Browsing by Subject "Infection"
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Item Open Access CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.(Dis Model Mech, 2015-12) Cronan, Mark R; Rosenberg, Allison F; Oehlers, Stefan H; Saelens, Joseph W; Sisk, Dana M; Jurcic Smith, Kristen L; Lee, Sunhee; Tobin, David MVisualization of infection and the associated host response has been challenging in adult vertebrates. Owing to their transparency, zebrafish larvae have been used to directly observe infection in vivo; however, such larvae have not yet developed a functional adaptive immune system. Cells involved in adaptive immunity mature later and have therefore been difficult to access optically in intact animals. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Recently, CLARITY and PACT (passive clarity technique) methodologies have enabled clearing and direct visualization of dissected organs. Here, we show that these techniques can be applied to image host-pathogen interactions directly in whole animals. CLARITY and PACT-based clearing of whole adult zebrafish and Mycobacterium tuberculosis-infected mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we were able to image normal and pathogenic structures and their surrounding host context at high resolution. We identified the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterized the induction of the cytokine tumor necrosis factor (TNF) within the granuloma using an established fluorescent reporter line. We observed heterogeneity in TNF induction within granuloma macrophages, consistent with an evolving view of the tuberculous granuloma as a non-uniform, heterogeneous structure. Broad application of this technique will enable new understanding of host-pathogen interactions in situ.Item Open Access Design and Development of an Anti-fouling Urinary Catheter Utilizing Active Surface Deformation(2015-01-01) Levering, Vrad WilsonThere are over 30 million Foley urinary catheters used annually, and the greatest problem with Foley catheters is catheter-associated urinary tract infections (CAUTIs). CAUTIs are the number one cause of hospital-acquired infections and make up to 40% of nosocomial infections. Biofilms on urinary catheters are critical to the progression of symptomatic CAUTIs, but are difficult to treat due to the protective effect of the biofilm matrix against antibiotics. The anti-fouling catheter technology proposed and demonstrated herein uses a mechanical, non-antibiotic approach to physically remove biofilms and thereby provide an appealing option for potentially stopping the progression of symptomatic infections. Additionally, because the anti-fouling technology is mechanical, it can circumvent the persistent failings of chemical and biological approaches that have failed to address catheter-associated urinary tract infections for the last 50+ years since Foley catheters were introduced.
We designed and optimized urinary catheter prototypes capable of on-demand removal of biofilms from the previously-inaccessible main drainage lumen of catheters. The concept uses pressure-actuated chambers in elastomer constructs to generate regio-selective strain and thereby remove biofilms. We first grew mature Proteus mirabilis crystalline biofilms on flat silicone elastomer substrates, and showed that application of strain to the substrate debonded the biofilm, and that increasing the strain rate increased biofilm detachment. A quantitative relationship between the applied strain rate and biofilm debonding was found through an analysis of the biofilm segment length and the calculated driving force for debonding. We then constructed proof-of-concept prototypes of sections of anti-fouling catheter shafts using silicone and 3D printed reverse molding in methods akin to those used for soft robotics. The proof-of-concept prototypes demonstrated release of mature P. mirabilis crystalline biofilms from their strained surfaces, and prompted our development of more advanced multi-lumen prototypes. The multi-lumen prototypes were designed and optimized using successive rounds of finite element modeling to adjust the number and postion of intra-wall inflation lumens. We then constructed prototypes based on the optimized design with clinically relevant dimensions and showed they were able to generate greater than 30% strain on the majority of the luminal surface, and along their full length. Those catheter prototypes were able to on-demand, and repeatedly, remove greater than 80% of a mixed community biofilm of P. mirabilis and E. coli. In summary, this study shows (1) strain in the elastomeric substrate actively debonds crystalline biofilms in vitro (2) modeling based on characterization of biofilm properties and understanding of substrate strain informs and facilitates prototype catheter design (3) urinary catheter prototypes utilizing inflation-induced substrate strain are capable of on-demand and repeatedly removing biofilms in vitro.
