Scholarly Works, Virginia-Maryland College of Veterinary Medicine

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Aligned nanofiber topography directs the tenogenic differentiation of mesenchymal stem cells
Popielarczyk, Tracee L.; Nain, Amrinder S.; Barrett, Jennifer G. (MDPI, 2017-01-06)
Tendon is commonly injured, heals slowly and poorly, and often suffers re-injury after healing. This is due to failure of tenocytes to effectively remodel tendon after injury to recapitulate normal architecture, resulting in poor mechanical properties. One strategy for improving the outcome is to use nanofiber scaffolds and mesenchymal stem cells (MSCs) to regenerate tendon. Various scaffold parameters are known to influence tenogenesis. We designed suspended and aligned nanofiber scaffolds with the hypothesis that this would promote tenogenesis when seeded with MSCs. Our aligned nanofibers were manufactured using the previously reported non-electrospinning Spinneret-based Tunable Engineered Parameters (STEP) technique. We compared parallel versus perpendicular nanofiber scaffolds with traditional flat monolayers and used cellular morphology, tendon marker gene expression, and collagen and glycosaminoglycan deposition as determinants for tendon differentiation. We report that compared with traditional control monolayers, MSCs grown on nanofibers were morphologically elongated with higher gene expression of tendon marker scleraxis and collagen type I, along with increased production of extracellular matrix components collagen (p = 0.0293) and glycosaminoglycan (p = 0.0038). Further study of MSCs in different topographical environments is needed to elucidate the complex molecular mechanisms involved in stem cell differentiation.
Antibiotics ameliorate lupus-like symptoms in mice
Mu, Qinghui; Tavella, Vincent J.; Kirby, Jay L.; Cecere, Thomas E.; Chung, Matthias; Lee, Jiyoung; Li, Song; Ahmed, Sattar Ansar; Eden, Kristin; Allen, Irving C. (Nature, 2017-10-20)
Gut microbiota and the immune system interact to maintain tissue homeostasis, but whether this interaction is involved in the pathogenesis of systemic lupus erythematosus (SLE) is unclear. Here we report that oral antibiotics given during active disease removed harmful bacteria from the gut microbiota and attenuated SLE-like disease in lupus-prone mice. Using MRL/lpr mice, we showed that antibiotics given after disease onset ameliorated systemic autoimmunity and kidney histopathology. They decreased IL-17-producing cells and increased the level of circulating IL-10. In addition, antibiotics removed Lachnospiraceae and increased the relative abundance of Lactobacillus spp., two groups of bacteria previously shown to be associated with deteriorated or improved symptoms in MRL/lpr mice, respectively. Moreover, we showed that the attenuated disease phenotype could be recapitulated with a single antibiotic vancomycin, which reshaped the gut microbiota and changed microbial functional pathways in a time-dependent manner. Furthermore, vancomycin treatment increased the barrier function of the intestinal epithelium, thus preventing the translocation of lipopolysaccharide, a cell wall component of Gram-negative Proteobacteria and known inducer of lupus in mice, into the circulation. These results suggest that mixed antibiotics or a single antibiotic vancomycin ameliorate SLE-like disease in MRL/lpr mice by changing the composition of gut microbiota.
