Elsa Bodier-Montagutelli

Alexie Mayor

Laurent Vecellio

Renaud Respaud

Nathalie Heuzé-Vourc'h

Designing inhaled protein therapeutics for topical lung delivery: what are the next steps?

Respiratory diseases (RDs) constitute a major public health issue worldwide, and some RDs represent a significant unmet medical need. Inhaled protein therapeutics are now attracting increasing interest for the topical treatment of RDs, with one drug on the market and almost 20 others in clinical development.

Here, we review advances in inhaled protein therapeutics and comment on the development strategies that can be considered. To date, the transformation of a therapeutic protein into an inhalable drug has often been performed on a case-by-case basis by taking account of specific protein-, inhaler-, and patient-related factors. A rational design process for both the drug formulation and the delivery device is crucial for maintaining the protein's integrity and optimizing deposition in the lung, which in turn are decisive for clinically successful aerosol therapy. Despite significant advances in this field, there are still a number of outstanding questions about the stability of nebulized proteins and the consequences of nebulization on the biological activity, safety and pharmacokinetics of these innovative therapeutics. A better understanding of these parameters should promote the implementation of more rational development strategies, and thus accelerate progression towards clinical applications.

Advances in inhaled protein therapeutics

Respiratory diseases (RDs) constitute a major public health issue worldwide. Indeed, there are four RDs (pneumonia, tuberculosis, lung cancer, and chronic obstructive pulmonary disease (COPD)) among the ten most common causes of death. Additionally, RDs represent a major socio-economic burden through disability, healthcare costs, and loss of productivity.

In view of their advantageous pharmacological features, protein therapeutics (including antibodies) have ushered in a new era in respiratory medicine; they provide significant clinical benefit and are becoming increasingly important tools in the therapeutic armamentarium [START_REF] Sécher | Therapeutic antibodies: A new era in the treatment of respiratory diseases?[END_REF]. Given the limited diffusion of protein therapeutics from the bloodstream to the lungs (partly due to their high molecular weight), pulmonary delivery of aerosolized proteins seems an obvious choice -provided that the therapeutic target operates in the lungs, of course [START_REF] Dall'acqua | Properties of Human IgG1s engineered for enhanced binding to the neonatal Fc Receptor (FcRn)[END_REF]. Approval of the first inhaled protein (the enzyme dornase alpha, marketed as Pulmozyme®) in 1993 reshaped the management of patients with cystic fibrosis; when combined with standard medications, the protein significantly improved lung function. After two decades, a number of promising new inhaled protein therapeutics with topical lung delivery are finally reaching the clinicespecially since 2010 (Table 1). To the best of our knowledge, 18 inhaled protein therapeutics have entered clinical trials and are being tested in either major RDs (asthma and pneumonia) or in rare/orphan lung diseases (e.g. cystic fibrosis, pulmonary alveolar proteinosis, and alpha-1 antitrypsin deficiency). It is noteworthy that two fatal RDs are missing from the list: lung cancer and COPD. Firstly, lung cancer is often detected late (i.e. with large, metastatic tumours), and inhaled protein therapeutics may not pass well into both the tumour tissue and the systemic circulation. However, in situ lung carcinoma with lepidic growth may be a relevant target for inhalation therapy [START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system[END_REF]. Secondly, COPD is a complex disease with poorly elucidated underlying mechanisms and thus a lack of clear drug targets. To date, most protein therapeutics have countered infection-triggered acute exacerbations of COPD, which are correlated with disease progression and a decline in lung function. It is noteworthy that Synairgen has announced a Phase II trial of inhaled interferon beta (SNG001) to boost antiviral defences in patients with COPD.

Strategies for designing inhaled protein therapeutics

Although proteins can be formulated as dry powders for aerosolization, 75% of the inhaled protein therapeutics in clinical development have been developed as liquids for nebulization (Table 1).

Accordingly, this editorial will mainly comment on advances in protein nebulization for topical lung delivery. Indeed, protein nebulization is often the first step in the formulation process because it offers several advantages over dry powders: it avoids drying steps, it is suitable for all clinical situations, and it enables greater pulmonary deposition than dry powder inhalers. Nebulizers have been significantly improved in recent years, and can deliver a 70% lung dose vs a maximum of 30% with current dry powder inhalers or the 50% expected with next-generation devices [START_REF] Fink | Good Things in Small Packages: an Innovative Delivery Approach for Inhaled Insulin[END_REF][START_REF] Yang | Pulmonary drug delivery by powder aerosols[END_REF][START_REF] De Boer | Dry powder inhalation: past, present and future[END_REF]. Indeed, dornase alpha is administered daily (2.5 mg per session) by nebulization. Nevertheless, nebulization exposes proteins to stressful conditions by generating a huge air-liquid interface (ALI) and, in some cases, high temperatures and/or shear forces. These conditions may cause protein unfolding, aggregation, oxidation, deamidation or glycation, which may lead to changes in biological activity and safety concerns. In this prospect, nebulizers and formulations constitute two relevant tools to optimize protein stability during nebulization.