Item Open Access EBV-Associated Gastric Cancer: From Initial Infection to Unique Therapeutic Approaches(2023) Stanland, Lyla JuneEpstein-Barr virus (EBV) is a ubiquitous human herpesvirus that infects over 95% of the adult population. While infection is typically asymptomatic, in some individuals, particularly the immunocompromised, EBV is the causative agent of several cancers including lymphomas and epithelial cancers. Specifically, gastric carcinoma, nasopharyngeal carcinoma, and lymphoepithelioma-like carcinoma which occurs across multiple sites in the body, most notably, in the lung. EBV-associated gastric cancer (EBVaGC) is a unique subset of gastric cancer that makes up 10% of all GCs worldwide. EBVaGCs display an 80% rate of activating PIK3CA mutation and are also the most hypermethylated of any tumor type, displaying what is known as a CpG island hypermethylator phenotype (CIMP). EBV infection of B cells can be easily modeled in vitro using primary B cells and lymphoblastoid cell lines (LCLs). However, epithelial cell infection models have proven much more difficult to develop. Given these difficulties, EBV infection and outgrowth in epithelial cells is comparatively understudied and the process of tumorigenesis in vivo is poorly understood. In this dissertation, I developed methods to generate EBV infected epithelial cell lines derived from both gastric cancer and lung adenocarcinoma using diverse strains of EBV. I used these models to complete a CRISPR/Cas9 whole genome knockout screen to identify cellular restriction factors of infection and outgrowth. Together, these data will provide novel insights into the process of EBV infection and the dynamic interplay between virus and host during tumorigenesis. Furthermore, I have used these models to explore unique therapeutic approaches for EBV+ epithelial cancers. Specifically, I have characterized the lytic reactivation potential to histone deacetylase (HDAC) inhibitors and generated preclinical data supporting the use of HDAC inhibitors and the anti-viral ganciclovir for treatment of EBV+ epithelial tumors. Lastly, I have identified modulators of the response to a PI3Ka inhibitor in PIK3CA mutant gastric cancers. I found that loss of NEDD9 or inhibition of BCL-XL rendered cells hyper-sensitive to the PI3Ka inhibitor BYL719. Additionally, I found that loss of CBFB conferred resistance to BYL719 through up- regulation of the protein kinase PIM1 and defined the clinical utility of our data in the context of PI3K inhibition more broadly. The work outlined in this dissertation contributes to the study of EBV infection and tumorigenesis in the stomach as well as provides mechanistic insights into novel therapeutic approaches for EBV+ epithelial cancers and PIK3CA mutant gastric cancers.
Item Open Access Epstein-Barr virus infection phenocopies apoptosis regulation in germinal center B cells(2019) Dai, JoanneThe Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects more than >95% of the global adult population. In immunocompetent individuals, EBV infection is asymptomatic and takes place in the oral cavity, where EBV establishes a life-long latent infection in memory B cells by temporally regulating viral gene expression to mimic B cell maturation. In immunocompromised individuals, however, EBV infection can give rise to infectious mononucleosis, epithelial carcinomas, and lymphomas. To model EBV-mediated lymphomagenesis, infection of EBV in vitro generates growth-transformed and immortalized lymphoblastoid cell lines (LCLs), which allows for the characterization of dynamic viral and host gene expression. Our lab has found that the early phase after infection is transcriptionally distinct from the late phase when infected B cells are fully growth-transformed. We also found that apoptosis regulation in each phase of infection is uniquely regulated by a single viral nuclear protein that regulates host gene expression through epigenetic mechanisms. To determine if apoptosis regulation in EBV-infected B cells is virus-specific, I have characterized apoptosis regulation in uninfected maturing B cells and mitogen-stimulated B cells. For the upregulation of one anti-apoptotic protein, EBV infection promotes a chromatin structure resembling that in germinal center light zone B cells, indicating that EBV phenocopies germinal center chromatin regulation to promote apoptosis resistance. In addition to apoptosis regulation, EBV infection phenocopies various aspects of GC B cells and plasmablasts, where the inhibition of plasma cell differentiation increases the efficiency of immortalization and growth-transformation of B cells infected in vitro. The work outlined in this dissertation demonstrate that viral and host genes cooperate in mediating apoptosis regulation, differentiation, and ultimately fate-determination of EBV-infected B cells.