Bursts of Bipolar Microsecond Pulses Inhibit Tumor Growth
Sano, Michael B.; Arena, Christopher B.; Bittleman, Katelyn Rose; DeWitt, Matthew R.; Cho, Hyung J.; Szot, Cchristopher S.; Saur, Dieter; Cissell, James M.; Robertson, John L.; Lee, Yong Woo; Davalos, Rafael V. (Nature Publishing Group, 2015-10-13)
Irreversible electroporation (IRE) is an emerging focal therapy which is demonstrating utility in the treatment of unresectable tumors where thermal ablation techniques are contraindicated. IRE uses ultra-short duration, high-intensity monopolar pulsed electric fields to permanently disrupt cell membranes within a well-defined volume. Though preliminary clinical results for IRE are promising, implementing IRE can be challenging due to the heterogeneous nature of tumor tissue and the unintended induction of muscle contractions. High-frequency IRE (H-FIRE), a new treatment modality which replaces the monopolar IRE pulses with a burst of bipolar pulses, has the potential to resolve these clinical challenges. We explored the pulse-duration space between 250 ns and 100 μs and determined the lethal electric field intensity for specific H-FIRE protocols using a 3D tumor mimic. Murine tumors were exposed to 120 bursts, each energized for 100 μs, containing individual pulses 1, 2, or 5 μs in duration. Tumor growth was significantly inhibited and all protocols were able to achieve complete regressions. The H-FIRE protocol substantially reduces muscle contractions and the therapy can be delivered without the need for a neuromuscular blockade. This work shows the potential for H-FIRE to be used as a focal therapy and merits its investigation in larger pre-clinical models.
Chemokines and Chemokine Receptors in the Development of Lupus Nephritis
Liao, Xiaofeng; Pirapakaran, Tharshikha; Luo, Xin M. (Hindawi, 2016-06-14)
Lupus nephritis (LN) is a major cause of morbidity and mortality in the patients with systemic lupus erythematosus (SLE), an autoimmune disease with damage to multiple organs. Leukocyte recruitment into the inflamed kidney is a critical step to promote LN progression, and the chemokine/chemokine receptor system is necessary for leukocyte recruitment. In this review, we summarize recent studies on the roles of chemokines and chemokine receptors in the development of LN and discuss the potential and hurdles of developing novel, chemokine-based drugs to treat LN.
A comparative genomics perspective on the genetic content of the alkaliphilic haloarchaeon Natrialba magadii ATCC 43099T
Siddaramappa, Shivakumara; Challacombe, Jean F.; DeCastro, Rosana E.; Pfeiffer, Friedhelm; Sastre, Diego E.; Giménez, María I.; Paggi, Roberto A.; Detter, John C.; Davenport, Karen W.; Goodwin, Lynne A.; Kyrpides, Nikos; Tapia, Roxanne; Pitluck, Samuel; Lucas, Susan; Woyke, Tanja; Maupin-Furlow, Julie A. (2012-05-04)
Background Natrialba magadii is an aerobic chemoorganotrophic member of the Euryarchaeota and is a dual extremophile requiring alkaline conditions and hypersalinity for optimal growth. The genome sequence of Nab. magadii type strain ATCC 43099 was deciphered to obtain a comprehensive insight into the genetic content of this haloarchaeon and to understand the basis of some of the cellular functions necessary for its survival. Results The genome of Nab. magadii consists of four replicons with a total sequence of 4,443,643 bp and encodes 4,212 putative proteins, some of which contain peptide repeats of various lengths. Comparative genome analyses facilitated the identification of genes encoding putative proteins involved in adaptation to hypersalinity, stress response, glycosylation, and polysaccharide biosynthesis. A proton-driven ATP synthase and a variety of putative cytochromes and other proteins supporting aerobic respiration and electron transfer were encoded by one or more of Nab. magadii replicons. The genome encodes a number of putative proteases/peptidases as well as protein secretion functions. Genes encoding putative transcriptional regulators, basal transcription factors, signal perception/transduction proteins, and chemotaxis/phototaxis proteins were abundant in the genome. Pathways for the biosynthesis of thiamine, riboflavin, heme, cobalamin, coenzyme F420 and other essential co-factors were deduced by in depth sequence analyses. However, approximately 36% of Nab. magadii protein coding genes could not be assigned a function based on Blast analysis and have been annotated as encoding hypothetical or conserved hypothetical proteins. Furthermore, despite extensive comparative genomic analyses, genes necessary for survival in alkaline conditions could not be identified in Nab. magadii. Conclusions Based on genomic analyses, Nab. magadii is predicted to be metabolically versatile and it could use different carbon and energy sources to sustain growth. Nab. magadii has the genetic potential to adapt to its milieu by intracellular accumulation of inorganic cations and/or neutral organic compounds. The identification of Nab. magadii genes involved in coenzyme biosynthesis is a necessary step toward further reconstruction of the metabolic pathways in halophilic archaea and other extremophiles. The knowledge gained from the genome sequence of this haloalkaliphilic archaeon is highly valuable in advancing the applications of extremophiles and their enzymes.
