EMIM 2018 ControlCenter

Online Program Overview Session: PW-04

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Imaging Infection

Session chair: Greetje Vande Velde - Leuven, Belgium; Wiktor Szymanski - Groningen, The Netherlands
 
Shortcut: PW-04
Date: Thursday, 22 March, 2018, 11:30 AM
Room: Banquet Hall | level -1
Session type: Poster Session

Abstract

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# 040

Controlled delivery of antimicrobial peptides in the lungs by aerosol administration: towards personalised therapies to combat antibiotic-resistant lung infections (#156)

U. Cossío1, V. Gómez-Vallejo1, C. Falciani2, A. Pini3, 4, M. Strandh5, Y. M. te Welscher6, C. F. van Nostrum6, I. Loinaz7, M. Marradi7, J. Llop1

1 CIC biomaGUNE, Radiochemistry and Nuclear Imaging Group, San Sebastian, Gipuzkoa, Spain
2 SetLance srl, Siena, Italy
3 University of Siena, Department of Medical Biotechnologies, Siena, Italy
4 Azienda Ospedaliera Universitaria Senese, Clinical Pathology Laboratory, Siena, Italy
5 Adenium Biotech ApS, Copenhagen N, Denmark
6 Utrecht University, Department of Pharmaceutics, Utrecht, Netherlands
7 CIDETEC Nanomedicine, San Sebastian, Gipuzkoa, Spain

Introduction

Delivery of drugs by inhalation exhibits numerous advantages compared to oral or intravenous administration; however, small natural peptides can be subjected to enzymatic hydrolysis unless they are chemically modified. To prevent rapid metabolism of peptides after lung administration, they can be attached/entrapped to/into nanocarriers (NCs). Such modification can prolong residence time in the lungs and improve therapeutic efficacy. Here, we investigate the residence time in the lungs of two labelled antimicrobial peptides (AMPs), both in their “free” form and when attached to a polymeric NC.

Methods

The radio-iodination of the peptides (AMP1 and AMP2) was carried out by aromatic substitution on the tyrosine residues using Na[124I] in the presence of Iodobeads®. Formation of the nanosystems was achieved by electrostatic interaction between the peptides and the NC. The labelled peptides or peptide-loaded nanosystems were administered to anesthetized rats using the Penn-Century MicroSprayer® nebulizer. Immediately after administration, dynamic whole body PET images were acquired for 40 min using an eXplore Vista PET-CT system. Imaging sessions were repeated at t= 3, 6, 9, 15 and 24 hours. Images were reconstructed by filtered back projection and analyzed using PMOD analysis tool.

Results/Discussion

The AMPs were efficiently labelled with radioiodine with decay-corrected radiochemical yields of 42±5% (for AMP1) and 63±2% (for AMP2). Specific activities of 0.5-1.5 GBq/µmol were obtained at the end of the synthesis with both peptides. Stability studies showed that more than 95% of [124I]AMPs remained unchanged during 48 hours in moderate acidic conditions (sodium acetate buffer, pH=5.5). Incorporation of the [124I]AMPs to the NC was achieved in >50% radiochemical yield. Stability studies in water showed no significant dissociation of [124I]AMP after 24 hours. Imaging studies revealed a steady clearance of the activity from the lungs over 24 hours and increased residence time of the peptides in the lungs when NCs were used.

Conclusions

The residence time in the lungs of [124I]AMP increases when the peptide is attached to NCs.

Acknowledgement

This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 604434.

Keywords: Nanotherapy, Lung Imaging, Antimicrobial Peptides, Nanocarriers
# 041

Sensitive Bioluminescent Imaging of viral infection using NanoLuc Binary Technology (#436)

N. Gašpar1, 3, G. Zambito1, 2, C. Löwik4, 1, A. Chan3, L. Mezzanotte1

1 Erasmus Medical Center, Dept of Radiology, Rotterdam, Netherlands
2 Medres, Cologne, Germany
3 Percuros B.V., Enschede, Netherlands
4 Institute of Chemical Sciences and Engineering, Lausanne, Switzerland

Introduction

Optical Molecular Imaging has been successfully applied to track and determine viral infection in vivo and in vitro1. To date split-bioluminescence complementation assays have been mainly used to study protein-protein interaction, due to the low affinity of the two luciferase subunits.The NanoBit system is characterized by 2 subunits of the NanoLuc Luciferase2: LargeBiT (LgBiT) and the Small HighBiT peptide (HiBiT)3. These NanoLuc subunits have been optimized for stability and high self-association which enables to track virus-cell infection in real-time in living cells.

