Publications by authors named "Kevin P Francis"

78 Publications

Comparison of two fluorescent probes in preclinical non-invasive imaging and image-guided debridement surgery of Staphylococcal biofilm implant infections.

Sci Rep 2021 Jan 15;11(1):1622. Epub 2021 Jan 15.

Department of Orthopaedic Surgery, David Geffen School of Medicine at University of California Los Angeles, 1250 16th Street, Suite 2100, Santa Monica, CA, 90404, USA.

Implant-associated infections are challenging to diagnose and treat. Fluorescent probes have been heralded as a technologic advancement that can improve our ability to non-invasively identify infecting organisms, as well as guide the inexact procedure of surgical debridement. This study's purpose was to compare two fluorescent probes for their ability to localize Staphylococcus aureus biofilm infections on spinal implants utilizing noninvasive optical imaging, then assessing the broader applicability of the more successful probe in other infection animal models. This was followed by real-time, fluorescence image-guided surgery to facilitate debridement of infected tissue. The two probe candidates, a labelled antibiotic that targets peptidoglycan (Vanco-800CW), and the other, a labelled antibody targeting the immunodominant Staphylococcal antigen A (1D9-680), were injected into mice with spine implant infections. Mice were then imaged noninvasively with near infrared fluorescent imaging at wavelengths corresponding to the two probe candidates. Both probes localized to the infection, with the 1D9-680 probe showing greater fidelity over time. The 1D9-680 probe was then tested in mouse models of shoulder implant and allograft infection, demonstrating its broader applicability. Finally, an image-guided surgery system which superimposes fluorescent signals over analog, real-time, tissue images was employed to facilitate debridement of fluorescent-labelled bacteria.
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http://dx.doi.org/10.1038/s41598-020-78362-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810895PMC
January 2021

Fighting Staphylococcus aureus infections with light and photoimmunoconjugates.

JCI Insight 2020 11 19;5(22). Epub 2020 Nov 19.

Department of Medical Microbiology and.

Infections caused by multidrug-resistant Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), are responsible for high mortality and morbidity worldwide. Resistant lineages were previously confined to hospitals but are now also causing infections among healthy individuals in the community. It is therefore imperative to explore therapeutic avenues that are less prone to raise drug resistance compared with today's antibiotics. An opportunity to achieve this ambitious goal could be provided by targeted antimicrobial photodynamic therapy (aPDT), which relies on the combination of a bacteria-specific targeting agent and light-induced generation of ROS by an appropriate photosensitizer. Here, we conjugated the near-infrared photosensitizer IRDye700DX to a fully human mAb, specific for the invariantly expressed staphylococcal antigen immunodominant staphylococcal antigen A (IsaA). The resulting immunoconjugate 1D9-700DX was characterized biochemically and in preclinical infection models. As demonstrated in vitro, in vivo, and in a human postmortem orthopedic implant infection model, targeted aPDT with 1D9-700DX is highly effective. Importantly, combined with the nontoxic aPDT-enhancing agent potassium iodide, 1D9-700DX overcomes the antioxidant properties of human plasma and fully eradicates high titers of MRSA. We show that the developed immunoconjugate 1D9-700DX targets MRSA and kills it upon illumination with red light, without causing collateral damage to human cells.
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http://dx.doi.org/10.1172/jci.insight.139512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710284PMC
November 2020

In vivo Mouse Model of Spinal Implant Infection.

J Vis Exp 2020 06 23(160). Epub 2020 Jun 23.

Department of Orthopaedic Surgery, University of California Los Angeles;

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections. In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 10 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden. To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia. While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.
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http://dx.doi.org/10.3791/60560DOI Listing
June 2020

Novel in vivo mouse model of shoulder implant infection.

J Shoulder Elbow Surg 2020 Jul 31;29(7):1412-1424. Epub 2020 Jan 31.

David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA; Department of Orthopedic Surgery, University of California, Los Angeles, Santa Monica, CA, USA. Electronic address:

Background: Animal models are used to guide management of periprosthetic implant infections. No adequate model exists for periprosthetic shoulder infections, and clinicians thus have no preclinical tools to assess potential therapeutics. We hypothesize that it is possible to establish a mouse model of shoulder implant infection (SII) that allows noninvasive, longitudinal tracking of biofilm and host response through in vivo optical imaging. The model may then be employed to validate a targeting probe (1D9-680) with clinical translation potential for diagnosing infection and image-guided débridement.

Methods: A surgical implant was press-fit into the proximal humerus of c57BL/6J mice and inoculated with 2 μL of 1 × 10 (e3), or 1 × 10 (e4), colony-forming units (CFUs) of bioluminescent Staphylococcus aureus Xen-36. The control group received 2 μL sterile saline. Bacterial activity was monitored in vivo over 42 days, directly (bioluminescence) and indirectly (targeting probe). Weekly radiographs assessed implant loosening. CFU harvests, confocal microscopy, and histology were performed.