Comparison of the Effects of Interleukin-1 on Equine Articular Cartilage Explants and Cocultures of Osteochondral and Synovial Explants
Byron, Christopher R.; Trahan, Richard A. (Frontiers, 2017-09-20)
Osteoarthritis (OA) is a ubiquitous disease affecting many horses. The disease causes chronic pain and decreased performance for patients and great cost to owners for diagnosis and treatment. The most common treatments include systemic non-steroidal anti-inflammatory drugs and intra-articular injection of corticosteroids. There is excellent support for the palliative pain relief these treatments provide; however, they do not arrest progression and may in some instances hasten advancement of disease. Orthobiologic treatments have been investigated as potential OA treatments that may not only ameliorate pain but also prevent or reverse pathologic articular tissue changes. Clinical protocols for intra-articular use of such treatments have not been optimized; the high cost of in vivo research and concerns over humane use of research animals may be preventing discovery. The objective of this study was to evaluate a novel in vitro articular coculture system for future use in OA treatment research. Concentrations and fold increases in various markers of inflammation (prostaglandin E2 and tumor necrosis factor-alpha), degradative enzyme activity [matrix metalloproteinase-13 (MMP-13)], cartilage and bone metabolism (bone alkaline phosphatase and dimethyl-methylene blue), and cell death (lactate dehydrogenase) were compared between IL-1-stimulated equine articular cartilage explant cultures and cocultures comprised of osteochondral and synovial explants (OCS). Results suggested that there are differences in responses of culture systems to inflammatory stimulation. In particular, the IL-1-induced fold changes in MMP-13 concentration were significantly different between OCS and cartilage explant culture systems after 96 h. These differences may be relevant to responses of joints to inflammation in vivo and could be important to the biological relevance of in vitro research findings.
Computed Tomographic Features in a Case of Bilateral Neoplastic Cryptorchidism with Suspected Torsion in a Dog.
Stokowski, Scott; Ruth, Jeffrey D.; Lanz, Otto I.; Ziglioli, Vincent (2016)
An 11-year-old male German Shepherd dog presented for inappetence and weight loss. Physical examination and initial bloodwork revealed palpable abdominal masses, mild non-regenerative anemia, and thrombocytopenia. Survey radiography and abdominal ultrasonography confirmed the presence of bilateral abdominal masses and lymphadenopathy. Contrast-enhanced computed tomography (CT) was performed in order to further investigate the origin of the intraabdominal masses, confirming two enlarged cryptorchid testes, one of which had an associated CT "whirl sign." Histopathology of the testes and lymph nodes revealed bilateral malignant Sertoli cell tumors and seminomas with lymph node metastasis of both neoplasms. The purpose of this case report is to discuss the benefits of CT in the diagnosis of cryptorchid testes and describe an additional organ that may display CT "whirl sign."