 

Methods

HT-29 colon cancer cells and PC-3 prostate cancer cells have been seeded in a 96-well plate and transfected using a plasmid for constitutive expression of the large subunit (LgBit) of NanoLuc Luciferase in the cytosol. A self-inactivating non-replicating lentivirus expressing the small part of NanoLuc Luciferase, the HiBiT unit, has been chosen as model virus. After infection of HT-29 and PC-3 transfected cells structural complementation will occur between two enzyme subunits. After addition of the Nanoluc specific substrate Furimazine total light emission and spectrum were detected using the IVIS spectrum imager and different assay conditions (e.g. the use of polybrene and/or cell lysis). The photon flux (ph/s) was collected and a time course analysis was performed.

Results/Discussion

Signal was clearly visible already 2h post-infection time point and peaking at 24h, using both cell lines. No significant difference in light output could be detected using live cells or lytic assay buffer. On the other hand addition of polybrine, which facilitate infection, resulted in increased light signal.

Conclusions

Our results indicate that this novel imaging platform will allow tracking and can be used to study the efficacy of viral infections in live cells. Moreover, due to the small size of HiBiT peptide also viruses with a genome of limited size and those that tolerate limited genetic manipulation (e.g. small RNA viruses) may be efficiently imaged. Further testing in preclinical animal models will reveal its sensitivity for in vivo imaging.

References

1. In Vivo Molecular Bioluminescence Imaging: New Tools and Applications. Laura Mezzanotte, Moniek van ‘t Root, Hacer Karatas, Elena A. Goun, Clemens W.G.M. Löwik.

2. NanoLuc reporter for dual luciferase imaging in living animals. Stacer AC, Nyati S, Moudgil P, Iyengar R, Luker KE, Rehemtulla A, Luker GD.

3. NanoLuc reporter for dual luciferase imaging in living animals. Stacer AC, Nyati S, Moudgil P, Iyengar R, Luker KE, Rehemtulla A, Luker GD.

Acknowledgement

This research was supported by the FP7 program by the project grant H2020-MSCA-ITN-2015-ISPIC grant number 675743.

 

NanoLuc complementation assay for real time imaging of viral infection.

The use of polybrene significantly enhance the infection rate.

( ** p value < 0.01; * p value < 0.05; Student's T test (n=3))

Imaging viral infection using NanoBit technology.
Illustration of the concept.
Keywords: NanoLuc Luciferase, Optical Molecular Imaging, LgBiT, HiBiT, Imaging of Viral Infection
# 042

In vivo imaging for monitoring infections, host response and treatments in non-human primate models of infectious diseases (#565)

R. Le Grand1, 2, C. Chapon1, 3

1 CEA-Université Paris Sud-Inserm U1184, Fontenay-aux-Roses, France
2 CEA/DRF/Jacob, IDMIT, Fontenay-aux-Roses, France
3 CEA/DRF/Jacob, IDMIT/L3i, Fontenay-aux-Roses, France

Introduction

Although non-human primates (NHP) provide a good model for infectious diseases, their exploration for viral transmission and dissemination by in vivo imaging has not been used extensively. This can be explained by the limited access to adequate structures for imaging large animals with a high resolution while having specific regulations according to the level of confinement for the human pathogens. Our main objectives are to develop minimally invasive technologies for the longitudinal monitoring of infections, host response and treatments in NHP.

Methods

IDMIT (Infectious disease models for innovative therapies) is a national infrastructure (www.idmitcenter.fr) founded by five major academic institutions in France (the CEA, the INSERM, ANRS, the Université Paris Sud and the Institut Pasteur) which is located at Fontenay-aux-Roses (France). The infrastructure develops NHP models for immune related disorders and human infectious diseases (HIV, pertussis, yellow fever...) for preclinical evaluation of human vaccines, immunotherapies and anti-microbial treatments. IDMIT has implemented a unique set of facilities for in vivo imaging in BSL2 and BSL3 constrains (near infrared fluorescence, probe based confocal endomicroscopy, echography, X-ray and PET-CT).

Results/Discussion

So far, we have developed non-invasive and longitudinal fluorescence imaging approaches using probe based confocal endomicroscopy to study the behavior of skin antigen presenting cells following intradermal immunization with different vaccine vectors in order to better understand the mechanisms leading to the induction of cellular and humoral immune responses after vaccination against HIV in macaques1,2. Furthermore, non-invasive in vivo imaging procedures using probe based confocal endomicroscopy coupled with bronchoscopy were also developed to track bacterial localization and cellular interactions with host cells in the lower respiratory tract of challenged and naturally infected animals in a model of bordetella pertussis in baboons.

Conclusions

These in vivo imaging approaches could now be used for extended characterization of diverse cell types in vivo and their interactions with other vaccine antigens and/or pathogens. Furthermore the implementation of the PET-CT system will allow us to study whole body dissemination of pathogens and the distribution of antigens and treatment against infectious diseases with a high sensitivity. 