Many marketed or investigational technologies have given encouraging preclinical or clinical results

when used to aerosolize protein therapeutics (Table 2). A number of trends can be identified: as with other new inhaled therapeutics, vibrating-mesh nebulizers are now frequently used for therapeutic protein delivery in humans (86% of the cases in which the nebulizer technology is disclosed) [START_REF] Pritchard | Mesh nebulizers have become the first choice for new nebulized pharmaceutical drug developments[END_REF]. Indeed, vibrating-mesh nebulizers allow the delivery of high drug amounts (often required for protein therapeutics) and are less harsh with regard to chemical and physical constraints [START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF]. For example, the ALX-0171 Nanobody™ aerosols displayed satisfactory aerodynamic properties and biologic potency when produced by vibrating-mesh nebulization, with no protein aggregation [START_REF] Van Heeke | Nanobodies as inhaled biotherapeutics for lung diseases[END_REF].

Formulation is also an important parameter in protein nebulization; it modulates the aerosol's aerodynamic profiles but also offers a unique opportunity to stabilize the protein. Since the mechanisms underlying protein degradation during nebulization are not always well understood, formulation design often relies on an empirical screening of excipients already known to stabilize proteins and/or improve aerosols. To prevent aggregation (occurring mainly at the ALI), surfactants (e.g. polysorbates) have been successfully included in formulations of monoclonal antibodies and aviscumine [START_REF] Hertel | Protein stability in pulmonary drug delivery via nebulization[END_REF][START_REF] Respaud | Effect of formulation on the stability and aerosol performance of a nebulized antibody[END_REF].

Polyethylene glycols have also been used to stabilize proteins by generating steric repulsion between monomers [START_REF] Hertel | Protein stability in pulmonary drug delivery via nebulization[END_REF]. Likewise, protein therapeutics can be stabilized by other excipients (buffers, sugars, polyols and amino acids, Table 1). However, due to intellectual property concerns, formulation details are rarely disclosed for inhaled proteins in clinical development (i.e. for only six proteins in Table 1). This clearly limits the extrapolation of general principles to the nebulization of protein therapeutics.

Formulation may also make it possible to enhance a protein's pharmacological properties. For instance, modified-release formulations (such as microparticles, nanoparticles or liposomes) might customize pharmacokinetic profiles while avoiding rapid protein degradation [START_REF] Loira-Pastoriza | Delivery strategies for sustained drug release in the lungs[END_REF]. Nevertheless, few FDA-approved excipients for inhalation (i.e. endogenous lung compounds or inactive formulation ingredients) are currently available. Thus, progress in formulation advances may require the safety of new excipients to be assessed.

Expert opinion

Nebulization is now a leading approach for administering proteins to the airways, as evidenced by the number of ongoing clinical trials in this field. Today's success stories highlight the advantages and potential of nebulization for topical lung delivery -as long as the inhalation system ensures protein stability and effective drug deposition within the respiratory tract. However, a number of molecule-and/or administration-route-related challenges remain (Figure 1).

At present, there are several relevant device and formulation options for effective, safe protein aerosolization. The significant interplay between these two parameters has prompted the development of unique drug-device combinations tailored to the protein of interest (in which the device and the formulation are engineered in tandem). Indeed, four of the inhaled proteins in clinical trials are delivered with a customized nebulizer (Table 1). However, redesigning the device and the formulation might not be feasible or relevant for every new protein. If rapid development is required or if the protein is only moderately sensitive to aerosolization stresses, it may be more straightforward and affordable to adjust the formulation to a marketed device.

One of the particularities of the inhaled route relates to the mechanisms of protein degradation, which are still partially elucidated. Although all proteins will be exposed to a large ALI (the magnitude of which depends on the droplet size), the level of applied stress probably differs from one device to another. For example, aerosolization with two different mesh nebulizers generated distinct aggregate populations with the same protein [START_REF] Respaud | Effect of formulation on the stability and aerosol performance of a nebulized antibody[END_REF]. Hence, aerosol development approaches might need to be reconsidered for each new protein. Gaining insight into the physical/chemical mechanisms contributing to protein degradation will be crucial for protecting the active compound and making formulation design more rational and time-efficient.

It is still not clear how to assess protein stability. Although aggregation and biological activity are common read-outs, other protein alterations (e.g. oxidation or deamidation) are less frequently studied -despite their obvious links to protein activity and immunogenicity [START_REF] Filipe | Immunogenicity of different stressed IgG monoclonal antibody formulations in immune tolerant transgenic mice[END_REF]. In the literature, a range of analytical techniques are employed to assess protein stability and aggregation, with various levels of performance. This complicates inter-study comparisons and the ability to highlight key parameters in protein stability. In addition, most of today's analytical methods are not applicable to aerosol droplets.

Hence, droplets must be pooled into a bulk liquid before analysis, which might modify protein stability and thus induce pre-analytical bias. Hence, there is a need for in-depth, relevant assessments of the physicochemical and biological aspects of protein stability following aerosolization.

Given the specific characteristics of the inhalation route, dedicated studies of aerosol-specific stresses, subsequent protein modifications, and their impacts on efficacy, pharmacokinetics and safety are required. For example, immunogenicity appears to vary markedly among the four proteins studied to date, and is difficult to predict (Table 1). As evidenced by several in vivo studies, the protein aggregates formed in response to various stresses may raise immunogenicity, and prompt the production of potentially neutralizing anti-drug antibodies. This issue has not yet been well addressed for aerosolization stress and airway delivery.