Control of lupus nephritis by changes of gut microbiota
Mu, Qinghui; Zhang, Husen; Liao, Xiaofeng; Lin, Kaisen; Liu, Hualan; Edwards, Michael R.; Ahmed, Sattar Ansar; Yuan, Ruoxi; Li, Liwu; Cecere, Thomas E.; Branson, David B.; Kirby, Jay L.; Goswami, Poorna; Leeth, Caroline M.; Read, Kaitlin A.; Oestreich, Kenneth J.; Vieson, Miranda D.; Reilly, Christopher M.; Luo, Xin M. (2017-07-11)
Background: Systemic lupus erythematosus, characterized by persistent inflammation, is a complex autoimmune disorder with no known cure. Immunosuppressants used in treatment put patients at a higher risk of infections. New knowledge of disease modulators, such as symbiotic bacteria, can enable fine-tuning of parts of the immune system, rather than suppressing it altogether. Results: Dysbiosis of gut microbiota promotes autoimmune disorders that damage extraintestinal organs. Here we report a role of gut microbiota in the pathogenesis of renal dysfunction in lupus. Using a classical model of lupus nephritis, MRL/lpr, we found a marked depletion of Lactobacillales in the gut microbiota. Increasing Lactobacillales in the gut improved renal function of these mice and prolonged their survival. We used a mixture of 5 Lactobacillus strains (Lactobacillus oris, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus gasseri), but L. reuteri and an uncultured Lactobacillus sp. accounted for most of the observed effects. Further studies revealed that MRL/lpr mice possessed a “leaky” gut, which was reversed by increased Lactobacillus colonization. Lactobacillus treatment contributed to an anti-inflammatory environment by decreasing IL-6 and increasing IL-10 production in the gut. In the circulation, Lactobacillus treatment increased IL-10 and decreased IgG2a that is considered to be a major immune deposit in the kidney of MRL/lpr mice. Inside the kidney, Lactobacillus treatment also skewed the Treg-Th17 balance towards a Treg phenotype. These beneficial effects were present in female and castrated male mice, but not in intact males, suggesting that the gut microbiota controls lupus nephritis in a sex hormone-dependent manner. Conclusions: This work demonstrates essential mechanisms on how changes of the gut microbiota regulate lupusassociated immune responses in mice. Future studies are warranted to determine if these results can be replicated in human subjects.
Effects of antemortem ingestion of ethanol on insect successional patterns and development of Phormia regina (Diptera : Calliphoridae)
Tabor, Kimberly L.; Fell, Richard D.; Brewster, Carlyle C.; Pelzer, Kevin D.; Behonick, George S. (Oxford University Press, 2005-05-01)
The effects of antemortem ingestion of ethanol by domestic pigs, Sus scrofa L., on postmortem insect successional patterns and the development of Phormia regina (Meigen) were studied during summer 2003 in Blacksburg, VA. Insect samples were collected from the carcasses of ethanol-treated and untreated pigs for 10 d postmortem during two successional studies. In total, 32 insect taxa were collected during the two studies, with 29 and 27 taxa observed on the carcasses of ethanol-treated and untreated pigs, respectively. The earliest arrivers to both carcass types were dipterans. This group was represented by six families, with P. regina and Phaenicia coeruleiviridis (Macquart) being the most common calliphorids. Beetles in six families were collected on the carcasses of ethanol-treated pigs, but only three of the families were collected on carcasses of the untreated pigs. Permutation analyses to test the null hypothesis of no similarity between successional patterns of insect taxa from carcasses of ethanol-treated and untreated pigs showed that the successional patterns were similar between carcass types in the first (P = 0.003) and the second (P = 0.01) studies. The results of the development study of P. regina maggots in the field show that there was a significant difference between the distributions of length for maggots reared on loin tissue from ethanol-treated and untreated pigs. Maggots that fed on tissue from ethanol-treated pigs took approximate to 11.9 11 longer to reach the pupal stage than maggots that fed on tissue from untreated pigs. The longer developmental time for maggots on tissue from ethanol-treated pigs was due mainly to the longer postfeeding period of the third instar.