References

1. Todorova, B., Salabert, N., Tricot, S., Boisgard, R., Rathaux, M., Le Grand, R. et al. Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques. Contrast Media Mol Imaging. 2017, 3127908  (2017).

2. Todorova, B., Adam, L., Culina, S., Boisgard, R., Martinon, F., Cosma, A. et al. Electroporation as a vaccine delivery system and a natural adjuvant to intradermal administration of plasmid DNA in macaques. Sci Rep. 7, 4122  (2017).

# 043

In vivo imaging of lung inflammation with neutrophil-specific 68Ga nano-radiotracer” (#294)

J. Pellico1, 2, A. V. Lechuga-Vieco1, 2, I. Fernández-Barahona1, J. Ruiz-Cabello3, 4, 2, F. Herranz1, 2

1 Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain
2 Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Madrid, Spain
3 CIC biomaGUNE. Ikerbasque, Basque Foundation for Science, Donostia-San Sebastián, Donostia-San Sebastián, Spain
4 Departamento Química Física II. Facultad de Farmacia. Universidad Complutense de Madrid, Madrid, Spain

Introduction

In vivo detection and quantification of inflammation is a major goal in molecular imaging. Furthermore, cell-specific detection of inflammation would be a tremendous advantage in the characterization of many diseases. We show how this goal can be achieved through the synergistic combination of nanotechnology and nuclear imaging. A good example of this approach is the binding of a large amount of a neutrophil-specific, hydrophobic peptide on the surface of 68Ga core-doped nanoparticles. This new nano-radiotracer has been used for non-invasive in vivo detection of acute and chronic inflammation.

Methods

A mixture of FeCl3, sodium citrate, 68GaCl3 (eluted from a 68Ge/68Ga generator), and hydrazine was heated at 100˚C in a synthesis microwave oven. After purification by gel chromatography, 68Ga Nano-radiotracer (68Ga-NRT) was fully characterized. After characterizing, we covalently attached the neutrophil-specific peptide cFLFLF by EDC/Sulfo-NHS reaction, yielding 68Ga-NRT-cFLFL. Cytotoxicity analysis of 68Ga-NRT-cFLFLF were performed in primary hematopoietic progenitor cells and mature neutrophils. Finally, in vivo behavior was assessed in acute lung inflammation by endotoxin lipopolysaccharide (LPS) treated C57Bl/6 mice, neutrophils-depletion mice, macrophages-depleted mice and in chronic inflammation in ApoE -/- mice. Results were confirmed by gamma-counter, flow cytometry and histology.

Results/Discussion

we obtained extremely small nano-radiotracers, 2.7 ± 1.0 nm of core size and 14.5 ± 2.1 nm hydrodynamic size (HD). The yield of the synthesis was 26.0 % Fe and a 92% radiolabeling, for a final specific activity of 7.1 GBq/mmol Fe. Stability was check by the maintenance of hydrodynamic size and the absence of NRT aggregation up to 24 hours after dispersion in PBS, saline and mouse serum. We used the probe in a model of acute inflammation in the lung, in which the NRT particles selectively accumulate in the lungs of animals treated with LPS, producing very clear images and labelling about 15% of neutrophils in vivo. We compared this results with the obtained for macrophages depletion and neutrophils depletion corroborating the specificity towards neutrophils. For detection of chronic inflammation, the probe was evaluated in ApoE-/- mice. Radiotracer revealed clear differences between aged and young HFD-fed ApoE-/- mice and between aged ApoE-/- mice fed the HFD and those fed a normal diet

Conclusions

The findings presented here demonstrate how nanotechnology and nuclear imaging can be combined to overcome the limitations of traditional approaches, yielding a new tool for the non-invasive detection of inflammation with in vivo selectivity towards neutrophils. These results pave the way to the non-invasive identification of neutrophils in a number of highly relevant inflammatory disorders.

References

Pellico, J. et al. In vivo imaging of lung inflammation with neutrophil-specific 68Ga nano-radiotracer. Sci. Rep. 7, 13242 (2017).

Pellico, J. et al. Fast synthesis and bioconjugation of 68Ga core-doped extremely small iron oxide nanoparticles for PET/MR imaging. Contrast Media Mol. Imaging 2016, 11, 203-210.

Acknowledgement

Tis study was supported by a grant from the Spanish Ministry for Economy and Competitiveness (MEyC) (grant number: SAF2016-79593-P) and from Carlos III Health Research Institure (grant number: DTS16/00059). We thank Simon Bartlett for editorial assistance and manuscript preparation. Te CNIC is supported by the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) and the Pro CNIC Foundation, and is a Severo Ochoa Centre of Excellence (MEIC award SEV-2015-0505).