The use of animal models to analyze the efficacy, pharmacokinetics and safety of inhaled drugs has been informative but remains cumbersome and ethically problematic. Indeed, it is difficult to extrapolate animal data on inhaled protein therapeutics to humans because of physiological, anatomical, biological and respiratory differences between species; the use of dissimilar aerosol devices induces distinct stresses on protein therapeutics and unequal drug deposition within the respiratory tract [START_REF] Guillon | Insights on animal models to investigate inhalation therapy: Relevance for biotherapeutics[END_REF]. The recent implementation of promising in vitro models (such as multilayer cell-based models in an aerosol chamber) may simplify inhaled drug development by integrating advanced functional studies in the early formulation steps [START_REF] Röhm | A comprehensive screening platform for aerosolizable protein formulations for intranasal and pulmonary drug delivery[END_REF]. Aerosol delivery in complex organ-mimicking systems (such as "lungs-on-a-chip") might also generate other options [START_REF] Doryab | Advances in pulmonary therapy and drug development: Lung tissue engineering to lung-on-a-chip[END_REF].

In conclusion, the development of proteins for nebulization is eminently possible, in view of the current state of scientific knowledge and recent successes in this field. However, addressing unresolved questions about protein stability (Figure 1) might help to standardize the development processes, facilitate the elaboration of dedicated regulatory guidelines, and accelerate progression towards clinical applications. Some of these concerns are also relevant for the development of dry protein aerosols; as already observed for anti-infectives, the use of these dry inhaled formulations is expected to increase over the coming years and decades.

investigational devices with at one feasibility study reported for proteins Aradigm AERx® (slow-velocity mist)

