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|2:15 p.m.||PS 07-01||
This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.
|2:33 p.m.||PS 07-02||
Translational immunoPET/MR imaging of invasive pulmonary aspergillosis. (#365)
Nicolas Beziere1, Andreas Maurer1, Anna-Maria Wild1, Johannes Schwenck2, Sophie Henneberg3, Genna Davies4, Gerald Reischl1, Philipp R. Spycher5, Joeri Wehrmueller5, Frederic Boschetti6, Stefan Wiehr1, Roger Schibli5, Matthias Gunzer3, Christopher Thornton4, Bernd J. Pichler1
1 Eberhard Karls University, Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Tübingen, Germany
The ubiquitous fungus Aspergillus fumigatus can cause the life-threatening lung disease Invasive Pulmonary Aspergillosis (IPA) in immunocompromised patients. Current non-invasive diagnostics lack specificity and sensitivity, while invasive biopsy or bronchoalveolar lavage and subsequent culture of the fungus is slow and carries risks for the patient. Through radiolabeling of an A. fumigatus specific antibody (JF5), we propose a highly specific non-invasive diagnostic and therapy monitoring method for IPA.
Humanized full length JF5 (hJF5) was generated from the variable regions of murine JF5 (1) and human IgG1 framework, conjugated to NODAGA and rabiolabeled using [64Cu]CuCl2. Neutropenic C57BL/6 mice were infected using 4x106 A. fumigatus spores deposited intra-tracheally and subsequently injected with 13 MBq of the radiotracer. Treated groups received Voriconazole or Caspofungin. Simultaneous in vivo PET/MR was performed 3, 24 and 48h after infection using a small animal PET insert and a 7T MRI (Bruker Biospin GmbH). In vivo results were validated using ex vivo biodistribution by gamma counting, autoradiography and fluorescence microscopy. For human application the chelator conjugation and radiolabeling were upscaled within a GMP setting, and toxicity testing was performed in rodents.
Radiolabeled hJF5 constructs revealed high uptake in vivo to A. fumigatus infection sites, in particular 48h after injection, and negligible accumulation in control animals. Specific uptake was not observed with [64Cu]CuCl2 nor radiolabeled isotype antibody. [64Cu]NODAGA-hJF5 was found to provide the overall best uptake ratio 48h after injection in the diseased lungs (17.1 ± 2.5 %ID/cc) compared to PBS treated lungs (9.4 ± 1.1 %ID/cc) (2). When injected with the antifungal drug Voriconazole, almost complete suppression of 64Cu accumulation in the lungs could be seen (10.0 ± 1.1 %ID/cc). Toxicity testing in rodents showed no adverse effects of repeated dosing. Conjugation procedure and radiolabeling were successfully validated according to GMP guidelines and we expect to obtain first human data in early 2019.
[64Cu]NODAGA-hJF5 proved to be a highly specific and sensitive tracer for immunoPET diagnosis of aspergillosis, showing very promising results in preclinical models for therapy monitoring of classical antifungal drugs. After recently establishing the GMP compliant synthesis of this tracer and successful systemic toxicity evaluations, we are now progressing to first-in-human trials.
(1) Rolle et al. PNAS 2016; 113(8); 1026-1033.
(2) Davies et al., Theranostics. 2017; 7(14): 3398–3414.
Figure 1: Therapy monitoring of Aspergillosis with Voriconazole
Sagittal PET maximum intensity projection (MIP), magnetic resonance imaging (MRI) and coregistration (Fusion) of representative PBS controls (control), treated (A. fumigatus + Voriconazole) and infected (A. fumigatus) animals 48h after [64Cu]NODAGA-JF5 injection.