Figure 1
Figure 2
Keywords: Lung inflammation, Nano-radiotracer, Neutrophils, Macrophages, PET Imaging
# 044

Age-dependent course of S. aureus induced subcutaneous skin infection (#482)

J. Höhn1, L. Fischer-Riepe2, Y. Ozegowski1, K. Dlubatz3, J. Roth2, K. Barczyk-Kahlert2, B. Löffler1, V. Hoerr1

1 Jena University Hospital, Institute of Medical Microbiology, Jena, Thuringia, Germany
2 University Hospital Münster, Institute of Immunology, Münster, North Rhine-Westphalia, Germany
3 Jena University Hospital, Center for Sepsis Control and Care, Jena, Thuringia, Germany

Introduction

Staphylococcus aureus is a Gram-positive, commensal bacterium and serves as an endogenous reservoir for clinical infections. It is the leading cause of bacterial skin infection which can either be found locally or can result in severe deep soft tissue infections. Thereby the course depends both on virulence factors of the bacterial strain as well as on the host response [1].
In our study we investigated the impact of aging in two different S. aureus induced footpad infections using microbiological tissue analysis, blood cell counts and fluorescence imaging.

Methods

Bacterial strain: the two S. aureus strains 6850 [2] and LS1 were used as infectious agents. The strain 6850 was originally isolated from a septic osteomyelitis infection while LS1 is an isolate from septic arthritis.
Infection model: 8-12 (young mice) and 77-90 (aged mice) weeks old C57/BL6 mice were inoculated subcutaneously into the right hind footpad using 2 x 107 CFUs/50 µl of S. aureus 6850 (young n= 21; aged: n=18) and LS1 (young n= 17; aged: n=16).
Imaging: Fluorescence reflectance imaging (FRI) was performed 24h post infection (pi) at an In-vivo Xtreme imaging system (Bruker, Ettlingen, Germany), using the two fluorescence markers Transferrin-Vivo (Tf) and Bacterial detection kit (BDK) (Perkin Elmer, excitation: 730; emission: 790; 2h post marker application).

Results/Discussion

24h pi, semi-quantitative FRI showed localized fluorescence signal of BDK in the footpad of young mice infected with S. aureus LS1, while the signal spread towards the lymphnodes in S. aureus-6850-infected mice. Bacterial dissemination was accompanied by strong accumulation of fluorescent Tf in the footpad and ankle (Figure 1; n=5), as well as by a fast and intensive foot swelling (foot swelling in young mice on day 2 pi: 6850: 1,54 +/- 0,35 mm; LS1: 0,86 +/- 0,20 mm). In aged mice, bacterial spread and Tf accumulation were less pronounced in both infection models (Figure 1, n=5). The animals however showed significantly higher values of footpad swelling (foot swelling in aged mice on day 2 pi: 6850: 1,88 +/- 0,28 mm; LS1: 0,99 +/- 0,53 mm) compared to young mice. 10 days pi tissue swelling was still present in mice infected with S. aureus 6850. Especially aged mice showed high bacterial counts in the footpad as well as substantially elevated leukocyte levels (Figure 2).

Conclusions

  • Both in young and aged mice, the course of skin infections induced by S. aureus 6850 is more severe than in infections induced by LS1.
  • S. aureus 6850 infections are more pronounced in aged mice than in young mice showing reduced Transferrin-Vivo accumulation but increased microbiological and immunological readouts.

References

[1] Nippe N. et al., J Invest Dermatol. 2011 Jan;131(1):125-132. [2] Balwit JM. et al., J Infect Dis. 1994 Oct;170(4):1033-1037.

Acknowledgement

We thank Katrin Schmuck for technical assistance.

Figure 1
Figure 2
Keywords: S. aureus skin infection, host response, aging
# 045

Investigating MRI-CEST pH mapping for assessing sepsis-induced Acute Kidney Injury (#440)

L. Consolino1, A. Antonello1, J. C. Cutrin1, S. Aime1, D. L. Longo1

1 University of Torino, Department of Molecular Biotechnology and Health Sciences, Torino, Italy

Introduction

Sepsis is a state of systemic inflammation to a suspected infection that lead to multiorgans failure, including acute kidney injury (AKI), with dramatic clinical outcomes [1]. Diagnosis of AKI commonly measures a decrease of glomerular filtration rate (GFR) and rise of serum creatinine. However, sepsis demonstrated to reduce the creatinine production, leading to an underestimation of renal injury [2]. Therefore, identification of novel biomarkers for sepsis-induced AKI are extremely required. In our work we exploited the MRI-CEST approach to map renal pH variations in a sepsis-induced model.