Engineering Tendon: Scaffolds, Bioreactors, and Models of Regeneration
Youngstrom, Daniel W.; Barrett, Jennifer G. (Hindawi, 2016)
Tendons bridge muscle and bone, translating forces to the skeleton and increasing the safety and efficiency of locomotion. When tendons fail or degenerate, there are no effective pharmacological interventions. The lack of available options to treat damaged tendons has created a need to better understand and improve the repair process, particularly when suitable autologous donor tissue is unavailable for transplantation. Cells within tendon dynamically react to loading conditions and undergo phenotypic changes in response to mechanobiological stimuli. Tenocytes respond to ultrastructural topography and mechanical deformation via a complex set of behaviors involving force-sensitive membrane receptor activity, changes in cytoskeletal contractility, and transcriptional regulation. Effective ex vivo model systems are needed to emulate the native environment of a tissue and to translate cell-matrix forces with high fidelity. While early bioreactor designs have greatly expanded our knowledge of mechanotransduction, traditional scaffolds do not fully model the topography, composition, and mechanical properties of native tendon. Decellularized tendon is an ideal scaffold for cultivating replacement tissue and modeling tendon regeneration. Decellularized tendon scaffolds (DTS) possess high clinical relevance, faithfully translate forces to the cellular scale, and have bulk material properties that match natural tissue. This review summarizes progress in tendon tissue engineering, with a focus on DTS and bioreactor systems.
Enzymatic, immunological and phylogenetic characterization of Brucella suis urease
Contreras-Rodriguez, Araceli; Quiroz-Limon, Jose; Martins, Ana M.; Peralta, Humberto; Avila-Calderon, Eric Daniel; Sriranganathan, Nammalwar; Boyle, Stephen M.; Lopez-Merino, Ahidé (2008-07-19)
Background The sequenced genomes of the Brucella spp. have two urease operons, ure-1 and ure-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from Brucella suis strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined. Results Urease encoded by the ure-1 operon of Brucella suis strain 1330 was purified to homogeneity using ion exchange and hydrophobic interaction chromatographies. The urease was purified 51-fold with a recovery of 12% of the enzyme activity and 0.24% of the total protein. The enzyme had an isoelectric point of 5, and showed optimal activity at pH 7.0 and 28-35°C. The purified enzyme exhibited a Michaelis-Menten saturation kinetics with a Km of 5.60 ± 0.69 mM. Hydroxyurea and thiourea are competitive inhibitors of the enzyme with Ki of 1.04 ± 0.31 mM and 26.12 ± 2.30 mM, respectively. Acetohydroxamic acid also inhibits the enzyme in a competitive way. The molecular weight estimated for the native enzyme was between 130-135 kDa by gel filtration chromatography and 157 ± 7 kDa using 5-10% polyacrylamide gradient non-denaturing gel. Only three subunits in SDS-PAGE were identified: two small subunits of 14,000 Da and 15,500 Da, and a major subunit of 66,000 Da. The amino terminal sequence of the purified large subunit corresponded to the predicted amino acid sequence encoded by ureC1. The UreC1 subunit was recognized by sera from patients with acute and chronic brucellosis. By phylogenetic and cluster structure analyses, ureC1 was related to the ureC typically present in the Rhizobiales; in contrast, the ureC2 encoded in the ure-2 operon is more related to distant species. Conclusion We have for the first time purified and characterized an active urease from B. suis. The enzyme was characterized at the kinetic, immunological and phylogenetic levels. Our results confirm that the active urease of B. suis is a product of ure-1 operon.
Evaluating Checklist Use in Companion Animal Wellness Visits in a Veterinary Teaching Hospital: A Preliminary Study
Nappier, Michael T.; Corrigan, Virginia Kiefer; Bartl-Wilson, Lara; Freeman, Mark D.; Werre, Stephen R.; Tempel, Eric (2017)
The number of companion animal wellness visits in private practice has been decreasing, and one important factor cited is the lack of effective communication between veterinarians and pet owners regarding the importance of preventive care. Checklists have been widely used in many fields and are especially useful in areas where a complex task must be completed with multiple small steps, or when cognitive fatigue is evident. The use of checklists in veterinary medical education has not yet been thoroughly evaluated as a potential strategy to improve communication with pet owners regarding preventive care. The authors explored whether the use of a checklist based on the American Animal Hospital Association/American Veterinary Medical Association canine and feline preventive care guidelines would benefit senior veterinary students in accomplishing more complete canine and feline wellness visits. A group of students using provided checklists was compared to a control group of students who did not use checklists on the basis of their medical record notes from the visits. The students using the checklists were routinely more complete in several areas of a wellness visit vs. those who did not use the checklists. However, neither group of students routinely discussed follow-up care recommendations such as frequency or timing of follow-up visits. The study authors recommend considering checklist use for teaching and implementing wellness in companion animal primary care veterinary clinical teaching settings.