Keywords: Positron Emission Tomography, Aspergillosis, Infection
|2:45 p.m.||PS 07-03||
In vivo imaging of B. pertussis colonization and interactions with the host by Fibered Confocal Fluorescence Microscopy in the baboon respiratory tract (#57)
Thibaut Naninck1, Loïc Coutte2, Céline Mayet1, Vanessa Contreras1, Sébastien Langlois1, Camille Locht2, Roger Le Grand1, Catherine Chapon1
1 CEA, JACOB/IDMIT, Fontenay-aux-roses, France
Whooping cough, due to Bordetella pertussis infection, is today re-emerging in many countries despite high vaccination coverage1,2. Studies in the baboon model of B. pertussis infection have risen the hypothesis of bacterial asymptomatic carriage and transmission3. It is therefore crucial to develop tools to assess bacterial colonization in the whole respiratory tract of primates. Here we developed in vivo Fibered Confocal Fluorescence Microscopy (FCFM)4 to explore the respiratory tract and assess colonization and interactions between B.pertussis and antigen presenting cells (APCs) in baboons.
FCFM in airways was first evaluated ex vivo on NHP explants. A solution of anti-HLA-DR mouse antibody labelled with AlexaFluor647 and a solution of acriflavine were dropped topically in the bronchus of a lung lobe and in a tracheal ring to label specifically the APCs and the other cellular structures, respectively. For in vivo studies, GFP-expressing B. pertussis was inoculated in young baboons by intranasal and intra-tracheal routes or intranasal route only. Besides, monoclonal anti-HLA-DR-AF647 antibody was administered by topical application in the trachea to specifically target and label APCs. FCFM, coupled with bronchoscopy, was performed in the lower respiratory tract weekly post-infection. Bacterial colonization was quantified using GFP positive signal area detected by FCFM.
Animals infected with a GFP-expressing B. pertussis B1917 strain by intranasal and intra tracheal routes developed the classical clinical symptoms for whooping cough as previously described in baboons infected with wild-type strains5. We were also able to specifically label and detect cells of interest like APCs in the airways of non-human primates ex vivo using FCFM (Figure 1). Furthermore, in vivo FCFM coupled with bronchoscopy allowed us to detect and quantify bacterial colonization kinetics and APC interactions in the lower respiratory tract of young baboons after B. pertussis-GFP infection in baboons (Figure 2). Ex vivo co-culture analyses also confirmed the interactions between B. pertussis and APCs in lungs explants. We were also able to detect by FCFM bacterial tracheal colonization in animals challenged by intranasal route only providing new insights concerning bacterial localization after natural infection.
This approach using fluorescence imaging will then be a useful tool to describe the mechanisms of action of the bacteria at a cellular resolution during infection to develop more effective vaccines against pertussis. Other in vivo imaging modalities such as PET-CT will be used to study the dissemination of the pathogen in the whole body. Moreover, this imaging protocol may also be implemented to study many diverse respiratory infectious diseases.
1. WHO | Diphtheria-tetanus-pertussis (DTP3) immunization coverage. WHO (2018). Available at: http://www.who.int/gho/immunization/dtp3/en/. (Accessed: 22nd January 2018)
2. WHO | WHO-recommended surveillance standard of pertussis. WHO (2018). Available at: http://www.who.int/immunization/monitoring_surveillance/burden/vpd/surveillance_type/passive/pertussis_standards/en/. (Accessed: 29th March 2018)
3. Warfel, J. M., Zimmerman, L. I. & Merkel, T. J. Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model. Proc. Natl. Acad. Sci. U. S. A. 111, 787–792 (2014).
4. Thiberville, L. et al. In Vivo Imaging of the Bronchial Wall Microstructure Using Fibered Confocal Fluorescence Microscopy. Am. J. Respir. Crit. Care Med. 175, 22–31 (2007).
5. Warfel, J. M., Beren, J., Kelly, V. K., Lee, G. & Merkel, T. J. Nonhuman Primate Model of Pertussis. Infect. Immun. 80, 1530–1536 (2012).