Results: Both inoculated groups established chronic infections. CFUs on postoperative day (POD) 42 were increased in the infected groups compared with the sterile group (P < .001). By POD 14, osteolysis was visualized in both infected groups. The e4 group developed catastrophic bone destruction by POD 42. The e3 group maintained a congruent shoulder joint. Targeting probes helped to visualize low-grade infections via fluorescence.

Discussion: Given bone destruction in the e4 group, a longitudinal, noninvasive mouse model of SII and chronic osteolysis was produced using e3 of S aureus Xen-36, mimicking clinical presentations of chronic SII.

Conclusion: The development of this model provides a foundation to study new therapeutics, interventions, and host modifications.
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http://dx.doi.org/10.1016/j.jse.2019.10.032DOI Listing
July 2020

Development of a Staphylococcus aureus reporter strain with click beetle red luciferase for enhanced in vivo imaging of experimental bacteremia and mixed infections.

Sci Rep 2019 11 13;9(1):16663. Epub 2019 Nov 13.

Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

In vivo bioluminescence imaging has been used to monitor Staphylococcus aureus infections in preclinical models by employing bacterial reporter strains possessing a modified lux operon from Photorhabdus luminescens. However, the relatively short emission wavelength of lux (peak 490 nm) has limited tissue penetration. To overcome this limitation, the gene for the click beetle (Pyrophorus plagiophtalamus) red luciferase (luc) (with a longer >600 emission wavelength), was introduced singly and in combination with the lux operon into a methicillin-resistant S. aureus strain. After administration of the substrate D-luciferin, the luc bioluminescent signal was substantially greater than the lux signal in vitro. The luc signal had enhanced tissue penetration and improved anatomical co-registration with infected internal organs compared with the lux signal in a mouse model of S. aureus bacteremia with a sensitivity of approximately 3 × 10 CFU from the kidneys. Finally, in an in vivo mixed bacterial wound infection mouse model, S. aureus luc signals could be spectrally unmixed from Pseudomonas aeruginosa lux signals to noninvasively monitor the bacterial burden of both strains. Therefore, the S. aureus luc reporter may provide a technological advance for monitoring invasive organ dissemination during S. aureus bacteremia and for studying bacterial dynamics during mixed infections.
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http://dx.doi.org/10.1038/s41598-019-52982-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853927PMC
November 2019

Preclinical Models and Methodologies for Monitoring Staphylococcus aureus Infections Using Noninvasive Optical Imaging.

Methods Mol Biol 2020 ;2069:197-228

Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

In vivo whole-animal optical (bioluminescence and fluorescence) imaging of Staphylococcus aureus infections has provided the opportunity to noninvasively and longitudinally monitor the dynamics of the bacterial burden and ensuing host immune responses in live anesthetized animals. Herein, we describe several different mouse models of S. aureus skin infection, skin inflammation, incisional/excisional wound infections, as well as mouse and rabbit models of orthopedic implant infection, which utilized this imaging technology. These animal models and imaging methodologies provide insights into the pathogenesis of these infections and innate and adaptive immune responses, as well as the preclinical evaluation of diagnostic and treatment modalities. Noninvasive approaches to investigate host-pathogen interactions are extremely important as virulent community-acquired methicillin-resistant S. aureus strains (CA-MRSA) are spreading through the normal human population, becoming more antibiotic resistant and creating a serious threat to public health.
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http://dx.doi.org/10.1007/978-1-4939-9849-4_15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745539PMC
December 2020

Cell-specific image-guided transcriptomics identifies complex injuries caused by ischemic acute kidney injury in mice.

Commun Biol 2019 2;2:326. Epub 2019 Sep 2.

1Division of Nephrology, Department of Medicine, University of Washington, Seattle, WA 98195 USA.

The kidney's inherent complexity has made identifying cell-specific pathways challenging, particularly when temporally associating them with the dynamic pathophysiology of acute kidney injury (AKI). Here, we combine renal cell-specific luciferase reporter mice using a chemoselective luciferin to guide the acquisition of cell-specific transcriptional changes in C57BL/6 background mice. Hydrogen peroxide generation, a common mechanism of tissue damage, was tracked using a peroxy-caged-luciferin to identify optimum time points for immunoprecipitation of labeled ribosomes for RNA-sequencing. Together, these tools revealed a profound impact of AKI on mitochondrial pathways in the collecting duct. In fact, targeting the mitochondria with an antioxidant, ameliorated not only hydrogen peroxide generation, but also significantly reduced oxidative stress and the expression of the AKI biomarker, LCN2. This integrative approach of coupling physiological imaging with transcriptomics and drug testing revealed how the collecting duct responds to AKI and opens new venues for cell-specific predictive monitoring and treatment.
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http://dx.doi.org/10.1038/s42003-019-0571-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718519PMC
April 2020

Corrigendum: Elevated Gut Microbiome-Derived Propionate Levels Are Associated With Reduced Sterile Lung Inflammation and Bacterial Immunity in Mice.