	Protein therapeutic Name(s) Molecular characteristics Alteplase Tissue plasminogen activator (tPA) (70 kDa) Inhaler technology Precise, Companies / Sponsors University of Michigan, Genentech Peking Union Medical College Hospital reproducible dosing Dornase alpha Deoxyribonuclease I (37 kDa) Roche ALX-0171 / VR465 Trivalent Nanobody™ (42 kDa) targets RSV F-protein Ablynx ALX-009: lactoferrin (+ hypothiocyanite) Antibacterial glycoprotein (80 kDa) Alaxia Pharma Dai Huaping Marketed nebulizers Molgramostim Recombinant human GM-CSF (14 kDa) University of Giessen Savara Pharmaceuticals Interferon beta-1a Synairgen Jet nebulizers SNG001 (22 kDa) Synairgen, AstraZeneca AZD1402 / PRS-060 Anticalin protein (~18 kDa) Target: IL-4Rα Pieris Pharmaceuticals, Ultrasonic nebulizers AstraZeneca Ansun Biopharma Mesh nebulizers VR942 / UCB4144 / CDP7766 Anti-IL-13 mAb fragment UCB Pharma Alpha-1 antitrypsin (Kamada-API) Lung serine protease inhibitor (52 kDa) Kamada Milton S. Hershey Medical Center Children's Hospital Medical Center, (Cincinnati), DAS181 (Fludase®) Recombinant neuraminidase (46 kDa) National Institute of Allergy and Infectious Diseases (NIAID) Soft mist inhalers Emerging/Aerosolization profile affected by the Clinical development Inhalation system Immunoge nicity (ADAs) Stage Therapeutic Dosing regimen Formulation Inhaler Phase II ongoing Plastic bronchitis 5 mg every 6h x 3 d Exc (Activase®): arginine, phosphoric acid, polysorbate 80 Nebulizer N/A Phase II (status unknown) aPAP 150 µg bid* N/A Nebulizer N/A Compatibility with high doses Minimal drug loss, and high Maintenance of Limitations/remarks Reference Reference / Clinical trial # (NCT)* / Patent # (EP/WO) NCT02315898 NCT02243228 protein integrity lung deposition indication Marketed Marketed Cystic fibrosis 2.5 mg/d C =1 mg/mL Exc: sodium chloride, calcium chloride dehydrate (pH 6.3) Jet or mesh nebulizers ADAs in <5% of patients (n=643) Pulmozyme® SmPC [17] Under clinical evaluation Phase IIb ongoing RSV infection 3 different doses Once daily x 3 d C = 50 mg/mL Exc: sodium chloride, phosphate buffer (among others) Mesh nebulizer (customized Vectura FOX-Flamingo® system) ADAs in 23% of patients NCT02979431 [9,18] Phase I ongoing Cystic fibrosis Single / multiple C = 4 mg/mL Nebulizer N/A NCT02598999 Phase II ongoing aPAP 150 µg bid* N/A N/A N/A NCT03316651 administrations Phase II ongoing ARDS 150-450 µg/d x 3 d N/A N/A N/A NCT02595060 Phase II / Phase III ongoing APAP 300 µg/d x 24 w continuously or intermittently N/A Mesh nebulizer (investigational PARI eFlow®) N/A NCT02702180 NCT03482752 Phase IIa ongoing NTM infections 300 µg/d x 24 w N/A Phase II completed (2012) Asthma 6 mIU/d x 14 d Neutral pH, no mannitol, no human serum albumin Mesh nebulizer (Philips I-neb®) NCT01126177 N/A [22] Phase II completed (2016) Asthma 24 μg /d x 14 d C = 48 µg/mL Mesh nebulizer N/A NCT02491684 Device performance affected by the (Philips I-neb®) Phase I ongoing Asthma Single dose N/A Nebulizer N/A drug solution's physicochemical properties (viscosity, surface tension, NCT03384290 [23] etc.) [8,10] Mesh nebulizer (investigational PARI eFlow®) N/A NCT03421743 Phase II Influenza virus infection 10 mg/d x 3 d N/A (named F02) DPI NCT01037205 N/A EP2841092A1 (n=30) Phase I completed (2016) Asthma 0.5-20 mg, single-or repeated dose (10 d) N/A DPI No clinical evidence of immunogeni city NCT02473939 [19] Phase II/III (Eur) completed (2015) Alpha-1 antitrypsin deficiency N/A C = 20 mg/mL Mesh nebulizer: customized PARI eFlow® N/A NCT01217671 Phase I ongoing Paediatric ARDS 25-250 µg/m² x 7 d Exc (Leukine®): mannitol, sucrose, tromethamine ± benzylalcohol Nebulizer N/A NCT02601365 Leukine® PI Phase II completed (2014) aPAP 250 µg/w* Exc (Leukine®): mannitol, sucrose, tromethamine ± benzylalcohol Nebulizer N/A NCT01511068 Leukine® PI Phase I completed (2011) Influenza virus infection 20-30 mg single dose or 20 mg/d x 10 d C = 70% (w/w) Exc: histidine, trehalose, citric acid, acetate buffer (named F03) NCT01173224 Relatively unsuitable with dispersed systems DPI [24] IgA or IgG in 88% of patients (n=18) Phase I completed (2012) Influenza virus infection 20 mg/d x 3d C = 65% (w/w) Exc: histidine, trehalose, MgSO4, citric acid, acetate DPI NCT01651494 [28] drug solution's properties [24] [20] Phase II (US) completed (2015) Alpha-1 antitrypsin deficiency 80-160 mg/d N/A Mesh nebulizer: customized PARI eFlow® N/A NCT02001688 Alpha-1 antitrypsin (Alpha-1 HC) Lung serine protease inhibitor (52 kDa) Grifols Therapeutics Phase II completed (2013) Cystic fibrosis 100-200 mg/d x 21d C = 50 mg/mL Exc: PBS, polysorbate Nebulizer N/A NCT01684410 Lyophilizate, reconstitution Sargramostim (Leukine®) Recombinant human GM-CSF (14 kDa) Genzyme Children's Hospital Medical Center (Cincinnati) Phase I ongoing aPAP 125-250 µg, single dose Exc (Leukine®): mannitol, sucrose, tromethamine ± benzylalcohol N/A N/A NCT03006146 Leukine® PI IRCCS Policlinico S. Matteo Phase II/III (status unknown) aPAP 250 µg/d* Exc (Leukine®): mannitol, sucrose, tromethamine ± benzylalcohol N/A N/A Leukine® PI Phase II completed (2016) virus infection 4.5 mg/d x 10 d F02 (undefined) DPI N/A NCT01644877 Parainfluenza NCT00901511 buffer (named F04) DAS181 (Paradase®) Recombinant neuraminidase (46 kDa) Ansun BioPharma Phase II completed (2017) Parainfluenza virus infection DPI: 10 mg/d x 7 d 4.5 mg/d x 7 d dose = 28.9 mg) saline (maximum total Nebulization: F02 dissolved in 10 mL normal DPI (Teva Cyclohaler®) Nebulizer [25] N/A NCT01924793 Nebulizer: 3.2-DPI: F02 (undefined) Surface acoustic wave (SAW) devices Not available Lack of in vivo data [28,29]
	GSK2862277 AIR DNase™ / Alidornase alfa / PRX-110 GM-CSF CSJ117	Anti-TNF-R1 mAb VH domain (13 kDa) Actin-inhibition-resistant deoxyribonuclease I (37 kDa) Recombinant human GM-CSF (14 kDa) Anti-TSLP mAb fragment (46 kDa)	GSK BioTherapeutics Medical & Dental Hospital Niigata University Protalix Barbara Ann Karmanos Cancer Institute Children's Oncology Group Novartis	Phase II terminated (2018) Phase II ongoing Cystic fibrosis Acute lung injury completed (2017) aPAP Phase II Phase I competed (2016) Cystic fibrosis Phase II completed (2010) Lung metastases / cancer Phase II spreading completed (2013) Osteosarcoma with lung Phase I ongoing Asthma	26 mg, single dose 2.5 mg/d x 28 d 125 µg bid* 1.25 -5 mg/d x 5 d 250 µg bid* 240-1750 µg bid* 3 different doses 1 dose/d x 12 w	before nebulization Exc (Phase I trial): sucrose, glycine, sodium dihydrogen phosphate, polysorbate 80 N/A N/A N/A Exc (Leukine®): mannitol, sucrose, tromethamine ± benzylalcohol Exc: from among leucine, ± benzylalcohol Exc (Leukine®): mannitol, sucrose, tromethamine glycine trileucine, HCl, trehalose, mannitol. Buffers: from among histidine and	Mesh nebulizer (PARI eFlow® with s30 mesh) Mesh nebulizer (Philips I-neb AAD®) N/A N/A N/A N/A N/A	N/A N/A N/A N/A N/A N/A N/A	NCT02221037 [21] NCT02722122 NCT02835742 NCT02605590 NCT00514215 Leukine® PI Leukine® PI Arndt 2010 NCT00066365 NCT03138811 WO2017042701A 1
									6

Trends are based on our best knowledge of literature reports.