Methods

Male C57BL/6J mice (n=4) were intraperitoneally administered 10 mg/kg body weight LPS from E. coli serotype O111:B4 [3]. MR images were acquired on kidney sections before and 18 hours after LPS injection on a 7T Bruker MRI scanner (FOV: 3 cm, in-plane resolution: 234 µm, slice thickness: 1.5 mm). MRI-CEST pH mapping [4] was performed using a fast spin-echo sequence preceded by a RF saturation pulse (3 μT x 5s) and acquiring Z-spectra before and after the injection of 4 g I/kg b.w. iopamidol into the tail vein. After MRI acquisition, mice were sacrificed and blood serum obtained for BUN and creatinine quantification. For the histological assessment, multiple organs (kidneys, liver, lung, spleen) were collected, fixed in 10% formalin and slices stained with H&E.

Results/Discussion

Based on in vivo anatomical MR images, any structural changes were observed in renal cortex and medulla sections, thus indicating that LPS-induced injury did not induce macroscopic, visible lesions (Fig.1A). By contrast, pH-CEST mapping was able to detect organ dysfunctional changes by significantly measuring increased extracellular pH values upon LPS administration in the whole kidney section (6.36 and 6.85 before and after LPS, p<0.0001) and in both renal compartments (cortex: 6.39 and 6.89; medulla: 6.25 and 6.8 before and after LPS, p<0.0001) (Fig.1A,B). Similar results were observed by considering pH variations of sepsis-induced mice in comparison to control mates (LPS: ΔpH=+0.50,0.51,0.56; vehicle: ΔpH = +0.23,0.17,0.16 for kidney, cortex and medulla sections, Fig.1C). This trend could be associated to reduced GFR and functional renal impairment, that may affect the regulation of of acid-base balance in kidneys with subsequent influences on renal pH changes [4].

Conclusions

Functional changes in kidneys may be predictable of renal injury/lesion due to sepsis-associated AKI. The proposed MRI-pH-CEST approach reported a shift of extracellular pH values towards more basic values, although morphological differences between pre and post LPS treatment were not detectable. Further investigations will be performed to correlate the obtained MRI results with conventional physiological parameters. To conclude, we surmise that mapping pH changes by MRI may be considered a promising approach for detecting sepsis-induced renal injuries.

References

  1. Zarjou et al., J am Soc Nephrol 2011, 22:999
  2. Doi et al., J am Soc Nephrol 2009, 20:1217
  3. Tran et al., J Clin Invest 2011, 121:4003
  4. Longo et al., NMR Biomed 2017, 17:e3720

Acknowledgement

This work was supported by Compagnia di San Paolo.

Figure 1.

A) Representative T2w-anatomical images and corresponding pH map before LPS (left panel) and after LPS injection of kidneys with coronal orientation. B) Bar graph showing extracellular pH values of the whole kidney, cortex and medulla section before LPS (white) and after LPS (grey) injection. C) Delta pH calculated in kidney section for mice injected with LPS (red) and control mates (blue).

Keywords: sepsis, MRI-CEST, pH
# 046

MRI of Staphylococcus aureus-induced infective endocarditis mouse models to reproduce human pathology (#474)

C. Schwarz1, Y. Töre1, V. Hoerr1, 2, S. Niemann3, C. Faber1

1 University Hospital Muenster, Department of Clinical Radiology, Muenster, North Rhine-Westphalia, Germany
2 University Hospital Jena, Institute of Medical Microbiology, Jena, Thuringia, Germany
3 University Hospital Muenster, Institute of Medical Microbiology, Muenster, North Rhine-Westphalia, Germany

Introduction

Staphylococcus aureus induced infective Endocarditis (IE) is a life threatening disease. To investigate and characterize IE in vivo, we have recently established a mouse model and appropriate MRI techniques [1], [2]. IE is induced by placing a permanent catheter into the right carotid artery to irritate the aortic valves and provide a seed for formation of bacterial vegetations. Pathology of IE in this model was found to differ between bacterial strains. Here, we investigate variations of this model to decipher the initial steps in formation of endocarditis or sepsis.

Methods

Three different surgical procedures were performed: the catheter was placed in the aortic arch (a), or on the valves and removed after 24 h (b) or left in place (c) (Fig. 1). MRI at 9.4T was performed 24h after surgery to monitor placement of the catheter, using a self-gated cardiac ultra-short echo time (UTE) sequence (TR/TE, 5/0.31 ms; in-plane/slice, 0.125/1 mm; duration: 12:08min) [1]. Next, C57Bl/6 mice were infected with 105 colony-forming units (CFU) of S. aureus 6850 or S. aureus Newman which is deficient in the adhesive molecule FnbpA/B. 24 h after infection, mice were sacrificed, organs (heart, lung, spleen, kidneys, liver, aortic valves, aortic arch) were prepared for histology or electron microscopy, or homogenized and plated on blood agar, to count the bacterial load.

