-mucin or mucin alone induced a significant but comparable reduction of peritoneal macrophages and lymphocytes, accompanied by a significant neutrophil recruitment and early interleukin-10 (IL-10) responses, suggesting that the resulting inflammatory cellular and cytokine responses were largely induced by the mucin. Depletion of peritoneal macrophages or neutralization of endogenous IL-10 activities showed no effect on the mucin-enhanced infectivity. However, pretreatment of mucin with iron chelator DIBI, but not deferoxamine, partially abolished its virulence enhancement ability, and replacement of mucin with iron significantly enhanced the bacterial burdens in the peritoneal cavity and lung. Taken together, our results favor the hypothesis that iron at least partially contributes to the mucin-enhanced infectivity of A. baumannii in this model.

Profiling of Cytokine and Chemokine Responses Using Multiplex Bead Array Technology.

Multiplex bead array technology expands upon the principles of the enzyme-linked immunosorbent assay by allowing the simultaneous quantification of a large number of cytokines and chemokines within a single sample. This allows researchers more freedom and opportunities to investigate complex immune responses both in vivo and in vitro. Here we describe and update the detailed assay protocol and technical tips for simultaneous quantification of multiple cytokines and chemokines in mouse biological fluids such as sera, bronchoalveolar lavage fluid, tissue homogenate supernatant, and tissue culture supernatant, using a multiplex bead array assay.

Acute intraperitoneal infection with a hypervirulent Acinetobacter baumannii isolate in mice.

Acinetobacter baumannii infection has become a major cause of healthcare-associated infection and a critical pathogen in the WHO antimicrobial resistance research and development priority list. Catheter-related septicemia is one of the major clinical manifestations of A. baumannii infection associated with high morbidity and mortality. In this study, we used a clinical A. baumannii strain (LAC-4) that is hypervirulent to immunocompetent C57BL/6 and BALB/c mice and established a mouse model of intraperitoneal (i.p.) A. baumannii infection. Our study showed that i.p. LAC-4 infection of C57BL/6 and BALB/c mice induces a lethal or sublethal infection with high bacterial burdens in peritoneal cavity, blood and tissues and the infected mice either succumbed to the infection within 24 hours or completely recovered from the infection. The infection induces acute peritoneal recruitment of neutrophils and other innate immune cells, and the local and systemic production of proinflammatory cytokines and chemokines (IL-1ß, IL-5, IL-6, TNF-α, RANTES, MIP-1ß, MCP-1, KC and IL-10). Mechanistic studies suggest an important role of macrophages in the host innate defense in this model in that in vitro stimulation of peritoneal macrophages with killed LAC-4 induced a similar pattern of cytokine/chemokine responses to those in the infected mice, and depletion of peritoneal macrophages rendered the mice significantly more susceptible to the infection. Thus, this mouse infection model will provide an alternative and useful tool for future pathogenesis studies of A. baumannii-associated septicemia and identification and characterization of important virulence factors, as well as serve as a surrogate model for rapid evaluation of novel therapeutics and vaccines for this emerging infectious agent.