We warmly thank the IDMIT infrastructure staff for excellent technical assistance, especially Laetitia Bossevot, Naya Sylla and Sebastien Langlois. The IDMIT facility is supported by the French government “Programme d’Investissements d’Avenir” (PIA), under grants ANR-11-INBS-0008 and ANR-10-EQPX-02-01. This work was supported by PERISCOPE. PERISCOPE has received funding from the Innovative Medicines Initiative 2 Joint Undertaking, under grant agreement no. 115910. This Joint Undertaking received support from the European Union’s Horizon 2020 research and innovation Program and European Federation of Pharmaceutical Industries and Associations (EFPIA) and Bill and Melinda Gates Foundation (BMGF).
In vivo imaging by FCFM coupled with bronchoscopy in B. pertussis B1917-GFP infected baboons
Images (A-F) extracted from FCFM acquisition in the trachea at D2 (A-C) and D14 (D-F) post-infection of one animal, representative of the three challenged baboons. B1917-GFP bacteria (aggregates and biofilms) are detected in 488-nm channel (green) (A, D) and APCs are labeled with anti-HLA-DR AF647 antibody and detected in the 660-nm channel (red) (B, E). (G) Image taken with the bronchoscope camera showing the pCLE probe (arrow) in the trachea. (H) Evaluation of B. pertussis B1917-GFP colonization in the trachea determined by the area of GFP signal in the 488-nm channel (n = 3 animals) and comparison of the data between timepoints (****: p< 0.0001).
Keywords: Fibered Confocal Fluorescence Microscopy, bronchoscopy, baboon, pertussis, whooping cough
|2:57 p.m.||PS 07-04||
Evaluating antifungal treatment efficacy in cerebral infections: a multimodal approach using MRI, MRS and BLI (#360)
Liesbeth Vanherp1, 2, Jennifer Poelmans1, 2, Matthias Brock3, Guilhem Janbon4, Katrien Lagrou5, Greetje Vande Velde1, 2, Uwe Himmelreich1, 2
1 KU Leuven, Biomedical MRI, Leuven, Belgium
Brain lesions caused by Cryptococcus typically require long-term treatment with antifungals as these have limited efficacy. To quantify the response of cryptococcomas to antifungal therapy, we evaluated different preclinical imaging techniques in mouse models. Bioluminescence imaging (BLI) and MR imaging (MRI) were used to monitor cell viability and lesion growth. Our previous results have identified the presence of trehalose, a possible storage carbohydrate, in cryptococcomas1. We studied the potential of this biomarker for treatment follow-up using in vitro and in vivo MR spectroscopy (MRS).
In vitro 1H-NMR spectra of Cryptococcus cells in PBS-D2O/H2O were acquired using a Bruker Avance 400MHz NMR spectrometer. Cryptococcomas were induced by stereotactic injection of 104 firefly luciferase-expressing C. neoformans KN99α cells2 in the mouse striatum (n=4 per group). Starting from 3 days post inoculation (p.i.), mice received daily intraperitoneal (i.p.) injections of saline, fluconazole (FLU, 75 mg/kg/day) or liposomal amphotericin-B (AMB, 10 mg/kg/day i.p. or 1x20 mg/kg intravenously). Anatomical MRI (2D RARE), MRS (PRESS, 2x2x2 mm³ voxel, TR/TE 1800/20 ms) and BLI were performed on day 3 (pre-treatment), 6 and 9 p.i. using a 9.4T Bruker Biospec MRI scanner or IVIS Spectrum system, respectively. After the last scan, animals were sacrificed for fungal load analysis.
