A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response1. Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques (Macaca fascicularis), and-concomitantly-protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model2 and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate.

Single cell RNA sequencing reveals endothelial cell killing and resolution pathways in experimental malaria-associated acute respiratory distress syndrome.

Plasmodium parasites cause malaria, a global health disease that is responsible for more than 200 million clinical cases and 600 000 deaths each year. Most deaths are caused by various complications, including malaria-associated acute respiratory distress syndrome (MA-ARDS). Despite the very rapid and efficient killing of parasites with antimalarial drugs, 15% of patients with complicated malaria succumb. This stresses the importance of investigating resolution mechanisms that are involved in the recovery from these complications once the parasite is killed. To study the resolution of MA-ARDS, P. berghei NK65-infected C57BL/6 mice were treated with antimalarial drugs after onset of symptoms, resulting in 80% survival. Micro-computed tomography revealed alterations of the lungs upon infection, with an increase in total and non-aerated lung volume due to edema. Whole body plethysmography confirmed a drastically altered lung ventilation, which was restored during resolution. Single-cell RNA sequencing indicated an increased inflammatory state in the lungs upon infection, which was accompanied by a drastic decrease in endothelial cells, consistent with CD8+ T cell-mediated killing. During resolution, anti-inflammatory pathways were upregulated and proliferation of endothelial cells was observed. MultiNicheNet interactome analysis identified important changes in the ligand-receptor interactions during disease resolution that warrant further exploration in order to develop new therapeutic strategies. In conclusion, our study provides insights in pro-resolving pathways that limit inflammation and promote endothelial cell proliferation in experimental MA-ARDS. This information may be useful for the design of adjunctive treatments to enhance resolution after Plasmodium parasite killing by antimalarial drugs.

Profiling Bacteria in the Lungs of Patients with Severe Influenza Versus COVID-19 with or without Aspergillosis.

RATIONALE: The influence of the lung bacterial microbiome, including potential pathogens, in patients with influenza- or COVID-19-associated pulmonary aspergillosis (IAPA or CAPA) is yet to be explored. OBJECTIVES: To explore the composition of the lung bacterial microbiome and its association with viral and fungal infection, immunity and outcome in severe influenza versus COVID-19 with or without aspergillosis. METHODS: We performed a retrospective study in mechanically ventilated influenza and COVID-19 patients with or without invasive aspergillosis in whom bronchoalveolar lavage (BAL) for bacterial culture (with or without PCR) was obtained within two weeks after ICU admission. Additionally, 16S rRNA gene sequencing data and viral and bacterial load of BAL samples from a subset of these patients, and of patients requiring non-invasive ventilation, were analyzed. We integrated 16S rRNA gene sequencing data with existing immune parameter datasets. MEASUREMENTS AND MAIN RESULTS: Potential bacterial pathogens were detected in 20% (28/142) of influenza and 37% (104/281) of COVID-19 patients, while aspergillosis was detected in 38% (54/142) of influenza and 31% (86/281) of COVID-19 patients. A significant association between bacterial pathogens in BAL and 90-day mortality was found only in influenza patients, particularly IAPA patients. COVID-19 but not influenza patients showed increased pro-inflammatory pulmonary cytokine responses to bacterial pathogens. CONCLUSIONS: Aspergillosis is more frequently detected in lungs of severe influenza patients than bacterial pathogens. Detection of bacterial pathogens associates with worse outcome in influenza patients, particularly in those with IAPA, but not in COVID-19 patients. The immunological dynamics of tripartite viral-fungal-bacterial interactions deserve further investigation. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

A Pathology-based Case Series of Influenza- and COVID-19-associated Pulmonary Aspergillosis: The Proof Is in the Tissue.