Functional Characterization of Detergent-Decellularized Equine Tendon Extracellular Matrix for Tissue Engineering Applications
Youngstrom, Daniel W.; Barrett, Jennifer G.; Jose, Rod R.; Kaplan, David L. (PLOS, 2013-05-17)
Natural extracellular matrix provides a number of distinct advantages for engineering replacement orthopedic tissue due to its intrinsic functional properties. The goal of this study was to optimize a biologically derived scaffold for tendon tissue engineering using equine flexor digitorum superficialis tendons. We investigated changes in scaffold composition and ultrastructure in response to several mechanical, detergent and enzymatic decellularization protocols using microscopic techniques and a panel of biochemical assays to evaluate total protein, collagen, glycosaminoglycan, and deoxyribonucleic acid content. Biocompatibility was also assessed with static mesenchymal stem cell (MSC) culture. Implementation of a combination of freeze/thaw cycles, incubation in 2% sodium dodecyl sulfate (SDS), trypsinization, treatment with DNase-I, and ethanol sterilization produced a non-cytotoxic biomaterial free of appreciable residual cellular debris with no significant modification of biomechanical properties. These decellularized tendon scaffolds (DTS) are suitable for complex tissue engineering applications, as they provide a clean slate for cell culture while maintaining native three-dimensional architecture.
Herpes Simplex Virus 1 Reactivates from Autonomic Ciliary Ganglia Independently from Sensory Trigeminal Ganglia To Cause Recurrent Ocular Disease
Lee, Sungseok; Ives, Angela M.; Bertke, Andrea S. (American Society for Microbiology, 2015-08-01)
Herpes simplex virus 1 (HSV-1) and HSV-2 establish latency in sensory and autonomic neurons after ocular or genital infection, but their recurrence patterns differ. HSV-1 reactivates from latency to cause recurrent orofacial disease, and while HSV-1 also causes genital lesions, HSV-2 recurs more efficiently in the genital region and rarely causes ocular disease. The mechanisms regulating these anatomical preferences are unclear. To determine whether differences in latent infection and reactivation in autonomic ganglia contribute to differences in HSV-1 and HSV-2 anatomical preferences for recurrent disease, we compared HSV-1 and HSV-2 clinical disease, acute and latent viral loads, and viral gene expression in sensory trigeminal and autonomic superior cervical and ciliary ganglia in a guinea pig ocular infection model. HSV-2 produced more severe acute disease, correlating with higher viral DNA loads in sensory and autonomic ganglia, as well as higher levels of thymidine kinase expression, a marker of productive infection, in autonomic ganglia. HSV-1 reactivated in ciliary ganglia, independently from trigeminal ganglia, to cause more frequent recurrent symptoms, while HSV-2 replicated simultaneously in autonomic and sensory ganglia to cause more persistent disease. While both HSV-1 and HSV-2 expressed the latency-associated transcript (LAT) in the trigeminal and superior cervical ganglia, only HSV-1 expressed LAT in ciliary ganglia, suggesting that HSV-2 is not reactivation competent or does not fully establish latency in ciliary ganglia. Thus, differences in replication and viral gene expression in autonomic ganglia may contribute to differences in HSV-1 and HSV-2 acute and recurrent clinical disease.