In vitro NMR spectroscopy showed a conversion of trehalose to its subunits of glucose and potential other downstream carbohydrates upon exposure to AMB, indicating a potential for treatment follow-up. In in vivo studies, animals receiving 6 days of antifungal treatment had a slower lesion growth on MRI than controls, but treatment could not stop growth of the lesion. Using BLI, we observed lower brain signals in the FLU-treated group, but large inter-animal variability complicated a robust assessment of treatment effects. MRS showed lower trehalose concentrations in the lesions of treated animals with the largest effect seen in the FLU-treated group. Statistical analysis (repeated measures two-way ANOVA) detected a treatment effect for MRS (p = 0.0006), but not for MRI (p = 0.0809) or BLI (p = 0.3356). Fungal load analysis showed a 10-fold lower fungal load in the FLU-group and in some of the AMB-animals than in the control group, but no significant reduction could be demonstrated.
Despite only a limited treatment efficacy, the MRS-marker trehalose was used for assessment of the viable fungal load, while this was more challenging using BLI and MRI. This preclinical platform allows rapid evaluation of the in vivo efficacy of antifungal treatment, including promising novel therapies. Furthermore, the biomarker trehalose can potentially be translated to the clinic for diagnosis and non-invasive treatment follow-up using MRS.
We are thankful for financial support from the European Commission for the Infect-ERA project CryptoVIEW. LV is an SB PhD fellow at Research Foundation Flanders (FWO).
Figure 1: Representative images of animals in the saline- and fluconazole-treated group.
Animals receiving fluconazole had smaller lesions, lower trehalose concentrations in the lesion and a lower BLI signal. All images are from 9 days post inoculation (6 days post initiation of treatment). Tre: trehalose, Lip: lipids, Lac: lactate.
Keywords: Fungal infection, multimodal imaging, treatment
|3:09 p.m.||PS 07-05||
89Zr-alfaToxAb as novel immunoPET radiotracer for the detection of S. aureus in bacteremia model (#301)
Mª Isabel González1, 2, Martha Kestler1, Patricia Muñoz1, 5, 6, Emilio Bouza1, 5, 6, Manuel Desco1, 3, 4, Beatriz Salinas1, 2, 3
1 Instituto de Investig. Sanitaria Gregorio Marañón, Experimental Medicine and Surgery Unit, Madrid, Spain
Staphylococcus aureus (S. a) is a leading cause of community-acquired and hospital-acquired bacteremia. Patients with S. aureus bacteremia can develop a broad array of complications that may be difficult to recognize initially and can increase morbidity and mortality . Proper location of staphylococcal activity is essential for initial diagnosis and treatment duration. Due to the role of a-toxin as main virulent factor in the infectious process, we present a specific anti α-toxin antibody radiotracer for the selective detection of S. aureus infection by PET/CT imaging.
anti S. aureus α-toxin antibody (α-ToxAb; 33KDa, 1 mg/ml) was adjusted to pH 8.8 and conjugated for 30min at 37ºC with DFO-NCS, with a 5 fold molar excess of the chelator. Then, DFO- α-ToxAb was radiolabeled with 89Zr4+ for 1h at 25ºC at pH 5.5 and purified with centrifugal filters units. Quality control of 89Zr-αToxAb was stablished by radio HPLC (PBS, 0.2 ml/min). Pharmacokinetics of the tracer was evaluated by blood half-life and ex vivo biodistribution studies (48h and 72h post injection). In vivo PET/CT imaging was performed in local infection-inflammation model of SD rats 5h post injection of 89Zr-aToxAb. In this model, left hind leg received intramuscular injection of alive S. aureus (0.4mL, 10*8CFU, infection side) and right hind leg of an inactive strain of S. a (0.4 mL, 10*8 CFU)
Pure 89Zr-DFO-α-toxin antibody was synthetized with a radiochemical yield of 21.4 % and purity > 95%, (Fig. 1A). Pharmacokinetic evaluation of the radiolabeled α-toxin antibody presents a blood half-life values of 160 min (Fig. 1B). Ex vivo biodistribution studies at 48 and 72h (Fig. 1C) confirms hepatobiliary metabolism, with main accumulation in spleen (4.7 %ID/g) and kidneys (4.0 %ID/g). In vivo PET-CT evaluation of the 89Zr-DFO-α-toxin in local infection model (100mCi, 150 mL, PBS) shown high uptake in the infected region, with no significant uptake in the non-infected one (Fig. 2). These results were confirmed by ex vivo studies, (Fig. 1D) with values of 95,46 ID%/g in the infected muscle vs. 4.53% of the inflamed one after 48h.