Rationale: Invasive pulmonary aspergillosis has emerged as a frequent coinfection in severe coronavirus disease (COVID-19), similarly to influenza, yet the clinical invasiveness is more debated. Objectives: We investigated the invasive nature of pulmonary aspergillosis in histology specimens of influenza and COVID-19 ICU fatalities in a tertiary care center. Methods: In this monocentric, descriptive, retrospective case series, we included adult ICU patients with PCR-proven influenza/COVID-19 respiratory failure who underwent postmortem examination and/or tracheobronchial biopsy during ICU admission from September 2009 until June 2021. Diagnosis of probable/proven viral-associated pulmonary aspergillosis (VAPA) was made based on the Intensive Care Medicine influenza-associated pulmonary aspergillosis and the European Confederation of Medical Mycology (ECMM) and the International Society for Human and Animal Mycology (ISHAM) COVID-19-associated pulmonary aspergillosis consensus criteria. All respiratory tissues were independently reviewed by two experienced pathologists. Measurements and Main Results: In the 44 patients of the autopsy-verified cohort, 6 proven influenza-associated and 6 proven COVID-19-associated pulmonary aspergillosis diagnoses were identified. Fungal disease was identified as a missed diagnosis upon autopsy in 8% of proven cases (n = 1/12), yet it was most frequently found as confirmation of a probable antemortem diagnosis (n = 11/21, 52%) despite receiving antifungal treatment. Bronchoalveolar lavage galactomannan testing showed the highest sensitivity for VAPA diagnosis. Among both viral entities, an impeded fungal growth was the predominant histologic pattern of pulmonary aspergillosis. Fungal tracheobronchitis was histologically indistinguishable in influenza (n = 3) and COVID-19 (n = 3) cases, yet macroscopically more extensive at bronchoscopy in influenza setting. Conclusions: A proven invasive pulmonary aspergillosis diagnosis was found regularly and with a similar histological pattern in influenza and in COVID-19 ICU case fatalities. Our findings highlight an important need for VAPA awareness, with an emphasis on mycological bronchoscopic work-up.

Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2-infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

Two Functional Epithelial Sodium Channel Isoforms Are Present in Rodents despite Pronounced Evolutionary Pseudogenization and Exon Fusion.

The epithelial sodium channel (ENaC) plays a key role in salt and water homeostasis in tetrapod vertebrates. There are four ENaC subunits (α, ß, γ, δ), forming heterotrimeric αßγ- or δßγ-ENaCs. Although the physiology of αßγ-ENaC is well understood, for decades the field has stalled with respect to δßγ-ENaC due to the lack of mammalian model organisms. The SCNN1D gene coding for δ-ENaC was previously believed to be absent in rodents, hindering studies using standard laboratory animals. We analyzed all currently available rodent genomes and discovered that SCNN1D is present in rodents but was independently lost in five rodent lineages, including the Muridae (mice and rats). The independent loss of SCNN1D in rodent lineages may be constrained by phylogeny and taxon-specific adaptation to dry habitats, however habitat aridity does not provide a selection pressure for maintenance of SCNN1D across Rodentia. A fusion of two exons coding for a structurally flexible region in the extracellular domain of δ-ENaC appeared in the Hystricognathi (a group that includes guinea pigs). This conserved pattern evolved at least 41 Ma and represents a new autapomorphic feature for this clade. Exon fusion does not impair functionality of guinea pig (Cavia porcellus) δßγ-ENaC expressed in Xenopus oocytes. Electrophysiological characterization at the whole-cell and single-channel level revealed conserved biophysical features and mechanisms controlling guinea pig αßγ- and δßγ-ENaC function as compared with human orthologs. Guinea pigs therefore represent commercially available mammalian model animals that will help shed light on the physiological function of δ-ENaC.

Cobalt exposure via skin alters lung immune cells and enhances pulmonary responses to cobalt in mice.