Item Open Access Etiologies of illness among patients meeting integrated management of adolescent and adult illness district clinician manual criteria for severe infections in northern Tanzania: implications for empiric antimicrobial therapy.(Am J Trop Med Hyg, 2015-02) Rubach, Matthew P; Maro, Venance P; Bartlett, John A; Crump, John AWe describe the laboratory-confirmed etiologies of illness among participants in a hospital-based febrile illness cohort study in northern Tanzania who retrospectively met Integrated Management of Adolescent and Adult Illness District Clinician Manual (IMAI) criteria for septic shock, severe respiratory distress without shock, and severe pneumonia, and compare these etiologies against commonly used antimicrobials, including IMAI recommendations for emergency antibacterials (ceftriaxone or ampicillin plus gentamicin) and IMAI first-line recommendations for severe pneumonia (ceftriaxone and a macrolide). Among 423 participants hospitalized with febrile illness, there were 25 septic shock, 37 severe respiratory distress without shock, and 109 severe pneumonia cases. Ceftriaxone had the highest potential utility of all antimicrobials assessed, with responsive etiologies in 12 (48%) septic shock, 5 (14%) severe respiratory distress without shock, and 19 (17%) severe pneumonia illnesses. For each syndrome 17-27% of participants had etiologic diagnoses that would be non-responsive to ceftriaxone, but responsive to other available antimicrobial regimens including amphotericin for cryptococcosis and histoplasmosis; anti-tuberculosis therapy for bacteremic disseminated tuberculosis; or tetracycline therapy for rickettsioses and Q fever. We conclude that although empiric ceftriaxone is appropriate in our setting, etiologies not explicitly addressed in IMAI guidance for these syndromes, such as cryptococcosis, histoplasmosis, and tetracycline-responsive bacterial infections, were common.Item Open Access Evidence-Based Perioperative Medicine comes of age: the Perioperative Quality Initiative (POQI): The 1st Consensus Conference of the Perioperative Quality Initiative (POQI).(Perioper Med (Lond), 2016) Miller, Timothy E; Shaw, Andrew D; Mythen, Michael G; Gan, Tong J; Perioperative Quality Initiative (POQI) I WorkgroupThe 1st POQI Consensus Conference occurred in Durham, NC, on March 4-5, 2016, and was supported by the American Society of Enhanced Recovery (ASER) and Evidence-Based Perioperative Medicine (EBPOM). The conference focused on enhanced recovery for colorectal surgery and discussed four topics-perioperative analgesia, perioperative fluid management, preventing nosocomial infection, and measurement and quality in enhanced recovery pathways.Item Open Access Human endotoxin administration as an experimental model in drug development.(Clin Pharmacol Ther, 2014-10) Suffredini, AF; Noveck, RJLinking human physiology to inflammatory mechanisms discovered in vitro or in animal models is essential to determine their importance. Innate immunity underlies many of these inflammatory responses in health and disease. Bacterial endotoxin is the quintessential trigger of innate immune responses. When administered to humans, endotoxin has been an important means of demonstrating key inflammatory mechanisms in vivo. Furthermore, endotoxin challenges have provided opportunities to test the effects of novel inflammation-modifying agents in humans.Item Open Access Impact of early postoperative oral nutritional supplement utilization on clinical outcomes in colorectal surgery.(Perioperative medicine (London, England), 2020-01) Williams, David GA; Ohnuma, Tetsu; Krishnamoorthy, Vijay; Raghunathan, Karthik; Sulo, Suela; Cassady, Bridget A; Hegazi, Refaat; Wischmeyer, Paul EBackground:Small randomized trials of early postoperative oral nutritional supplementation (ONS) suggest various health benefits following colorectal surgery (CRS). However, real-world evidence of the impact of early ONS on clinical outcomes in CRS is lacking. Methods:Using a nationwide administrative-financial database (Premier Healthcare Database), we examined the association between early ONS use and postoperative clinical outcomes in patients undergoing elective open or laparoscopic CRS between 2008 and 2014. Early ONS was defined as the presence of charges for ONS before postoperative day (POD) 3. The primary outcome was composite infectious complications. Key secondary efficacy (intensive care unit (ICU) admission and gastrointestinal complications) and falsification (blood transfusion and myocardial infarction) outcomes were also examined. Propensity score matching was used to assemble