Results/Discussion

Both removal of the catheter and placement in the aortic arch resulted in lower bacterial load in heart and on the valves (p<0.001) (Fig. 2A). Also lower clinical scores were observed in both cases (Fig. 2B). Generally, the bacterial load differed significantly between 6850 and Newman with catheter in place or without catheter (p<0.001) (Fig. 2A). The clinical scores were significantly higher in 6850-infected animals compared to Newman for both surgical procedures (with and without catheter) (Fig. 2B). For infection with Newman, clinical scores were not dependent on the catheter, which may be attributed to the initial adherence being mediated by the bacterial protein FnbpA/B, deficient in Newman.

Conclusions

The catheter itself and its position are essential to induce IE in the mouse model. The catheter most likely initiates heart insufficiency, supports bacterial growth on the heart valves, and gives rise to bacterial vegetations. MRI-monitoring of catheter position and progression of infection and inflammation is essential to make use of the IE mouse model, which may be employed to further investigate IE pathology or novel therapeutic approaches.

References

[1] Ring J et al. (2014). PLoS ONE 9(9): e107179. [2] Hoerr V et al. (2013). J Cardiovasc Magn Reson.15:59.

Acknowledgement

This work was funded by the DFG (SFB/TR34).

Fig. 1: 9.4T MR images of mice hearts.

A) Healthy non- catheterized heart valves B) Position of catheter in the aortic arch (red arrows) C) Position of the catheter on aortic valves (red arrows).

Fig. 2

A) Bacterial load and B) clinical score of S. aureus infected C57Bl/6 mice with different surgical procedures and infected with or without different S. aureus strains (*=(p<0.05), **=(p<0.01),***=(p<0.001)).

Keywords: infection, S. aureus, endocarditis, MRI, mouse model
# 047

Clumping factor A plays important role in initiation of S. aureus-induced infective endocarditis (#471)

Y. Töre1, C. Schwarz1, V. Hoerr1, 2, S. Niemann3, C. Faber1

1 University Hospital Muenster, Department of Clinical Radiology, Muenster, North Rhine-Westphalia, Germany
2 University Hospital Jena, Institute of Medical Microbiology, Jena, Thuringia, Germany
3 University Hospital Muenster, Institute of Medical Microbiology, Muenster, North Rhine-Westphalia, Germany

Introduction

Staphylococcus aureus-induced infective Endocarditis (IE) is a life threatening disease. A variety of adhesive surface proteins (adhesins) makes S. aureus extremely flexible and difficult to treat. Identification of the role of individual adhesins and their interplay in vivo may open new avenues for efficient therapy of IE. A mouse model of IE and appropriate MRI techniques [1], [2] have recently been established. We use this model with different knockout (KO) strains lacking single adhesins to evaluate the role of adhesins in S. aureus induced IE.

Methods

IE was induced in C57Bl/6 mice by a catheter placed in the right carotid artery irritating the aortic valve and serving as host for formation of bacterial vegetation. 24h after surgery infection was performed with 105 colony-forming units (CFU) of S. aureus Newman (n=7) and three different KO strains (Newman background) Δeap (n=10), ΔClfA (n=9) or Δspa (n=4), lacking one of the adhesins. KO was confirmed by qPCR (after 3h of culture). Catheter placement and progress of pathology were monitored by MRI at 9.4T, using a cardiac self-gated ultra-short echo time sequence (TR/TE, 5/0.31 ms; in-plane/slice, 0.125/1 mm) [1] (Fig. 1a). After 24h of infection, organs (kidneys, liver, heart and aortic valve) were prepared for homogenization and plated on blood agar, to count the bacterial load (BL).

Results/Discussion

Largely different BL in organs and levels of weight loss were observed in dependence of the bacterial strain, while differences in valve morphology were not obvious (Fig. 1b-c). Mice infected with Δeap or Δspa showed a significantly higher weight loss than mice infected with wildtype Newman or ΔClfA (Fig. 2a). This correlated with higher BL in organs of Δeap or Δspa infected mice, especially in the heart and the kidneys (Fig. 2b).

qPCR showed that Δeap and Δspa strains still expressed ClfA at 63% and 82% of the wildtype level, respectively, while the ΔClfA showed strongly downregulated eap and spa levels (Fig. 2c). In summary, our results suggest a prominent role of ClfA for initial adhesion in our in vivo model of IE. This finding confirms recent in vitro results which identified an essential role of ClfA in adhesion to host von-Willebrand factor via von Willebrand-binding protein [3].

Conclusions

ClfA plays an important role in initial adhesion in IE. Besides the fibronectin binding proteins, which are not fully functional in Newman strains, ClfA seems to be the major adhesion mechanism. However, expression profiles of S. aureus adhesins seem to be highly mutually dependent and also to vary with time. Targeting these two particular adhesion mechanisms may provide targets for novel diagnostic agents for IE, based on MRI or other non-invasive imaging modalities.

References

[1] Ring J et al. (2014). PLoS ONE 9(9): e107179.

[2] Hoerr V et al. (2013). J Cardiovasc Magn Reson.15:59.