Front Microbiol 2019 26;10:518. Epub 2019 Mar 26.

Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, CA, United States.

[This corrects the article DOI: 10.3389/fmicb.2019.00159.].
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http://dx.doi.org/10.3389/fmicb.2019.00518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6443955PMC
March 2019

Elevated Gut Microbiome-Derived Propionate Levels Are Associated With Reduced Sterile Lung Inflammation and Bacterial Immunity in Mice.

Front Microbiol 2019 14;10:159. Epub 2019 Feb 14.

Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, CA, United States.

Short-chain fatty acids (SCFA) are important dietary and microbiome metabolites that can have roles in gut immunity as well as further afield. We previously observed that gut microbiome alteration via antibiotics led to attenuated lung inflammatory responses. The rationale for this study was to identify gut microbiome factors that regulate lung immune homeostasis. We first investigated key factors within mouse colonic lumen filtrates (CLF) which could elicit direct inflammatory effects . We identified lipopolysaccharide (LPS) and SCFAs as key CLF ingredients whose levels and inflammatory capacity changed after antibiotic exposure in mice. Specifically, the SCFA propionate appeared to be a key regulator of LPS responses . Elevated propionate: acetate ratios, as seen in CLF after antibiotic exposure, strongly blunted inflammatory responses . , exposure of lungs to high dose propionate, to mimic how prior antibiotic exposure changed SCFA levels, resulted in diminished immune containment of pneumonia. Finally, we discovered an enrichment of propionate-producing gut bacteria in mice with reduced lung inflammation following lung ischemia reperfusion injury . Overall, our data show that propionate levels can distinctly modulate lung immune responses and and that gut microbiome increased production of propionate is associated with reduced lung inflammation.
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http://dx.doi.org/10.3389/fmicb.2019.00159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413706PMC
February 2019

Multimodal imaging guides surgical management in a preclinical spinal implant infection model.

JCI Insight 2019 Feb 7;4(3). Epub 2019 Feb 7.

Department of Orthopedic Surgery, David Geffen School of Medicine at UCLA, Santa Monica, California, USA.

Spine implant infections portend disastrous outcomes, as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. Current imaging modalities can detect anatomical alterations and anomalies but cannot differentiate between infection and aseptic loosening, diagnose specific pathogens, or delineate the extent of an infection. Herein, a fully human monoclonal antibody 1D9, recognizing the immunodominant staphylococcal antigen A on the surface of Staphylococcus aureus, was assessed as a nuclear and fluorescent imaging probe in a preclinical model of S. aureus spinal implant infection, utilizing bioluminescently labeled bacteria to confirm the specificity and sensitivity of this targeting. Postoperative mice were administered 1D9 probe dual labeled with 89-zirconium (89Zr) and a bars represent SEM dye (NIR680) (89Zr-NIR680-1D9), and PET-CT and in vivo fluorescence and bioluminescence imaging were performed. The 89Zr-NIR680-1D9 probe accurately diagnosed both acute and subacute implant infection and permitted fluorescent image-guided surgery for selective debridement of infected tissue. Therefore, a single probe could noninvasively diagnose an infection and facilitate image-guided surgery to improve the clinical management of implant infections.
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http://dx.doi.org/10.1172/jci.insight.124813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413782PMC
February 2019

Mouse model of Gram-negative prosthetic joint infection reveals therapeutic targets.

JCI Insight 2018 09 6;3(17). Epub 2018 Sep 6.

Department of Orthopaedic Surgery.

Bacterial biofilm infections of implantable medical devices decrease the effectiveness of antibiotics, creating difficult-to-treat chronic infections. Prosthetic joint infections (PJI) are particularly problematic because they require prolonged antibiotic courses and reoperations to remove and replace the infected prostheses. Current models to study PJI focus on Gram-positive bacteria, but Gram-negative PJI (GN-PJI) are increasingly common and are often more difficult to treat, with worse clinical outcomes. Herein, we sought to develop a mouse model of GN-PJI to investigate the pathogenesis of these infections and identify potential therapeutic targets. An orthopedic-grade titanium implant was surgically placed in the femurs of mice, followed by infection of the knee joint with Pseudomonas aeruginosa or Escherichia coli. We found that in vitro biofilm-producing activity was associated with the development of an in vivo orthopedic implant infection characterized by bacterial infection of the bone/joint tissue, biofilm formation on the implants, reactive bone changes, and inflammatory immune cell infiltrates. In addition, a bispecific antibody targeting P. aeruginosa virulence factors (PcrV and Psl exopolysaccharide) reduced the bacterial burden in vivo. Taken together, our findings provide a preclinical model of GN-PJI and suggest the therapeutic potential of targeting biofilm-associated antigens.
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http://dx.doi.org/10.1172/jci.insight.121737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171808PMC
September 2018

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer.