NB: Staccato™ technology was not included in this table, given its incompatibility with peptides and proteins.

Table 2 .

 2 Desirable characteristics for protein inhalers, and trends in nebulization devices. The development of nebulized protein formulations: several key questions have yet to be addressed.

	Poor	Moderate Excellent


Pierre Couprie

Designing Sound Representation for Musicology

Introduction

There are many forms of representation of the sound signal: from the waveform to higher-level representations such as FFT visualization, audio descriptors or the chromagram. Some of them are commonly used by musicians or musicologists, others are used more rarely. Using a sound representation in musicology does not only mean using technology coming from acoustics and computer science, but also means using a digital method of musical analysis.

Challenges

The transfer of methods from exact sciences to musicology poses serious challenges:

• Musicologists are generally limited to using tools developed for other sciences that are basically inadequate when applied to their scientific methods.

• The production of software is often limited to a few Python scripts that are difficult to use for a musicologist.

• The application of these techniques is therefore not to be found in a simple transfer between the exact sciences and musicology but in the development of new musicological methods. For the last fifteen years, we have developed the software iAnalyse which allows the musicologist to use complex technologies with representations adapted to their field of research.

Augmented Waveform

The representation of the sound amplitude or the waveform facilitates identification of the temporal relationship between sound characteristics such as attacks, musical intensity, morphologies or occurrence of silences.

• Two channels and their deviation.

• Two channels on different layers, it facilitates the visualization of their differences.

• Augmented waveform based on BStD (Malt & Jourdan 2015): Spectral centroid (y-axis), RMS amplitude (height) and the zero-crossing rate (colors)

Multilayered Chromagram with

Amplitude Envelope

The goal is to study the behavior of OMax software (played by Benjamin Levy) with the sheng improvisations (played by Li Lin Chin). We recorded the audio in multitrack and then developed representations to visualize interactions between the musician and the OMax software. The interaction between the musician (in red) and the software (the other colors) is clearly visible whereas the spectral texture is somewhat complex: response of the musician on some morphologies, morphological differentiation of pitches, improvisation strategies between the musician and each iteration of the software, software fragmentation of sounds.

5 Self-Similarity Matrix Interpretation-

Assistance System

Self-similarity matrices (computed from the FFT data or from a set of audio descriptors) are particularly relevant in the analysis of musical form or microstructures. They also facilitate the exploration or navigation of long audio files. However, interpretation is difficult for the musicologist, which is why we have developed an interpretation-assistance system based on Foote and Cooper segmentation and similarity visualization.

•

  Main similarity. • SSM + Novelty. • Similarity values at the green playhead position. • Result of novelty segmentation (Foote & Cooper 2003). • Manual segmentation from CD tracks. Download iAnalyse for macOS 10.13.6+


Antoine Guillon

Jeoffrey Pardessus

Pierre Lhommet

Christelle Parent

Renaud Respaud

Denis Marchand

Jerome Montharu

Michele De Monte

Philip Janiak

Christophe Boixel

Jérôme Montharu

Michèle De Monte

Héloïse Audat

Sylvain Huille

Etienne Guillot

Nathalie Heuze-Vourc'h
email: nathalie.vourch@med.univ-tours.fr

Dr Heuzé-Vourc'h Nathalie

Exploring the fate of inhaled monoclonal antibody in the lung parenchyma by microdialysis

Keywords: Monoclonal antibodies, Pulmonary delivery, Microdialysis, Pharmacokinetics

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Since the market release of the first therapeutic antibody (Ab) in 1986, Ab-based therapeutics have experienced a remarkable expansion and demonstrated therapeutic success in various diseases. The overall clinical pipeline was estimated to include 470 Abs in 2016, [START_REF] Reichert | Antibodies to watch in 2016[END_REF] mostly full-length IgGs (Mw ≈ 150 kDa). Respiratory diseases continue to represent major unmet medical needs and are a growing area of use for therapeutic Abs, with >40 molecules (excluding those used in lung cancer) studied in clinical trials. While Abs for cancer treatment are mainly delivered through the intravenous (i.v.) route, other routes may be beneficial for respiratory diseases because Abs do not passively diffuse through the different compartments of the body. Among these other routes, inhalation is a promising non-invasive method of delivery for Abs to treat respiratory diseases, as it has allowed for therapeutic responses in various models and leads to high Ab concentration in the lungs, while limiting Ab passage into the systemic circulation. [START_REF] Hervé | VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations[END_REF][START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system[END_REF][START_REF] Respaud | Development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin[END_REF][START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF] Prior to clinical trials, preclinical studies are mandatory to explore the pharmacodynamics, pharmacokinetics (PK) and toxicity profiles of Abs. More precisely, PK studies characterize the distribution and local concentrations of compounds and thereby guide toxicity studies; both study types generate critical data used to select the accurate doses and regimens for clinical trials. Classically, PK parameters are estimated by monitoring drug concentrations in the systemic circulation, from which the behavior of the drug at the target site is predicted after computation by mathematical compartmental models. However, this indirect estimation of tissue concentrations requires knowledge of intrinsic and extrinsic factors influencing Ab behavior, which should be previously determined experimentally. [START_REF] Kamath | Translational pharmacokinetics and pharmacodynamics of monoclonal antibodies[END_REF][START_REF] Liu | Pharmacokinetics of monoclonal antibodies and Fc-fusion proteins[END_REF][START_REF] Shah | Towards a platform PBPK model to characterize the plasma and tissue disposition of monoclonal antibodies in preclinical species and human[END_REF][START_REF] Wang | Projecting human pharmacokinetics of monoclonal antibodies from nonclinical data: comparative evaluation of prediction approaches in early drug development[END_REF][START_REF] Ferl | Physiologically based pharmacokinetic models of small molecules and therapeutic antibodies: a mini-review on fundamental concepts and applications[END_REF] Additionally, scaling across animal species is questionable for Abs because differences in antigen-Ab and FcRn-Ab binding exist, and may drive significant differences in Ab distribution and clearance. Simplified allometric scaling across species, in particular from monkey data, is only predictive (in humans) when the Ab kinetic profile is linear. In other cases, interpretations and translation of the PK results remain speculative. In the case of inhalation as a delivery route, the limited understanding of the processes and molecular drivers involved in absorption, as well as considerable species differences, makes interpreting PK results even more challenging.