Cobalt has been associated with allergic contact dermatitis and occupational asthma. However, the link between skin exposure and lung responses to cobalt is currently unknown. We investigated the effect of prior dermal sensitization to cobalt on pulmonary physiological and immunological responses after subsequent challenge with cobalt via the airways. BALB/c mice received epicutaneous applications (25 µL/ear) with 5% CoCl2*6H2O (Co) or the vehicle (Veh) dimethyl sulfoxide (DMSO) twice; they then received oropharyngeal challenges with 0.05% CoCl2*6H2O or saline five times, thereby obtaining four groups: Veh/Veh, Co/Veh, Veh/Co, and Co/Co. To detect early respiratory responses noninvasively, we performed sequential in vivo microcomputed tomography (µCT). One day after the last challenge, we assessed airway hyperreactivity (AHR) to methacholine, inflammation in bronchoalveolar lavage (BAL), innate lymphoid cells (ILCs) and dendritic cells (DCs) in the lungs, and serum IgE. Compared with the Veh/Veh group, the Co/Co group showed increased µCT-derived lung response, increased AHR to methacholine, mixed neutrophilic and eosinophilic inflammation, elevated monocyte chemoattractant protein-1 (MCP-1), and elevated keratinocyte chemoattractant (KC) in BAL. Flow cytometry in the Co/Co group demonstrated increased DC, type 1 and type 2 conventional DC (cDC1/cDC2), monocyte-derived DC, increased ILC group 2, and natural cytotoxicity receptor-ILC group 3. The Veh/Co group showed only increased AHR to methacholine and elevated MCP-1 in BAL, whereas the Co/Veh group showed increased cDC1 and ILC2 in lung. We conclude that dermal sensitization to cobalt may increase the susceptibility of the lungs to inhaling cobalt. Mechanistically, this enhanced susceptibility involves changes in pulmonary DCs and ILCs.

Automated computerized image analysis for the user-independent evaluation of disease severity in preclinical models of NAFLD/NASH.

Pathologists use a semiquantitative scoring system (NAS or SAF score) to facilitate the reporting of disease severity and evolution. Similar scores are applied for the same purposes in rodents. Histological scores have inherent inter- and intra-observer variability and yield discrete and not continuous values. Here we performed an automatic numerical quantification of NASH features on liver sections in common preclinical NAFLD/NASH models. High-fat diet-fed foz/foz mice (Foz HF) or wild-type mice (WT HF) known to develop progressive NASH or an uncomplicated steatosis, respectively, and C57Bl6 mice fed a choline-deficient high-fat diet (CDAA) to induce steatohepatitis were analyzed at various time points. Automated software image analysis of steatosis, inflammation, and fibrosis was performed on digital images from entire liver sections. Data obtained were compared with the NAS score, biochemical quantification, and gene expression. As histologically assessed, WT HF mice had normal liver up to week 34 when they harbor mild steatosis with if any, little inflammation. Foz HF mice exhibited grade 2 steatosis as early as week 4, grade 3 steatosis at week 12 up to week 34; inflammation and ballooning increased gradually with time. Automated measurement of steatosis (macrovesicular steatosis area) revealed a strong correlation with steatosis scores (r = 0.89), micro-CT liver density, liver lipid content (r = 0.89), and gene expression of CD36 (r = 0.87). Automatic assessment of the number of F4/80-immunolabelled crown-like structures strongly correlated with conventional inflammatory scores (r = 0.79). In Foz HF mice, collagen deposition, evident at week 20 and progressing at week 34, was automatically quantified on picrosirius red-stained entire liver sections. The automated procedure also faithfully captured and quantitated macrovesicular steatosis, mixed inflammation, and pericellular fibrosis in CDAA-induced steatohepatitis. In conclusion, the automatic numerical analysis represents a promising quantitative method to rapidly monitor NAFLD activity with high-throughput in large preclinical studies and for accurate monitoring of disease evolution.

The Added Value of Longitudinal Imaging for Preclinical In Vivo Efficacy Testing of Therapeutic Compounds against Cerebral Cryptococcosis.