J Vis Exp 2018 07 21(137). Epub 2018 Jul 21.

Division of Pulmonary and Critical Care Medicine, University of California Los Angeles David Geffen School of Medicine;

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.
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http://dx.doi.org/10.3791/57167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126521PMC
July 2018

Can 3D imaging provide insights into spatial variations in bacterial infection patterns?

Future Microbiol 2018 06 7;13:897-901. Epub 2018 Jun 7.

PerkinElmer, Hopkinton, MA 01748, USA.

Recently developed 3D noninvasive in vivo optical imaging is providing fresh insights into the understanding of the pathogenesis of invasive bacteria in small animal experimental models. Here, we describe the advantages of 3D diffuse light imaging tomography with integrated micro-computed tomography (DLIT-μCT) over more traditional 2D systems, in particular with regard to precise localization of infectious foci within tissues in 3D space. We highlight data from rodent studies that employ experimental infections replicating the course of naturally occurring bacterial disease, such as invasive Escherichia coli infections that arise following colonization of the GI tract in neonatal rats. It is argued that this technology will find increasing utility in the study and diagnosis of infectious disease.
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http://dx.doi.org/10.2217/fmb-2017-0272DOI Listing
June 2018

Noninvasive optical and nuclear imaging of Staphylococcus-specific infection with a human monoclonal antibody-based probe.

Virulence 2018 01 26;9(1):262-272. Epub 2017 Dec 26.

a Department of Medical Microbiology , University of Groningen, University Medical Center Groningen , Hanzeplein 1, Groningen , RB , The Netherlands.

Staphylococcus aureus infections are a major threat in healthcare, requiring adequate early-stage diagnosis and treatment. This calls for novel diagnostic tools that allow noninvasive in vivo detection of staphylococci. Here we performed a preclinical study to investigate a novel fully-human monoclonal antibody 1D9 that specifically targets the immunodominant staphylococcal antigen A (IsaA). We show that 1D9 binds invariantly to S. aureus cells and may further target other staphylococcal species. Importantly, using a human post-mortem implant model and an in vivo murine skin infection model, preclinical feasibility was demonstrated for 1D9 labeled with the near-infrared fluorophore IRDye800CW to be applied for direct optical imaging of in vivo S. aureus infections. Additionally, Zirconium-labeled 1D9 could be used for positron emission tomography imaging of an in vivo S. aureus thigh infection model. Our findings pave the way towards clinical implementation of targeted imaging of staphylococcal infections using the human monoclonal antibody 1D9.
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http://dx.doi.org/10.1080/21505594.2017.1403004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955194PMC
January 2018

NLRP3 Inflammasome Mediates Dormant Neutrophil Recruitment following Sterile Lung Injury and Protects against Subsequent Bacterial Pneumonia in Mice.

Front Immunol 2017 31;8:1337. Epub 2017 Oct 31.

Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, CA, United States.

Sterile lung injury is an important clinical problem that complicates the course of severely ill patients. Interruption of blood flow, namely ischemia-reperfusion (IR), initiates a sterile inflammatory response in the lung that is believed to be maladaptive. The rationale for this study was to elucidate the molecular basis for lung IR inflammation and whether it is maladaptive or beneficial. Using a mouse model of lung IR, we demonstrate that sequential blocking of inflammasomes [specifically, NOD-, LRR-, and pyrin domain-containing 3 (NLRP3)], inflammatory caspases, and interleukin (IL)-1β, all resulted in an attenuated inflammatory response. IL-1β production appeared to predominantly originate in conjunction with alveolar type 2 epithelial cells. Lung IR injury recruited unactivated or dormant neutrophils producing less reactive oxygen species thereby challenging the notion that recruited neutrophils are terminally activated. However, lung IR inflammation was able to limit or reduce the bacterial burden from subsequent experimentally induced pneumonia. Notably, inflammasome-deficient mice were unable to alter this bacterial burden following IR. Thus, we conclude that the NLRP3 inflammasome, through IL-1β production, regulates lung IR inflammation, which includes recruitment of dormant neutrophils. The sterile IR inflammatory response appears to serve an important function in inducing resistance to subsequent bacterial pneumonia and may constitute a critical part of early host responses to infection in trauma.
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http://dx.doi.org/10.3389/fimmu.2017.01337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5671513PMC
October 2017

Development of a Click Beetle Luciferase Reporter System for Enhanced Bioluminescence Imaging of : Analysis in Cell Culture and Murine Infection Models.