[3] Claes et al. (2017). J Thromb Haemost DOI: 10.1111/jth.13653

Acknowledgement

This work was supported by the MedK Münster.

Fig. 1 The aortic valve in placement monitoring MRI and during preparation

A) The preinfectious cardiac MRI shows mechanical irritation of the valve by the catheter Stars mark the course of the catheter in the aorta.

B) 24h after injection of S. aureus Newman Δeap bacterial vegetation is found on the swollen valve.

C) Macroscopically, the aortic valve of a wildtype infected mouse does not show a remarkable difference to KO infected mice.

Fig. 2 Analysis of different S. aureus Newman KO strains

A) Weight loss of mice infected with Newman KO and wildtype rel. to the weight at infection (24h after surgery).

B) BL of mice infected with Newman KO rel. to Newman wildtype (logCFU/ml: kidneys 4.3, liver 3.8, heart 4.8, catheter 2.7, aortic valve 3.2).

C) qPCR after 3h of culture rel. to Newman wildtype (1.2 copies of ClfA, 1.9 copies of eap, 0.1 copies of spa per copy gyrB).

Keywords: Staphylococcus, Endocarditis, MRI, ClfA, Infection
# 048

Non-invasive assessment of treatment efficacy in a murine pulmonary fungal infection model by use of MRI and BLI (#141)

J. Poelmans1, U. Himmelreich1, L. Vanherp1, L. Zhai1, A. Hillen1, S. Belderbos1, J. Maertens2, K. Lagrou2, G. Vande Velde1

1 Biomedical MRI unit / MoSAIC, Department Imaging and Pathology, KU Leuven, Leuven, Belgium
2 Clinical bacteriology and Mycology, Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium

Introduction

Aspergillus fumigatus is an opportunistic fungal pathogen which can cause invasive pulmonary aspergillosis (IPA), especially in immunocompromised patients. Mouse models are extensively used to assess antifungal treatments. However, standard techniques to evaluate experimental infections are limited by their invasiveness. In this study, bioluminescence imaging (BLI) and magnetic resonance imaging (MRI) were combined to investigate IPA development in neutropenic mice and to assess the influence of antifungal treatment on the fungal load and lesion size.

Methods

BALB/c mice were rendered neutropenic by intraperitoneal (IP) injections of 150 mg/kg cyclophosphamide on day 4 and 1 prior to intranasal instillation of the bioluminescent A. fumigatus strain 2/7/1 (5.105 spores). Non-treated control animals (n = 10) underwent MRI and BLI scans daily until day 4. Treated animals received daily IP injections of 20 mg/kg voriconazole combined with grape fruit juice to prevent rapid metabolization. Treatment was initiated either on the day of infection (day 0; n = 5) or on day 1 (n = 5), day 2 (n = 5) or day 3 (n = 5) post infection (PI). The course of infection was monitored by acquiring MRI scans on day 1, 2, 3, 4, 5 and 14 PI and by BLI scans on day 3, 5 and 9 PI. After 4 or 14 days, the lungs were isolated for validation by CFU counts and histology.

Results/Discussion

Pulmonary MR images of non-treated mice revealed the presence of hyper intense lesions within the infected lungs on day 3 and 4 PI. On the corresponding BLI images, high signal intensities could be detected from the lung area. Similar results were obtained for the treatment day 3 group. Initiating treatment on day 2 resulted in an improved survival rate (figure 1), although a gradual increase of lung lesions and a transient increase in BLI signal were observed (figure 2). Animals receiving treatment from day 0 or 1 onwards showed reduced lesion formation on MRI and no BLI signal from the lung region. Both MRI and BLI results indicated that initiation of treatment during the early stages of infection has a beneficial effect on both survival and disease state.

Conclusions

BLI was successfully used to determine dynamic changes in the lung fungal load upon voriconazole treatment. In parallel, MRI provided 3-dimensional insights in the influence of voriconazole on the extent and distribution of lesions emerging within the infected lungs. Combining both imaging modalities improved our knowledge on the relationship between detected lesion and fungal growth in a completely non-invasive manner.

Acknowledgement

A. fumigatus strain 2/7/1 was kindly provided by prof. Matthias Brock (Fungal Biology Group, School of Life Sciences, University Park, Nottingham NG7 2RD, United Kingdom).

Survival of infected animals upon treatment with voriconazole

Graph representing the survival of mice receiving treatment from day 3, 2, 1 or 0 post infection. Initiating treatment at early time points resulted in improved survival rates.

Multimodal imaging of infected animals receiving voriconazole treatment

(A) MRI on day 4 and 14 after infection with A. fumigatus. Hyper intense signal can be detected within the lungs of mice in the treatment d3 and d2 groups, which are not present in the treatment d1 and d0 groups.  (B) BLI on day 3, 5 and 9 after infection with A. fumigatus. Early treatment start resulted in decreased BLI signal originating from the lung region.