Brain infections with Cryptococcus neoformans are associated with significant morbidity and mortality. Cryptococcosis typically presents as meningoencephalitis or fungal mass lesions called cryptococcomas. Despite frequent in vitro discoveries of promising novel antifungals, the clinical need for drugs that can more efficiently treat these brain infections remains. A crucial step in drug development is the evaluation of in vivo drug efficacy in animal models. This mainly relies on survival studies or postmortem analyses in large groups of animals, but these techniques only provide information on specific organs of interest at predefined time points. In this proof-of-concept study, we validated the use of noninvasive preclinical imaging to obtain longitudinal information on the therapeutic efficacy of amphotericin B or fluconazole monotherapy in meningoencephalitis and cryptococcoma mouse models. Bioluminescence imaging enabled the rapid in vitro and in vivo evaluation of drug efficacy, while complementary high-resolution anatomical information obtained by magnetic resonance imaging of the brain allowed a precise assessment of the extent of infection and lesion growth rates. We demonstrated a good correlation between both imaging readouts and the fungal burden in various organs. Moreover, we identified potential pitfalls associated with the interpretation of therapeutic efficacy based solely on postmortem studies, demonstrating the added value of this noninvasive dual imaging approach compared to standard mortality curves or fungal load endpoints. This novel preclinical imaging platform provides insights in the dynamic aspects of the therapeutic response and facilitates a more efficient and accurate translation of promising antifungal compounds from bench to bedside.

Preterm birth impairs postnatal lung development in the neonatal rabbit model.

BACKGROUND: Bronchopulmonary dysplasia continues to cause important respiratory morbidity throughout life, and new therapies are needed. The common denominator of all BPD cases is preterm birth, however most preclinical research in this area focusses on the effect of hyperoxia or mechanical ventilation. In this study we investigated if and how prematurity affects lung structure and function in neonatal rabbits. METHODS: Pups were delivered on either day 28 or day 31. For each gestational age a group of pups was harvested immediately after birth for lung morphometry and surfactant protein B and C quantification. All other pups were hand raised and harvested on day 4 for the term pups and day 7 for the preterm pups (same corrected age) for lung morphometry, lung function testing and qPCR. A subset of pups underwent microCT and dark field imaging on day 0, 2 and 4 for terms and on day 0, 3, 5 and 7 for preterms. RESULTS: Preterm pups assessed at birth depicted a more rudimentary lung structure (larger alveoli and thicker septations) and a lower expression of surfactant proteins in comparison to term pups. MicroCT and dark field imaging revealed delayed lung aeration in preterm pups, in comparison to term pups. Preterm birth led to smaller pups, with smaller lungs with a lower alveolar surface area on day 7/day 4. Furthermore, preterm birth affected lung function with increased tissue damping, tissue elastance and resistance and decreased dynamic compliance. Expression of vascular endothelial growth factor (VEGFA) was significantly decreased in preterm pups, however in the absence of structural vascular differences. CONCLUSIONS: Preterm birth affects lung structure and function at birth, but also has persistent effects on the developing lung. This supports the use of a preterm animal model, such as the preterm rabbit, for preclinical research on BPD. Future research that focuses on the identification of pathways that are involved in in-utero lung development and disrupted by pre-term birth, could lead to novel therapeutic strategies for BPD.

Staphylococcus aureus endocarditis: distinct mechanisms of bacterial adhesion to damaged and inflamed heart valves.