To follow the behavior of inhaled Abs in relevant tissues, i.e., the lungs, few methods are available, and each has specific strength and weakness. Iterative tissue sampling uses many animals and is ethically questionable. Broncho-alveolar lavage (BAL) cannot be repeated on the same animal and reflects only the airway compartment. Real time-imaging techniques rely on conjugation of Abs to isotopes/dyes that may modify Ab binding to their target antigen or Fc receptors. Alternatively, in vivo microdialysis appears to be an interesting semi-invasive PK sampling technique to measure unmodified drugs in the interstitial spaces of various tissues, including the lungs. [START_REF] Elmquist | Application of microdialysis in pharmacokinetic studies[END_REF][START_REF] Brunner | Microdialysis versus other techniques for the clinical assessment of in vivo tissue drug distribution[END_REF] Microdialysis enables continuous repetitive sampling of drugs in a single animal using a semi-permeable membrane inserted in the tissue and perfused with physiological buffer at constant flow rate. Although mainly described for small molecules, microdialysis of proteins and Abs has recently become possible due to the availability of large molecular weight cutoff membranes and the push-pull perfusion technique to avoid fluid loss. [START_REF] Jadhav | Microdialysis of Large Molecules[END_REF][START_REF] Jadhav | Tissue Distribution of a Therapeutic Monoclonal Antibody Determined by Large Pore Microdialysis[END_REF] Herein, we attempted to use microdialysis for the dynamic quantification of an Ab administered by inhalation in non-human primates (NHP); the Ab targets a soluble antigen in the interstitial lung space (optimized mAb provided by Sanofi Genzyme, target undisclosed for confidential reasons)..

Results

We first optimized the microdialysis flow to achieve a reliable and optimal diffusion rate of the antibody of interest, mAbX, by using the experimental setup illustrated in supplemental Figure S1. As previously reported, we observed an inverse relationship between the perfusion flow rate and the mAbX diffusion rate. [START_REF] Jadhav | Microdialysis of Large Molecules[END_REF][START_REF] Jadhav | Tissue Distribution of a Therapeutic Monoclonal Antibody Determined by Large Pore Microdialysis[END_REF] The recovery rate, corresponding to the percentage of mAbX diffusing into the perfusion fluid (Figure 1A), ranged from 15.5% (1 µL/min) to 40.25% (0.2 µL/min). A flow rate of 0.3 µL/min was selected based on an acceptable duration of time to collect 50 µL of microdialysate and achieve a median recovery rate value of 28.9% [4.5%], (3 probes, 30 h of observation). It is noteworthy that the recovery rate variability was very low over a two-day period (data not shown), which encompassed the duration of the PK study. In a second step, we qualified the reversibility of the probe in vitro using an irrelevant murine IgG1 and mAbX by retrodialysis. As shown in Figure 1B, the murine IgG1 (35.67% [15.33%]) displayed similar reverse diffusion to mAbX (33.40% [3.95%]), and may be considered as standard tracer for in vivo retrodialysis. The recovery rate of the murine IgG1 was investigated in vivo by retrodialysis and was equal to 27.1% [16.9%] (median value of 34 measurements performed in two animals). Overall, the recovery rate obtained in vitro with mAbX was considered accurate for correcting calculation of mAbX concentration in vivo.

On the basis of the in vitro results, we designed the experimental setup (Figure 2A) to carry out microdialysis sampling of mAbX in vivo. Non-human primates (macaques) were chosen because they are highly similar to humans in aspects relevant to aerosol therapy (lung anatomy, delivery method) and are recommended for preclinical studies of Ab development. [START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system[END_REF] Immediately after delivery of mAbX by inhalation [3 mL at 10 mg/mL for 15-20 min to obtain on average 2.5% of the dose, i.e., 0.75 mg deposited in the lungs (Figure 2B)] in spontaneously breathing animals, animals were anesthetized, and the microdialysis probe was implanted in the lung parenchyma by thoracic surgery. Animals were thereafter maintained under prolonged anesthesia and mechanical ventilation for up to 2 days (Figure 2A). The follow-up measurements of clinical parameters showed that all physiological parameters of the animals were maintained within normal levels throughout the experiments (Table 1).