Keywords: Fungal infections, multimodal imaging, Invasive pulmonary aspergillosis
# 049

White Blood Cell Tracking with Magnetic Particle Imaging Towards Sensitive and Radiation-Free Diagnosis of Infection and Inflammation (#118)

X. Y. Zhou1, 2, P. Chandrasekharan2, D. Mai2, K. E. Jeffris2, E. Y. Yu1, 2, B. Zheng2, S. M. Conolly2, 3

1 UC Berkeley - UCSF Graduate Program in Bioengineering, Bioengineering, Berkeley, California, United States of America
2 UC Berkeley, Bioengineering, Berkeley, California, United States of America
3 UC Berkeley, Electrical Engineering and Computer Science, Berkeley, California, United States of America

Introduction

White blood cell (WBC) scintigraphy has significant impact on clinical care for suspected osteomyelitis [1,2]. To create a clinically useful image, clinicians wait 24+ h for WBCs to clear from areas of nonspecific uptake to areas of infection, by which time image quality has degraded due to radioactive decay [3]. Magnetic particle imaging (MPI) is a sensitive medical imaging technique with zero ionizing radiation and long persistence for SPIO tracers [4]. We show time course MPI images of cultured macrophages in mice and MPI images of SPIO-labeled neutrophils isolated from mouse blood.

Methods

RAW 264.7 macrophages were cultured as described in [5] and labeled with VivoTrax superparamagnetic iron oxide (SPIOs) tracers by overnight incubation. Iron-labeled cells were visualized with Prussian Blue staining. Cell viability was determined with Trypan Blue staining. Neutrophils were extracted by lysis and isolated using negative selection via magnetic activated cell sorting from peripheral blood of C57BL/6 mice, then labeled with SPIOs via electroporation. C57BL/6 mice were injected with 8 million SPIO-labeled RAW 264.7 cells. MPI images were taken with the UCB home-built 6.3 T/m field free line scanner (shown in Fig. 2a) and reconstructed with x-space methods [6]. X-ray imaging was performed on a Kubtec Xpert 40.

Results/Discussion

Prussian Blue staining confirmed SPIO uptake by macrophages, as shown  in Fig. 1a and Fig. 1b. The MPI time course with x-ray anatomical reference in Fig. 1c shows that cultured macrophages were localized to mouse lungs and cleared slowly over time on account of the large cell size. 300,000 neutrophils were isolated from 1.2 mL of blood sample and were confirmed for CD11b and Ly6G surface marker via flow cytometry, shown in Fig. 2b. Neutrophils before electroporation were 66% viable according to Trypan Blue staining, whereas only 33.3% of cells were viable after electroporation in the presence of SPIO. Of the total cell sample 26.7% were labeled cells. MPI imaging of SPIO-labeled neutrophils in test tubes is shown in Fig. 2c. The MPI signal was found to be linear with cell number (R2 = 0.967), as expected. Increasing neutrophil viability post SPIO-labeling will allow for MPI imaging of autologous WBCs.

Conclusions

Time course MPI imaging of cultured macrophages and initial work on SPIO tracer labeling of neutrophils isolated from whole mouse blood demonstrates that MPI WBC imaging has potential to provide a radiation-free diagnostic complement to Indium-111 WBC and Technetium-99m WBC scans. Because there is no decay in magnetic signal over time, there is no tradeoff between radioactive half-life, SNR and time constant for optimal pathophysiologic contrast.

References

[1] Lewis et al., Open Forum Infect Dis, 2014

[2] Censullo and Vijayan, Open Forum Infect Dis, 2017

[3] Ziessman et al., 2014

[4] Goodwill et al., Adv Mater, 2012

[5] Maldiney et al., Nat Mater, 2014

[6]  Yu et al., ACS Nano, 2017

Acknowledgement

We would like to acknowledge funding from the National Institutes of Health and National Science Foundation Graduate Research Fellowship Program.

Fig. 1

(a) 10x and (b) 20x microscopic images, respectively, of Prussian Blue stained RAW 264.7 macrophages incubated with Vivotrax SPIOs. (c) MPI leukocyte scans in vivo mice over six hours. SPIO-labeled leukocytes are initially uptaken to the lung vasculature due to their large size; over time, the cells clear to the liver and spleen. X-ray anatomical reference.

Fig. 2

(a) UCB MPI 6.3 T/m FFL scanner. (b) Flow cytometry of isolated neutrophils.  Neutrophils were confirmed with flow cytometry to have CD11b and Ly6G surface markers. (c) MPI neutrophil phantom scans. Test tubes of SPIO-labeled were scanned and MPI signal was correlated to cell number (R2 = 0.967)

Keywords: magnetic particle imaging, infection, white blood cell, cell tracking, WBC scans