AIMS: The pathogenesis of endocarditis is not well understood resulting in unsuccessful attempts at prevention. Clinical observations suggest that Staphylococcus aureus infects either damaged or inflamed heart valves. Using a newly developed endocarditis mouse model, we therefore studied the initial adhesion of S. aureus in both risk states. METHODS AND RESULTS: Using 3D confocal microscopy, we examined the adhesion of fluorescent S. aureus to murine aortic valves. To mimic different risk states we either damaged the valves with a surgically placed catheter or simulated valve inflammation by local endothelium activation. We used von Willebrand factor (VWF) gene-deficient mice, induced platelet and fibrinogen depletion and used several S. aureus mutant strains to investigate the contribution of both host and bacterial factors in early bacterial adhesion. Both cardiac valve damage and inflammation predisposed to endocarditis, but by distinct mechanisms. Following valve damage, S. aureus adhered directly to VWF and fibrin, deposited on the damaged valve. This was mediated by Sortase A-dependent adhesins such as VWF-binding protein and Clumping factor A. Platelets did not contribute. In contrast, upon cardiac valve inflammation, widespread endothelial activation led to endothelial cell-bound VWF release. This recruited large amounts of platelets, capturing S. aureus to the valve surface. Here, neither fibrinogen, nor Sortase A were essential. CONCLUSION: Cardiac valve damage and inflammation predispose to S. aureus endocarditis via distinct mechanisms. These findings may have important implications for the development of new preventive strategies, as some interventions might be effective in one risk state, but not in the other.

A Label-free Multicolor Optical Surface Tomography (ALMOST) imaging method for nontransparent 3D samples.

BACKGROUND: Current mesoscale 3D imaging techniques are limited to transparent or cleared samples or require the use of X-rays. This is a severe limitation for many research areas, as the 3D color surface morphology of opaque samples-for example, intact adult Drosophila, Xenopus embryos, and other non-transparent samples-cannot be assessed. We have developed "ALMOST," a novel optical method for 3D surface imaging of reflective opaque objects utilizing an optical projection tomography device in combination with oblique illumination and optical filters. RESULTS: As well as demonstrating image formation, we provide background information and explain the reconstruction-and consequent rendering-using a standard filtered back projection algorithm and 3D software. We expanded our approach to fluorescence and multi-channel spectral imaging, validating our results with micro-computed tomography. Different biological and inorganic test samples were used to highlight the versatility of our approach. To further demonstrate the applicability of ALMOST, we explored the muscle-induced form change of the Drosophila larva, imaged adult Drosophila, dynamically visualized the closure of neural folds during neurulation of live Xenopus embryos, and showed the complementarity of our approach by comparison with transmitted light and fluorescence OPT imaging of a Xenopus tadpole. CONCLUSION: Thus, our new modality for spectral/color, macro/mesoscopic 3D imaging can be applied to a variety of model organisms and enables the longitudinal surface dynamics during development to be revealed.

Local pulmonary drug delivery in the preterm rabbit: feasibility and efficacy of daily intratracheal injections.

Recent clinical trials in newborns have successfully used surfactant as a drug carrier for an active compound, to minimize systemic exposure. To investigate the translational potential of surfactant-compound mixtures and other local therapeutics, a relevant animal model is required in which intratracheal administration for maximal local deposition is technically possible and well tolerated. Preterm rabbit pups (born at 28 days of gestation) were exposed to either hyperoxia or normoxia and randomized to receive daily intratracheal surfactant, daily intratracheal saline, or no injections for 7 days. At day 7, the overall lung function and morphology were assessed. Efficacy in terms of distribution was assessed by micro-PET-CT on both day 0 and day 7. Lung function as well as parenchymal and vascular structure were altered by hyperoxia, thereby reproducing a phenotype reminiscent of bronchopulmonary dysplasia (BPD). Neither intratracheal surfactant nor saline affected the survival or the hyperoxia-induced BPD phenotype of the pups. Using PET-CT, we demonstrate that 82.5% of the injected radioactive tracer goes and remains in the lungs, with a decrease of only 4% after 150 min. Surfactant and saline can safely and effectively be administered in spontaneously breathing preterm rabbits. The described model and method enable researchers to evaluate intratracheal pharmacological interventions for the treatment of BPD.

Monitoring of Fluconazole and Caspofungin Activity against In Vivo Candida glabrata Biofilms by Bioluminescence Imaging.