We present a new inmunePET tracer specific of S. aureus based on the radiolabeling of anti a-toxin antibody[3,4]. Our agent has demonstrated its efficiency in the detection of local infection caused by S. aureus. Furthermore, results achieved has confirmed evidently selectivity of the radio-antibody, allowing distinguishing between inflammatory and infectious processes and probing its potential as platform for early diagnosis of bacteremia.
 Baddour, L.M. et al. (2007) Mayo Clin. Proc., 82(10), p. 1163-1164;  De Simone, D.C., et al (2016) Journal of the American College of Cardiology, 67(13_S), p. 2184;  Lewis, J.S. et al. (2015) J. Vis. Exp., 96, e52521;  Van Dongen, G. A. M. S. et al (2010) Nature Protocols, 5 (4), p. 739-743
The Small Animal Imaging core, especially Yolanda Sierra, Alexandra de Francisco, Maria Felipe and Lorena Cussó, as well as the department of microbiology at HGGM. This work was partially supported by Comunidad de Madrid (S2017/BMD-3867 RENIM-CM, co-financed by European Structural and Investment Funds), and Ministry of Economy, Industry and Competitiveness (ISCIII-FIS grants PI16/02037, co-financed by ERDF, FEDER, Funds from the European Commission, “A way of making Europe”).
Keywords: S aureus, Bacteremia, tracer, infection disseases, immunoPET
|3:21 p.m.||PS 07-06||
Assessing bacteria-induced infective endocarditis by MRI and elemental mass spectrometric imaging (#273)
Christian Schwarz1, Rebecca Buchholz2, Uwe Karst2, Moritz Wildgruber1, Cornelius Faber1
1 University Hospital Muenster, Department of Clinical Radiology, Muenster, North Rhine-Westphalia, Germany
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 , . 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 accumulates different amounts of chemical elements. The competition for metals between host and pathogen is one of the most important factors for the outcome of infection. Here, we assess whether mass spectrometry can detect IE.
A 32G-catheter was placed in C57Bl/6 mice to irritate the aortic valves (Fig.1). Next, 24h after catheter placement, mice were infected with S. aureus or PBS (MOCK-infected). MRI at 9.4T was performed 24h after infection to monitor aortic valve morphology and function, using a self-gated cardiac ultra-short echo time (UTE) sequence (TR/TE, 5/0.31ms; in-plane/slice, 0.125/1mm; duration: 12:08min) . Mice were sacrificed and the hearts were embedded in paraffin. Aortic valves were sliced 10 µm thick to perform laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to determine the amount of Ca, Cu, Fe, Zn and Mn. A qualitative measurement was done for P. Consecutive tissue slices were stained for Gram or H&E to relate element concentrations to infection or inflammation.
IE was successfully induced as confirmed by MRI, macroscopic findings and Gram staining that revealed S. aureus vegetations on the aortic valves (Fig. 2). Focusing on this infected tissue, LA-ICP-MS showed a high concentration of Ca (maximum value: 800 µg/g) and a high qualitative intensity of P. The presence of P was assigned to pathogenic- and eukaryotic -cells containing phospholipids, ATP, RNA and DNA. High amount of Zn (maximum value: 250 µg/g) was detected where histology suggested an increased presence of immune cells and S. aureus. The competition for Zn between host and pathogen is described as nutritional immunity and induces an increased oxygen stress in bacteria. Increased Mn (maximum value: 4 µg/g) was observed where histology showed the presence of chondrocytes or was observed as unspecific elevated signal in muscle cells.