Front Microbiol 2017 26;8:1797. Epub 2017 Sep 26.

APC Microbiome Institute, University College Cork, Cork, Ireland.

is a Gram-positive facultative intracellular pathogen that is widely used as a model organism for the analysis of infection biology. In this context, there is a current need to develop improved reporters for enhanced bioluminescence imaging (BLI) of the pathogen in infection models. We have developed a click beetle red luciferase (CBR-) based vector (pPL2CBR) expressing codon optimized CBR- under the control of a highly expressed Listerial promoter (P) for and have compared this to a -based system expressing bacterial luciferase for BLI of the pathogen using growth experiments and models. The CBR- plasmid stably integrates into the chromosome and can be used to label field isolates and laboratory strains of the pathogen. Growth experiments revealed that CBR- labeled emits a bright signal in exponential phase that is maintained during stationary phase. In contrast, -labeled bacteria produced a light signal that peaked during exponential phase and was significantly reduced during stationary phase. Light from CBR- labeled bacteria was more efficient than the signal from -labeled bacteria in penetrating an artificial tissue depth assay system. A cell invasion assay using C2Bbe1 cells and a systemic murine infection model revealed that CBR- is suited to BLI approaches and demonstrated enhanced sensitivity relative to in the context of infection models. Overall, we demonstrate that this novel CBR reporter system provides efficient, red-shifted light production relative to and may have significant applications in the analysis of pathogenesis.
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http://dx.doi.org/10.3389/fmicb.2017.01797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622934PMC
September 2017

Non-invasive three-dimensional imaging of Escherichia coli K1 infection using diffuse light imaging tomography combined with micro-computed tomography.

Methods 2017 08 15;127:62-68. Epub 2017 May 15.

University College London School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK. Electronic address:

In contrast to two-dimensional bioluminescence imaging, three dimensional diffuse light imaging tomography with integrated micro-computed tomography (DLIT-μCT) has the potential to realise spatial variations in infection patterns when imaging experimental animals dosed with derivatives of virulent bacteria carrying bioluminescent reporter genes such as the lux operon from the bacterium Photorhabdus luminescens. The method provides an opportunity to precisely localise the bacterial infection sites within the animal and enables the generation of four-dimensional movies of the infection cycle. Here, we describe the use of the PerkinElmer IVIS SpectrumCT in vivo imaging system to investigate progression of lethal systemic infection in neonatal rats following colonisation of the gastrointestinal tract with the neonatal pathogen Escherichia coli K1. We confirm previous observations that these bacteria stably colonize the colon and small intestine following feeding of the infectious dose from a micropipette; invading bacteria migrate across the gut epithelium into the blood circulation and establish foci of infection in major organs, including the brain. DLIT-μCT revealed novel multiple sites of colonisation within the alimentary canal, including the tongue, oesophagus and stomach, with penetration of the non-keratinised oesophageal epithelial surface, providing strong evidence of a further major site for bacterial dissemination. We highlight technical issues associated with imaging of infections in new born rat pups and show that the whole-body and organ bioburden correlates with disease severity.
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http://dx.doi.org/10.1016/j.ymeth.2017.05.005DOI Listing
August 2017

Combinatory antibiotic therapy increases rate of bacterial kill but not final outcome in a novel mouse model of Staphylococcus aureus spinal implant infection.

PLoS One 2017 28;12(2):e0173019. Epub 2017 Feb 28.

Department of Orthopaedic Surgery, David Geffen School of Medicine at University of California Los Angeles, Santa Monica, California, United States of America.

Background: Management of spine implant infections (SII) are challenging. Explantation of infected spinal hardware can destabilize the spine, but retention can lead to cord compromise and biofilm formation, complicating management. While vancomycin monotherapy is commonly used, in vitro studies have shown reduced efficacy against biofilm compared to combination therapy with rifampin. Using an established in vivo mouse model of SII, we aim to evaluate whether combination therapy has increased efficacy compared to both vancomycin alone and infected controls.

Methods: An L-shaped, Kirschner-wire was transfixed into the L4 spinous process of 12-week-old C57BL/6 mice, and inoculated with bioluminescent Staphylococcus aureus. Mice were randomized into a vancomycin group, a combination group with vancomycin plus rifampin, or a control group receiving saline. Treatment began on post-operative day (POD) 7 and continued through POD 14. In vivo imaging was performed to monitor bioluminescence for 35 days. Colony-forming units (CFUs) were cultured on POD 35.