A History of the Development of Brucella Vaccines
Avila-Calderon, Eric Daniel; Lopez-Merino, Ahidé; Sriranganathan, Nammalwar; Boyle, Stephen M.; Contreras-Rodriguez, Araceli (Hindawi, 2013)
Brucellosis is a worldwide zoonosis affecting animal and human health. In the last several decades, much research has been performed to develop safer Brucella vaccines to control the disease mainly in animals. Till now, no effective human vaccine is available. The aim of this paper is to review and discuss the importance of methodologies used to develop Brucella vaccines in pursuing this challenge.
Horizontal gene transfer in Histophilus somni and its role in the evolution of pathogenic strain 2336, as determined by comparative genomic analyses
Siddaramappa, Shivakumara; Challacombe, Jean F.; Duncan, Alison J.; Gillaspy, Allison F.; Carson, Matthew; Gipson, Jenny; Orvis, Joshua; Zaitshik, Jeremy; Barnes, Gentry; Bruce, David; Chertkov, Olga; Detter, J. Chris; Han, Cliff S.; Tapia, Roxanne; Thompson, Linda S.; Dyer, David W.; Inzana, Thomas J. (2011-11-23)
Background Pneumonia and myocarditis are the most commonly reported diseases due to Histophilus somni, an opportunistic pathogen of the reproductive and respiratory tracts of cattle. Thus far only a few genes involved in metabolic and virulence functions have been identified and characterized in H. somni using traditional methods. Analyses of the genome sequences of several Pasteurellaceae species have provided insights into their biology and evolution. In view of the economic and ecological importance of H. somni, the genome sequence of pneumonia strain 2336 has been determined and compared to that of commensal strain 129Pt and other members of the Pasteurellaceae. Results The chromosome of strain 2336 (2,263,857 bp) contained 1,980 protein coding genes, whereas the chromosome of strain 129Pt (2,007,700 bp) contained only 1,792 protein coding genes. Although the chromosomes of the two strains differ in size, their average GC content, gene density (total number of genes predicted on the chromosome), and percentage of sequence (number of genes) that encodes proteins were similar. The chromosomes of these strains also contained a number of discrete prophage regions and genomic islands. One of the genomic islands in strain 2336 contained genes putatively involved in copper, zinc, and tetracycline resistance. Using the genome sequence data and comparative analyses with other members of the Pasteurellaceae, several H. somni genes that may encode proteins involved in virulence (e.g., filamentous haemaggutinins, adhesins, and polysaccharide biosynthesis/modification enzymes) were identified. The two strains contained a total of 17 ORFs that encode putative glycosyltransferases and some of these ORFs had characteristic simple sequence repeats within them. Most of the genes/loci common to both the strains were located in different regions of the two chromosomes and occurred in opposite orientations, indicating genome rearrangement since their divergence from a common ancestor. Conclusions Since the genome of strain 129Pt was ~256,000 bp smaller than that of strain 2336, these genomes provide yet another paradigm for studying evolutionary gene loss and/or gain in regard to virulence repertoire and pathogenic ability. Analyses of the complete genome sequences revealed that bacteriophage- and transposon-mediated horizontal gene transfer had occurred at several loci in the chromosomes of strains 2336 and 129Pt. It appears that these mobile genetic elements have played a major role in creating genomic diversity and phenotypic variability among the two H. somni strains.