Cardiac MRI gives the first hints to pathophysiological processes to IE. A selective detection of bacteria within the tissue stays challenging and histology is still necessary to confirm these findings. The application of LA-ICP-MS may become a valuable tool to differentiate between inflammation and infection. Calcium, phosphor and zinc in combination may be used to unambiguously identify bacterial vegetations in IE.
 Ring J et al. (2014). PLoS ONE 9(9): e107179.  Hoerr V et al. (2013). J Cardiovasc Magn Reson.15:59.
Keywords: cardiac MRI, LA-ICP-MS, S. aureus, infective endocarditis
|3:33 p.m.||PS 07-07||
Noncanonical NF-κB activation is essential for the development of chronic cutaneous delayed-type hypersensitivity reaction (#404)
Roman Mehling1, Johannes Schwenck1, 2, Marc Riemann3, Martin Röcken4, Bernd Pichler1, Manfred Kneilling1, 4
1 University of Tuebingen, Preclinical Imaging and Radiopharmacy, Tuebingen, Baden-Württemberg, Germany
As a crucial regulator of inflammation, NF-κB modulates expression of cytokines, chemokines, leukocyte infiltration and cellular reactive oxygen/nitrogen species (ROS/RNS) production thereby regulating various aspects of innate and adaptive immune responses. While canonical NF-κB activation is commonly linked to innate immune responses, the noncanonical NF-κB activation is more associated with the adaptive immunity. Nevertheless, the exact role of canonical and noncanonical NF-κB activation on T cell driven cutaneous delayed-type hypersensitivity reaction (DTHR) has not been described yet.
Wild-type mice (WT) NF-kB1-/- (canonical NF-κB pathway) and NF-κB2-/- (noncanonical NF-κB pathway) mice were sensitized with 5% TNCB at the abdomen and challenged with 1% TNCB at the right ear 7 days later to elicit acute DTHR. To induce chronic DTHR we challenged mice every 48h up to 5 times. We determined ear-swelling (ES) responses and conducted non invasive in vivo optical imaging (OI) measurements employing L-012 (a ROS/RNS-sensitive chemiluminescence probe) 0h, 4h, 12h, and 24h after the 1st, 3rd and 5th TNCB challenge (Ch). Additionally, we obtained the ear tissues for H&E histology and RT-PCR analysis of NF-κB driven genes and performed flow cytometry analysis of the draining lymph nodes and the spleen.
We determined normal ES in all strains during acute CHSR but strongly reduced ES response in the NF-κB2-/- mice and slightly reduced response in the NF-κB1-/- mice during chronic DTHR when compared to the WT mice. H&E staining of ears with chronic DTHR revealed strongly reduced ear thickness, edema, leucocyte infiltrate, acanthosis and hyperkeratosis in NF-κB2-/- mice when compared to WT and NF-κB1-/- mice. In vivo OI measurements uncovered an up to fifteen-fold decreased ROS/RNS production in ears of NF-κB2-/- mice 4h after the 3rd and 5th Ch and a two-fold reduction in the ears of NF-κB1-/- mice 4h after the 3rd Ch when compared to the WT mice. Flow cytometry analysis revealed strongly diminished B cells population but a significantly increased amount of CD4+ and CD8+ T cells in the draining lymph nodes and spleens of NF-kB2-/- mice. In addition, we determined strongly reduced mRNA expression of the NF-κB driven genes TNF, IL-1β and IL-6 in inflamed ears of NF-κB2-/- mice.
We uncovered the essential pro-inflammatory role of noncanonical NF-κB activation for the development of chronic DTHR, as NF-κB2-/- mice revealed strongly reduced ear-swelling responses, ROS/RNS production and genomic expression of pro-inflammatory cytokines. Thus, therapeutic targeting of NF-κB2 activation could represent a novel powerful tool for the treatment of T-cell-driven autoimmune diseases, such as psoriasis or rheumatoid arthritis.
Keywords: Inflammation, ROS, Optical Imaging, NF-κB