Results: Bioluminescence peaked around POD 7 for all groups. The combination group had a 10-fold decrease in signal by POD 10. The vancomycin and control groups reached similar levels on POD 17 and 21, respectively. On POD 25 the combination group dropped below baseline, but rebounded to the same level as the other groups, demonstrating a biofilm-associated infection by POD 35. Quantification of CFUs on POD 35 confirmed an ongoing infection in all three groups.

Conclusions: Although both therapies were initially effective, they were not able to eliminate implant biofilm bacteria, resulting in a rebound infection after antibiotic cessation. This model shows, for the first time, why histologic-based, static assessments of antimicrobials can be misleading, and the importance of longitudinal tracking of infection. Future studies can use this model to test combinations of antibiotic therapies to see if they are more effective in eliminating biofilm prior to human trials.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173019PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330510PMC
August 2017

Preclinical Evaluation of Photoacoustic Imaging as a Novel Noninvasive Approach to Detect an Orthopaedic Implant Infection.

J Am Acad Orthop Surg 2017 Feb;25 Suppl 1:S7-S12

From Washington University, St. Louis, MO (Dr. Wang), Johns Hopkins University School of Medicine, Baltimore, MD (Dr. Thompson), the University of California, Los Angeles, Los Angeles, CA (Ms. Ashbaugh), École Polytechnique Fédérale de Lausanne (Dr. Khodakivskyi and Dr. Dubikovskaya) and Intrace Medical SA (Dr. Budin and Dr. Sinisi), Lausanne, Switzerland, FUJIFILM VisualSonics, Toronto, Ontario, Canada (Mr. Heinmiller), the University of Groningen, Groningen, The Netherlands (Dr. van Oosten, Dr. van Dijl, and Dr. van Dam), PerkinsElmer, Waltham, MA (Dr. Francis), Ronald Reagan University of California, Los Angeles Medical Center, Santa Monica, CA (Dr. Bernthal), and the Division of infectious Diseases and Department of Orthopaedic Surgery, The Johns Hopkins Hospital, Baltimore, MD (Dr. Miller).

Introduction: Diagnosing prosthetic joint infection (PJI) poses significant challenges, and current modalities are fraught with low sensitivity and/or potential morbidity. Photoacoustic imaging (PAI) is a novel ultrasound-based modality with potential for diagnosing PJI safely and noninvasively.

Materials: In an established preclinical mouse model of bioluminescent Staphylococcus aureus PJI, fluorescent indocyanine green (ICG) was conjugated to β-cyclodextrin (CDX-ICG) or teicoplanin (Teic-ICG) and injected intravenously for 1 week postoperatively. Daily fluorescent imaging and PAI were used to localize and quantify tracer signals. Results were analyzed using 2-way analysis of variance.

Results: Fluorescence clearly localized to the site of infection and was significantly higher with Teic-ICG compared with CDX-ICG (P = 0.046) and ICG alone (P = 0.0087). With PAI, the photoacoustic signal per volumetric analysis was substantially higher and better visualized with Teic-ICG compared with CDX-ICG and ICG alone, and colocalized well with bioluminescence and fluorescence imaging.

Conclusion: Photoacoustic imaging successfully localized PJI in this proof-of-concept study and demonstrates potential for clinical translation in orthopaedics.
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http://dx.doi.org/10.5435/JAAOS-D-16-00630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056014PMC
February 2017

Novel in vivo mouse model of implant related spine infection.

J Orthop Res 2017 01 8;35(1):193-199. Epub 2016 May 8.

Department of Orthopaedic Surgery, David Geffen School of Medicine at University of California Los Angeles (UCLA), Santa Monica, 90404, California.

Post-operative spine infections are a challenge, as hardware must often be retained to prevent destabilization of the spine, and bacteria form biofilm on implants, rendering them inaccessible to antibiotic therapy, and immune cells. A model of posterior-approach spinal surgery was created in which a stainless steel k-wire was transfixed into the L4 spinous process of 12-week-old C57BL/six mice. Mice were then randomized to receive either one of three concentrations (1 × 10 , 1 × 10 , and 1 × 10 colony forming units (CFU)) of a bioluminescent strain of Staphylococcus aureus or normal saline at surgery. The mice were then longitudinally imaged for bacterial bioluminescence to quantify infection. The 1 × 10 CFU group had a decrease in signal down to control levels by POD 25, while the 1 × 10 and 1 × 10 CFU groups maintained a 10-fold higher signal through POD 35. Bacteria were then harvested from the pin and surrounding tissue for confirmatory CFU counts. All mice in the 1 × 10 CFU group experienced wound breakdown, while no mice in the other groups had this complication. Once an optimal bacterial concentration was determined, mice expressing enhanced green fluorescent protein in their myeloid cells (Lys-EGFP) were utilized to contemporaneously quantify bacterial burden, and immune response. Neutrophil fluorescence peaked for both groups on POD 3, and then declined. The infected group continued to have a response above the control group through POD 35. This study, establishes a noninvasive in vivo mouse model of spine implant infection that can quantify bacterial burden and host inflammation longitudinally in real time without requiring animal sacrifice. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:193-199, 2017.
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http://dx.doi.org/10.1002/jor.23273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5268448PMC
January 2017

Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo.