Next, the circulation of perfusion fluid was started, and microdialysate and blood were sampled on a preset schedule to quantify mAbX and endogenous/control markers. The stability of the blood/microdialysate urea ratio throughout the study (median [IQR]=1.1 [0.3], n=9 for each animal) underlined the correct permeability of the microdialysis probe (Figures 2C). Figure 3A showed the concentration-time profiles of "unbound" mAbX in the microdialysate (corrected by the in vitro recovery rate) and in the blood, which represent the lung interstitial fluid concentration and transfer of inhaled mAbX from the airways into the bloodstream, respectively. Overall, we observed: 1) a two-phase disappearance of mAbX in the microdialysate, with a linear/continuous decrease for 33 hours followed by a pseudoplateau thereafter ; and 2) low and slow passage of mAbX into the bloodstream (Figure 3A) as previously described. [START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system[END_REF][START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF] It is noteworthy that the disappearance profiles showed similar trends in each animal. Although heterogeneous between animals, the lung was more exposed than the systemic circulation to mAbX (Figure 3B) during the experiment, and this was more remarkable during the first 33 hours (ratio AUCmicrodialysate over AUCserum = 22.37, 1.58 and 57.55 for each animal). As shown in Figure 3A, the residual amount of "unbound" mAbX corresponded to 2.4% [1.3%] of the initial amount measured in the microdialysate. This value is consistent with the residual amounts of mAbX detected in the lungs after animal sacrifices (median [IQR]=4.9% [2.6%]) and normalized to the deposited mass estimated from the scintigraphy imaging (Figure 2B). Accordingly, mAbX was immunodetected in lung sections from animals after sacrifice up to 54 hours after the inhalation, and was mainly observed within airway epithelial cells and alveolar macrophages (Figure 4).

Discussion

Therapeutic Abs are emerging therapies for pulmonary diseases, including cancer and rare diseases like fibrosis. These pathologies would be better addressed with focused distribution of Ab, especially in diseases associated with marked rarefaction of blood vessels, [START_REF] Colombat | Pulmonary vascular lesions in end-stage idiopathic pulmonary fibrosis: Histopathologic study on lung explant specimens and correlations with pulmonary hemodynamics[END_REF][START_REF] Hanumegowda | Angiogenesis in pulmonary fibrosis: too much or not enough?[END_REF] which impedes the diffusion of systemic drugs into the lungs. Indeed, Abs are large macromolecules that exhibit limited distribution to the lungs after i.v. injection. [START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system[END_REF][START_REF] Hart | Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys[END_REF][START_REF] Dall'acqua | Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn)[END_REF] Because inhaled Ab passes slowly and poorly from the airways to the systemic circulation after pulmonary delivery, 2,3 the lung interstitial concentration of inhaled mAbX is expected to be higher than that in the blood compartment. Consequently, the concentration-profile in the blood compartment may not reflect the true kinetics of inhaled mAbX in the lungs. Determining the concentration of the Ab in the lung interstitium is crucial for obtaining a true representation of the inhaled mAbX exposure in the target organ and for helping in the development of more predictive PK models. In this study, we reported for the first time the dynamic quantification of an inhaled Ab (mAbX) that targets a soluble antigen in the lung interstitium by continuous lung microdialysis in a large animal model.

In vivo microdialysis is a semi-invasive method and a powerful technique to continuously measure soluble molecules from the interstitial fluid of one animal. Although it is a widely used and well-established technique for small molecules, only a few successful examples of the technique has involved Ab microdialysis. [START_REF] Jadhav | Microdialysis of Large Molecules[END_REF][START_REF] Jadhav | Tissue Distribution of a Therapeutic Monoclonal Antibody Determined by Large Pore Microdialysis[END_REF] Indeed, a major limitation of Ab microdialysis is the risk of low Ab recovery, mainly due to the high mass transport resistance and small aqueous diffusion coefficients typical of Abs. Herein, we used a large pore probe to favor Ab diffusion. After optimization, the selected parameters allowed a stable recovery rate of 28.9% during 30 hours of in vitro monitoring, which was remarkable for a high molecular weight protein. [START_REF] Jadhav | Microdialysis of Large Molecules[END_REF][START_REF] Jadhav | Tissue Distribution of a Therapeutic Monoclonal Antibody Determined by Large Pore Microdialysis[END_REF] Another issue is the calibration of the microdialysis probe membrane, which should maintain its efficiency throughout the experiment to ensure the consistent appropriate measurement of the mAbX diffusion rate. As previously reported, 11- 14,19 this can be achieved through different methods. Herein, we showed that measuring the disappearance of a murine IgG1, which was used as an internal standard with similar physicochemical properties as mAbX, in vivo was in the same range of the recovery rate obtained in vitro, thus validating the use of in vitro results to calculate the accurate concentration of the molecule of interest in animals. [START_REF] Chang | Antibody pharmacokinetics in rat brain determined using microdialysis[END_REF] Besides, the measurement of an endogenous molecule, such as urea in different compartments (circulation and lung) may act as a surrogate for qualification of probe permeability during the in vivo experiment.