Candida glabrata can attach to various medical implants and forms thick biofilms despite its inability to switch from yeast to hyphae. The current in vivoC. glabrata biofilm models only provide limited information about colonization and infection and usually require animal sacrifice. To gain real-time information from individual BALB/c mice, we developed a noninvasive imaging technique to visualize C. glabrata biofilms in catheter fragments that were subcutaneously implanted on the back of mice. Bioluminescent C. glabrata reporter strains (lucOPT 7/2/4 and lucOPT 8/1/4), free of auxotrophic markers, expressing a codon-optimized firefly luciferase were generated. A murine subcutaneous model was used to follow real-time in vivo biofilm formation in the presence and absence of fluconazole and caspofungin. The fungal load in biofilms was quantified by CFU counts and by bioluminescence imaging (BLI). C. glabrata biofilms formed within the first 24 h, as documented by the increased number of device-associated cells and elevated bioluminescent signal compared with adhesion at the time of implant. The in vivo model allowed monitoring of the antibiofilm activity of caspofungin against C. glabrata biofilms through bioluminescent imaging from day four after the initiation of treatment. Contrarily, signals emitted from biofilms implanted in fluconazole-treated mice were similar to the light emitted from control-treated mice. This study gives insights into the real-time development of C. glabrata biofilms under in vivo conditions. BLI proved to be a dynamic, noninvasive, and sensitive tool to monitor continuous biofilm formation and activity of antifungal agents against C. glabrata biofilms formed on abiotic surfaces in vivo.

A Multimodal Imaging Approach Enables In Vivo Assessment of Antifungal Treatment in a Mouse Model of Invasive Pulmonary Aspergillosis.

Aspergillus fumigatus causes life-threatening lung infections in immunocompromised patients. Mouse models are extensively used in research to assess the in vivo efficacies of antifungals. In recent years, there has been an increasing interest in the use of noninvasive imaging techniques to evaluate experimental infections. However, single imaging modalities have limitations concerning the type of information they can provide. In this study, magnetic resonance imaging and bioluminescence imaging were combined to obtain longitudinal information on the extent of developing lesions and fungal load in a leukopenic mouse model of invasive pulmonary aspergillosis (IPA). This multimodal imaging approach was used to assess changes occurring within lungs of infected mice receiving voriconazole treatment starting at different time points after infection. The results showed that IPA development depends on the inoculum size used to infect animals and that disease can be successfully prevented or treated by initiating intervention during early stages of infection. Furthermore, we demonstrated that a reduction in fungal load is not necessarily associated with the disappearance of lesions on anatomical lung images, especially when antifungal treatment coincides with immune recovery. In conclusion, multimodal imaging allows an investigation of different aspects of disease progression or recovery by providing complementary information on dynamic processes, which are highly useful for assessing the efficacy of (novel) therapeutic compounds in a time- and labor-efficient manner.

Irritant-induced asthma to hypochlorite in mice due to impairment of the airway barrier.

Inhalation of commonly present irritants, such as chlorine and chlorine derivatives, can cause adverse respiratory effects, including irritant-induced asthma (IIA). We hypothesize that due to airway barrier impairment, exposure to hypochlorite (ClO-) can result in airway hypersensitivity. C57Bl/6 mice received an intra-peritoneal (i.p.) injection of the airway damaging agent naphthalene (NA, 200 mg/kg body weight) or vehicle (mineral oil, MO). In vivo micro-computed tomography (CT) images of the lungs were acquired before and at regular time points after the i.p. TREATMENT: After a recovery period of 14 days an intranasal (i.n.) challenge with 0.003% active chlorine (in ClO-) or vehicle (distilled water, H2O) was given, followed by assessment of the breathing frequency. One day later, pulmonary function, along with pulmonary inflammation was determined. Lung permeability was assessed by means of total broncho-alveolar lavage (BAL) protein content and plasma surfactant protein (SP)-D levels. In vivo micro-CT imaging revealed enlargement of the lungs and airways early after NA treatment, with a return to normal at day 14. When challenged i.n. with ClO-, NA-pretreated mice immediately responded with a sensory irritant response. Twenty-four hours later, NA/ClO- mice showed airway hyperreactivity (AHR), accompanied by a neutrophilic and eosinophilic inflammation. NA administration followed by ClO- induced airway barrier impairment, as shown by increased BAL protein and plasma SP-D concentrations; histology revealed epithelial denudation. These data prove that NA-induced lung impairment renders the lungs of mice more sensitive to an airway challenge with ClO-, confirming the hypothesis that incomplete barrier repair, followed by irritant exposure results in airway hypersensitivity.