PLoS One 2016 2;11(3):e0149972. Epub 2016 Mar 2.

Department of Microbial Pathogenesis and Immunology, Texas A & M Health Science Center, Bryan, Texas, United States of America.

The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149972PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4774912PMC
July 2016

In Vivo Bioluminescence Imaging of Intratumoral Bacteria.

Methods Mol Biol 2016 ;1409:69-77

Cork Cancer Research Centre, University College Cork, Cork, Ireland.

This chapter describes the use of whole-body bioluminescent imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in therapy of cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and tumor sites concurrently. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor-bearing mice. This powerful, and inexpensive, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research. This protocol outlines the procedure for studying lux-tagged Escherichia coli and Bifidobacterium breve in mice, demonstrating the spatial and temporal readout from 2D and 3D BLI achievable with whole-body in vivo luminescence imaging.
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http://dx.doi.org/10.1007/978-1-4939-3515-4_7DOI Listing
November 2016

Multi-Modal Imaging with a Toolbox of Influenza A Reporter Viruses.

Viruses 2015 Oct 13;7(10):5319-27. Epub 2015 Oct 13.

Medical Microbiology and Immunology, University of Wisconsin Madison, Madison, WI 53706, USA.

Reporter viruses are useful probes for studying multiple stages of the viral life cycle. Here we describe an expanded toolbox of fluorescent and bioluminescent influenza A reporter viruses. The enhanced utility of these tools enabled kinetic studies of viral attachment, infection, and co-infection. Multi-modal bioluminescence and positron emission tomography-computed tomography (PET/CT) imaging of infected animals revealed that antiviral treatment reduced viral load, dissemination, and inflammation. These new technologies and applications will dramatically accelerate in vitro and in vivo influenza virus studies.
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http://dx.doi.org/10.3390/v7102873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632381PMC
October 2015

Development of a Bioluminescent Nitroreductase Probe for Preclinical Imaging.

PLoS One 2015 25;10(6):e0131037. Epub 2015 Jun 25.

School of Basic Sciences, Institute of Chemical Sciences and Engineering, Swiss Federal Institute of Technology of Lausanne, Lausanne, Switzerland.

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131037PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4482324PMC
April 2016

Targeted imaging of bacterial infections: advances, hurdles and hopes.

FEMS Microbiol Rev 2015 Nov 24;39(6):892-916. Epub 2015 Jun 24.

Department of Surgery, Division of Surgical Oncology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO Box 30001, 9700 RB Groningen, the Netherlands.

Bacterial infections represent an increasing problem in modern health care, in particular due to ageing populations and accumulating bacterial resistance to antibiotics. Diagnosis is rarely straightforward and consequently treatment is often delayed or indefinite. Therefore, novel tools that can be clinically implemented are urgently needed to accurately and swiftly diagnose infections. Especially, the direct imaging of infections is an attractive option. The challenge of specifically imaging bacterial infections in vivo can be met by targeting bacteria with an imaging agent. Here we review the current status of targeted imaging of bacterial infections, and we discuss advantages and disadvantages of the different approaches. Indeed, significant progress has been made in this field and the clinical implementation of targeted imaging of bacterial infections seems highly feasible. This was recently highlighted by the use of so-called smart activatable probes and a fluorescently labelled derivative of the antibiotic vancomycin. A major challenge remains the selection of the best imaging probes, and we therefore present a set of target selection criteria for clinical implementation of targeted bacterial imaging. Altogether, we conclude that the spectrum of potential applications for targeted bacterial imaging is enormous, ranging from fundamental research on infectious diseases to diagnostic and therapeutic applications.
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http://dx.doi.org/10.1093/femsre/fuv029DOI Listing
November 2015

Azithromycin inhibits nuclear factor-κB activation during lung inflammation: an in vivo imaging study.

Pharmacol Res Perspect 2014 Oct 2;2(5):e00058. Epub 2014 Jul 2.

Chiesi Farmaceutici S.p.A Parma, Italy.