patient groups that were comparable at baseline, and differences in outcomes were examined. Results:Overall, patients receiving early ONS were older with greater comorbidities and more likely to be Medicare beneficiaries with malnutrition. In a well-matched sample of early ONS recipients (n = 267) versus non-recipients (n = 534), infectious complications were significantly lower in early ONS recipients (6.7% vs. 11.8%, P < 0.03). Early ONS use was also associated with significantly reduced rates of pneumonia (P < 0.04), ICU admissions (P < 0.04), and gastrointestinal complications (P < 0.05). There were no significant differences in falsification outcomes. Conclusions:Although early postoperative ONS after CRS was more likely to be utilized in elderly patients with greater comorbidities, the use of early ONS was associated with reduced infectious complications, pneumonia, ICU admission, and gastrointestinal complications. This propensity score-matched study using real-world data suggests that clinical outcomes are improved with early ONS use, a simple and inexpensive intervention in CRS patients.Item Open Access Neuroimmune Signaling in the Hippocampus: Mechanisms of Risk and Resilience(2014) Williamson, Lauren LeshenThe interactions between the brain and the immune system are extensive and each has a profound influence on the other. The hippocampus is a brain region that is strongly impacted by the immune system, especially considering its large population of microglia, the resident immune cells of the brain. Cytokines and chemokines, the signaling molecules from immune cells, signal within the central nervous system (CNS) as well, and they are critical in hippocampal function. The relationship between the immune system and the hippocampus may underlie its particular vulnerability to diseases and disorders of the nervous system and the periphery. Conversely, immune signaling within the hippocampus is affected by alterations in hippocampal resilience and flexibility, such that increased hippocampal plasticity reduces vulnerability to immune challenges. The balance between risk and resilience in the hippocampus is modulated by immune signaling, especially by microglia.
The hippocampus is vulnerable to immune challenges, disease and injury, but it is simultaneously a region capable of profound plasticity and flexibility. The following dissertation experiments were designed to assess the roles of microglia and their signaling molecules, cytokines and chemokines, during normal hippocampal processes, such as learning and memory and response to immune challenge. The first set of experiments examined the effects of a neonatal bacterial infection in rats on hippocampal-dependent learning and memory as well as neuronal and microglial signaling in adulthood. In the first experiment, neonatally infected rats have impaired memory during fear conditioning following an immune challenge in adulthood. The impairment is caused by the exaggerated expression of the pro-inflammatory cytokine, interleukin (IL)-1β, within the hippocampus during learning. Hippocampal microglia are the primary source of IL-1β and the microglia in neonatally infected rats are "primed" by the infection into adulthood. In the second experiment, neonatally infected rats are more accurate on a Morris Water maze task following minimal training in adulthood, but have significantly impaired memory for a reversal platform location. In addition to improved accuracy, they have lower neural activation as measured by Arc protein expression within the dentate gyrus (DG) of the hippocampus. The next set of experiments assessed the effects of increasing hippocampal plasticity on immune signaling within the hippocampus. Following 7 weeks of environmental enrichment (EE), enriched rats had an attenuated pro-inflammatory response within the hippocampus in response to an in vivo peripheral immune challenge. The reduced immune response was specific to a subset of cytokines and chemokines and occurred only within the hippocampus and not adjacent cortical regions. Enrichment increased glial antigen expression within the DG as well. In another group of enriched rats, an ex vivo stimulation of isolated hippocampal microglia from EE rats demonstrated that the reduced microglial reactivity observed in vivo requires influence of other neural cell types on microglia phenotype, such that microglia within the DG of EE rats are smaller than controls. Taken together, these experiments define cellular and molecular mechanisms of hippocampal vulnerability and resilience as a function of interactions between the brain and the immune system.