Longitudinal microcomputed tomography-derived biomarkers for lung metastasis detection in a syngeneic mouse model: added value to bioluminescence imaging.

With more patients dying from metastasis than from primary cancers, metastasis is a very important area in cancer research. Investigators thereby heavily rely on animal models of metastasis to common organs such as the lung to improve our insight into the pathogenesis and to research novel therapeutic approaches to combat metastasis. In this experimental context, novel tools that allow longitudinal monitoring of lung metastasis in individual animals are highly needed. We have therefore evaluated for the first time microcomputed tomography (µCT) as a very efficient and crossvalidated means to noninvasively and repeatedly monitor metastasis to the lung in individual, free-breathing syngeneic mice. Two individual clones of KLN205 cancer cells were intravenously injected in syngeneic DBA/2 mice and lung metastasis was monitored weekly during 3 weeks using µCT, and was compared with the current gold standard histology and bioluminescence imaging (BLI). µCT enabled us to visualize diffuse tumor morphology and also to extract four different biomarkers that quantify not only tumor load but also aerated space in the lung as a marker of vital lung capacity and potential compensatory mechanisms. Complementary to BLI, applying this novel µCT-based approach enabled us to unravel sensitively and efficiently differences in metastatic potential between two cellular clones. In conclusion, µCT and BLI offer biomarkers that describe different and complementary aspects of lung metastasis, underlining the importance of multimodality follow-up. The added value of µCT findings is important to better assess lung metastasis and host/lung response in preclinical studies, which will be valuable for translational applications.

Longitudinal, in vivo assessment of invasive pulmonary aspergillosis in mice by computed tomography and magnetic resonance imaging.

Invasive aspergillosis is an emerging threat to public health due to the increasing use of immune suppressive drugs and the emergence of resistance against antifungal drugs. To deal with this threat, research on experimental disease models provides insight into the pathogenesis of infections caused by susceptible and resistant Aspergillus strains and by assessing their response to antifungal drugs. However, standard techniques used to evaluate infection in a preclinical setting are severely limited by their invasive character, thereby precluding evaluation of disease extent and therapy effects in the same animal. To enable non-invasive, longitudinal monitoring of invasive pulmonary aspergillosis in mice, we optimized computed tomography (CT) and magnetic resonance imaging (MRI) techniques for daily follow-up of neutropenic BALB/c mice intranasally infected with A. fumigatus spores. Based on the images, lung parameters (signal intensity, lung tissue volume and total lung volume) were quantified to obtain objective information on disease onset, progression and extent for each animal individually. Fungal lung lesions present in infected animals were successfully visualized and quantified by both CT and MRI. By using an advanced MR pulse sequence with ultrashort echo times, pathological changes within the infected lung became visually and quantitatively detectable at earlier disease stages, thereby providing valuable information on disease onset and progression with high sensitivity. In conclusion, these non-invasive imaging techniques prove to be valuable tools for the longitudinal evaluation of dynamic disease-related changes and differences in disease severity in individual animals that might be readily applied for rapid and cost-efficient drug screening in preclinical models in vivo.