We studied in vivo the potential involvement of nuclear factor-κB (NF-κB) pathway in the molecular mechanism of the anti-inflammatory and immunomodulatory activity of azithromycin in the lung. Mice transiently transfected with the luciferase gene under the control of a NF-κB responsive element were used to assess in vivo NF-κB activation by bioluminescence imaging. Bioluminescence as well as inflammatory cells and concentrations of proinflammatory cytokines in bronchoalveolar lavage fluids, were monitored in an acute model of pulmonary inflammation resulting from intratracheal instillation of lipopolysaccharide. Lipopolysaccharide (LPS) instillation induced a marked increase in lung bioluminescence in mice transiently transfected with the luciferase gene under the control of an NF-κB responsive element, with significant luciferase expression in resident cells such as endothelial and epithelial cells, as assessed by duoplex immunofluorescence staining. Activation of NF-κB and inflammatory cell lung infiltration linearly correlated when different doses of bortezomib were used to inhibit NF-κB activation. Pretreatment with azithromycin significantly decreased lung bioluminescence and airways cell infiltration induced by LPS, also reducing proinflammatory cytokines concentrations in bronchoalveolar lavages and inhibiting NF-κB nuclear translocation. The results obtained using a novel approach to monitor NF-κB activation, provided, for the first time, in vivo evidence that azithromycin treatment results in pulmonary anti-inflammatory activity associated with the inhibition of NF-κB activation in the lung.
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http://dx.doi.org/10.1002/prp2.58DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4186419PMC
October 2014

Combined in vivo optical and µCT imaging to monitor infection, inflammation, and bone anatomy in an orthopaedic implant infection in mice.

J Vis Exp 2014 Oct 16(92):e51612. Epub 2014 Oct 16.

Department of Dermatology, Johns Hopkins University School of Medicine; Department of Medicine, Division of Infectious Diseases, Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine;

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and μCT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, μCT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical μCT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with μCT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.
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http://dx.doi.org/10.3791/51612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480817PMC
October 2014

Real-time bioluminescence imaging of mixed mycobacterial infections.

PLoS One 2014 29;9(9):e108341. Epub 2014 Sep 29.

Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, Texas, United States of America.

Molecular analysis of infectious processes in bacteria normally involves construction of isogenic mutants that can then be compared to wild type in an animal model. Pathogenesis and antimicrobial studies are complicated by variability between animals and the need to sacrifice individual animals at specific time points. Live animal imaging allows real-time analysis of infections without the need to sacrifice animals, allowing quantitative data to be collected at multiple time points in all organs simultaneously. However, imaging has not previously allowed simultaneous imaging of both mutant and wild type strains of mycobacteria in the same animal. We address this problem by using both firefly (Photinus pyralis) and click beetle (Pyrophorus plagiophthalamus) red luciferases, which emit distinct bioluminescent spectra, allowing simultaneous imaging of two different mycobacterial strains during infection. We also demonstrate that these same bioluminescence reporters can be used to evaluate therapeutic efficacy in real-time, greatly facilitating our ability to screen novel antibiotics as they are developed. Due to the slow growth rate of mycobacteria, novel imaging technologies are a pressing need, since they can they can impact the rate of development of new therapeutics as well as improving our understanding of virulence mechanisms and the evaluation of novel vaccine candidates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108341PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4180448PMC
November 2015

Efficacy of enrofloxacin in a mouse model of sepsis.

J Am Assoc Lab Anim Sci 2014 Jul;53(4):381-6

Laboratory of Transfusion Biology, Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York, USA; Institute of Comparative Medicine, Columbia University, New York, New York, USA.

We examined the efficacy of enrofloxacin administered by 2 different routes in a mouse model of sepsis. Male CD1 mice were infected with a bioluminescent strain of enteropathogenic Escherichia coli and treated with enrofloxacin either by injection or in drinking water. Peak serum levels were evaluated by using HPLC. Mice were monitored for signs of clinical disease, and infections were monitored by using bioluminescence imaging. Serum levels of enrofloxacin and the active metabolite ciprofloxacin were greater in the group treated by injection than in controls or the groups treated by administration in drinking water. Survival of the group treated with enrofloxacin injection was greater than that of controls and groups treated with enrofloxacin in the drinking water. Bioluminescence in the group treated with enrofloxacin injection was less than that in the groups treated with oral administration at 12 h and in the groups treated orally and the control group at 16 h. According to these findings, we recommend the use of injectable enrofloxacin at 5 mg/kg SC for mice with systemic infections.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113238PMC
July 2014

α-Intercalated cells defend the urinary system from bacterial infection.

J Clin Invest 2014 Jul 17;124(7):2963-76. Epub 2014 Jun 17.

α-Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC-dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.
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http://dx.doi.org/10.1172/JCI71630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4071397PMC
July 2014