Item Restricted Pseudotumor with superimposed periprosthetic infection following metal-on-metal total hip arthroplasty: a case report.(J Bone Joint Surg Am, 2010-07-07) Watters, Tyler Steven; Eward, William C; Hallows, Rhett K; Dodd, Leslie G; Wellman, Samuel S; Bolognesi, Michael PItem Open Access Recovery from an acute infection in C. elegans requires the GATA transcription factor ELT-2.(PLoS Genet, 2014-10) Head, Brian; Aballay, AlejandroThe mechanisms involved in the recognition of microbial pathogens and activation of the immune system have been extensively studied. However, the mechanisms involved in the recovery phase of an infection are incompletely characterized at both the cellular and physiological levels. Here, we establish a Caenorhabditis elegans-Salmonella enterica model of acute infection and antibiotic treatment for studying biological changes during the resolution phase of an infection. Using whole genome expression profiles of acutely infected animals, we found that genes that are markers of innate immunity are down-regulated upon recovery, while genes involved in xenobiotic detoxification, redox regulation, and cellular homeostasis are up-regulated. In silico analyses demonstrated that genes altered during recovery from infection were transcriptionally regulated by conserved transcription factors, including GATA/ELT-2, FOXO/DAF-16, and Nrf/SKN-1. Finally, we found that recovery from an acute bacterial infection is dependent on ELT-2 activity.Item Open Access Skewing of the population balance of lymphoid and myeloid cells by secreted and intracellular osteopontin.(Nature immunology, 2017-09) Kanayama, Masashi; Xu, Shengjie; Danzaki, Keiko; Gibson, Jason R; Inoue, Makoto; Gregory, Simon G; Shinohara, Mari LThe balance of myeloid populations and lymphoid populations must be well controlled. Here we found that osteopontin (OPN) skewed this balance during pathogenic conditions such as infection and autoimmunity. Notably, two isoforms of OPN exerted distinct effects in shifting this balance through cell-type-specific regulation of apoptosis. Intracellular OPN (iOPN) diminished the population size of myeloid progenitor cells and myeloid cells, and secreted OPN (sOPN) increase the population size of lymphoid cells. The total effect of OPN on skewing the leukocyte population balance was observed as host sensitivity to early systemic infection with Candida albicans and T cell-mediated colitis. Our study suggests previously unknown detrimental roles for two OPN isoforms in causing the imbalance of leukocyte populations.Item Open Access Surgical technique for development of a clinically-representative ventral hernia repair infection rat model.(MethodsX, 2020-01) Anastasio, Albert Thomas; Van Eps, Jeffrey L; Fernandez-Moure, Joseph SThe animal model of infection following ventral hernia repair (VHR) has previously been utilized in exploring treatments and innovative therapies, such as implantation of biologic mesh imbedded with various anti-bacterial properties. The rat model has been utilized most commonly, but prior work has failed to recreate an adequately clinically representative model of infection following VHR. Additionally, there is lack of standardization of mesh infection severity across existing literature. Therefore, the aim of this paper is to describe the creation of a clinically representative VHR infection model utilizing an index procedure where a hernia defect is created followed by a VHR using biologic mesh and subsequent infectious agent inoculation. Additionally, we describe the development of a standardization index to quantify severity of mesh infection: the Mesh Infection Severity Index (MISI).•Our protocol involves two procedures, an index procedure where a hernia model is created, and a subsequent procedure where an infectious inoculant is introduced.•We describe the MISI, a standardization tool we hope will allow for ease of cross-institutional data assessment.•In summary, our protocol not only serves as a more clinically representative animal model, but also includes a novel metric to standardize mesh infection severity.