In Situ Real-Time Chemiluminescence Imaging of Reactive Oxygen Species Formation from Cardiomyocytes
Li, Yunbo; Shen, Haiou; Zhu, Hong; Trush, Michael A.; Jiang, Ming; Wang, Ge (Hindawi, 2009-02-25)
We have applied the highly sensitive chemiluminescence (CL) imagingtechnique to investigate the in situ ROS formation in cultured monolayers of rat H9c2 cardiomyocytes. Photon emission was detected via an innovative imaging system after incubation of H9c2 cells in culture with luminol and horseradish peroxidase (HRP), suggesting constitutive formation of ROS by the cardiomyocytes. Addition of benzo(a)pyrene-1,6-quinone(BPQ) to cultured H9c2 cells resulted in a 4-5-fold increase in the formation of ROS, as detected by the CL imaging. Both constitutive and BPQ-stimulated CL responses in cultured H9c2 cells were sustained for up to 1 hour. The CL responses were completely abolished in the presence of superoxide dismutase and catalase, suggesting the primary involvement of superoxide and hydrogen peroxide (). In contrast to BPQ-mediated redox cycling, blockage of mitochondrial electron transport chain by either antimycin A or rotenone exerted marginal effects on the ROS formation by cultured H9c2 cells. Upregulation of cellular antioxidants fordetoxifying both superoxide and by 3-1,2-dithiole-3-thione resulted in marked inhibition of both constitutive and BPQ-augmented ROS formation in cultured H9c2 cells. Taken together, we demonstrate the sensitive detection of ROS by CL imaging in cultured cardiomyocytes.
In vitro performance of lipid-PLGA hybrid nanoparticles as an antigen delivery system: lipid composition matters
Hu, Yun; Ehrich, Marion F.; Fuhrman, Kristel; Zhang, Chenming (Springer, 2014-08-27)
Due to the many beneficial properties combined from both poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and liposomes, lipid-PLGA hybrid NPs have been intensively studied as cancer drug delivery systems, bio-imaging agent carriers, as well as antigen delivery vehicles. However, the impact of lipid composition on the performance of lipid-PLGA hybrid NPs as a delivery system has not been well investigated. In this study, the influence of lipid composition on the stability of the hybrid NPs and in vitro antigen release from NPs under different conditions was examined. The uptake of hybrid NPs with various surface charges by dendritic cells (DCs) was carefully studied. The results showed that PLGA NPs enveloped by a lipid shell with more positive surface charges could improve the stability of the hybrid NPs, enable better controlled release of antigens encapsulated in PLGA NPs, as well as enhance uptake of NPs by DC.
In vitro susceptibility of Borrelia burgdorferi isolates to three antibiotics commonly used for treating equine Lyme disease
Caol, Sanjie; Divers, Thomas; Crisman, Mark V.; Chang, Yung-Fu (2017-09-29)
Background Lyme disease in humans is predominantly treated with tetracycline, macrolides or beta lactam antibiotics that have low minimum inhibitory concentrations (MIC) against Borrelia burgdorferi. Horses with Lyme disease may require long-term treatment making frequent intravenous or intramuscular treatment difficult and when administered orally those drugs may have either a high incidence of side effects or have poor bioavailability. The aim of the present study was to determine the in vitro susceptibility of three B. burgdorferi isolates to three antibiotics of different classes that are commonly used in practice for treating Borrelia infections in horses. Results Broth microdilution assays were used to determine minimum inhibitory concentration of three antibiotics (ceftiofur sodium, minocycline and metronidazole), for three Borrelia burgdorferi isolates. Barbour-Stoner-Kelly (BSK K + R) medium with a final inoculum of 106 Borrelia cells/mL and incubation periods of 72 h were used in the determination of MICs. Observed MICs indicated that all isolates had similar susceptibility to each drug but susceptibility to the tested antimicrobial agents varied; ceftiofur sodium (MIC = 0.08 μg/ml), minocycline hydrochloride (MIC = 0.8 μg/ml) and metronidazole (MIC = 50 μg/ml). Conclusions The MIC against B. burgorferi varied among the three antibiotics with ceftiofur having the lowest MIC and metronidazole the highest MIC. The MIC values observed for ceftiofur in the study fall within the range of reported serum and tissue concentrations for the drug metabolite following ceftiofur sodium administration as crystalline-free acid. Minocycline and metronidazole treatments, as currently used in equine practice, could fall short of attaining MIC concentrations for B. burgdorferi.
Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge
Herbert, Andrew S.; Heffron, C. Lynn; Sundick, Roy; Roberts, Paul C. (2009-04-24)
Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1), demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC). Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4) fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective.

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