The dynamic quantification of inhaled mAbX in the lung interstitium by microdialysis was conducted in NHP. Inhalation therapy is probably one of the most challenging delivery routes in animal models because of the disparities between human and animal model anatomy, respiratory patterns and physiology. [START_REF] Guillon | Insights on animal models to investigate inhalation therapy: Relevance for biotherapeutics[END_REF] Macaques are appropriate models for Ab inhalation because: 1) they allow accurate prediction of aerosol deposition in the lung and are comparable to human infants, since they are in the weight range of a full-term newborn human and display similar ventilation parameters; [START_REF] Pitance | Delivery efficacy of a vibrating mesh nebulizer and a jet nebulizer under different configurations[END_REF][START_REF] Albuquerque-Silva | Particle deposition in a child respiratory tract model: in vivo regional deposition of fine and ultrafine aerosols in baboons[END_REF][START_REF] Réminiac | Nasal high flow nebulization in infants and toddlers: An in vitro and in vivo scintigraphic study[END_REF] 2) they can breathe spontaneously through a face mask connected to an aerosol device, although they are mainly nose breathers like infants; and 3) they are the species recommended by regulatory authorities for preclinical development of Abs. Herein, the average lung deposition of mAbX, as measured by scintigraphy, was low, since the device is not intended for infants. The dose predicted to deposit in the lungs by cascade impaction (inhalable fraction (expressed as the % of loaded mAbX): 47.1% ±3.4%; respirable fraction of mAbX > 0.5 µm and < 3 µm: 71.8% ±1.8%) is higher than the deposited dose in macaques and is compatible with aerosol therapy in humans.

The PK of inhaled mAbX was not linear. The concentration-profile indicated a twophase absorption/elimination. This result may be explained by an alpha-phase distribution of mAbX from the lung to the systemic compartment, followed by pseudo-equilibrium of mAbX between the different compartments. The human antibody mAbX binds to a soluble protein: it cross-reacts with both the NHP antigen and endogenous neonatal Fc receptor (FcRn) recycling system. Thus, the first distribution and/or clearance phase may also be attributed to target mediated drug disposition (TMDD) [START_REF] Mager | Target-mediated drug disposition and dynamics[END_REF] or active IgG intracellular catabolism due to saturation of FcRn. Of note, FcRn is restricted to alveolar macrophages and epithelial cells of the upper airways in NHP airways. [START_REF] Roopenian | FcRn: the neonatal Fc receptor comes of age[END_REF] Additional experiments (e.g., with different doses) would be required to decipher further the mechanisms underlying non-linear PK profile of inhaled mAbX. After 33 hours, mAbX elimination was slower or null, and "soluble" mAbX was maintained at low but detectable concentrations in the lung interstitial fluid. We hypothesize that the low but sustained presence of mAbX in the interstitial fluid may be attributed to the continuous release of intracellular mAbX through FcRn recycling. This hypothesis is supported by the immunohistochemistry profile showing some staining within cells in the lungs at the end of the experiment. Of major importance from a therapeutic point of view, lung concentrations achieved during the first 24 h hours after inhalation were above the affinity constant of mAbX for its target. As anticipated, [START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system[END_REF][START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF] the amount of mAbX reaching the blood circulation was low and characterized by a slow passage. Based on the literature showing poor diffusion from the circulation to the lung compartment, [START_REF] Hart | Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys[END_REF][START_REF] Dall'acqua | Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn)[END_REF] it would have been necessary to administer a huge amount of mAbX via the i.v. route to achieve the equivalent lung exposition obtained by inhalation, thereby possibly raising toxicity concerns.

Overall, compared to classical PK studies, long-term lung microdialysis of Ab appears suitable to better understand the interstitial tissue distribution and behavior in situ of inhaled Abs targeting soluble antigens. Importantly, from an ethical point of view, this individual dynamic sampling and monitoring can reduce animal use by using each animal as its own control and follow-up. Nevertheless, even though microdialysis is considered a minimally invasive technique, complicated logistics are required, and a notably "human-like" surgical procedure is necessary to access the lungs and implant the probes without promoting tissue trauma and inflammation. In fact, the continuous lung microdialysis and the long half-life of Ab in the biological fluids necessitate expert anesthesiologist skills and dedicated animal facilities to maintain animals under adequate analgesia and sedation for prolonged mechanical ventilation. Altogether, these factors restrict the use of this promising technology to specialized teams with dedicated animal facilities.

To conclude, we achieved the dynamic quantification of an Ab in the interstitial lung space by microdialysis. By overcoming the technical, including zootechnical, challenges of microdialysis, we established, for the first time, the conditions for lung microdialysis in the lung parenchyma of NHP, thereby simultaneously evaluating the kinetics of an inhaled Ab in the lung compartment and in the bloodstream. Overall, we were able to quantify the local concentrations of a nebulized mAb and observed a complex non-linear elimination/distribution. Our dynamic results were highly supported by the excellent correspondence between the residual amounts of mAbX measured in the dialysate and the lungs after animal sacrifices more than 48 h after inhalation delivery. Altogether, our data open avenues for tests aiming to strengthen the local delivery, the residual/recycling concentration and the efficacy of therapeutic Abs.

Materials and Methods

In vitro microdialysis

The mAbX was an optimized mAb provided by Sanofi Genzyme (target undisclosed for confidential reasons). A 1 MDa cut-off semipermeable membrane (CMA 20) was incubated with the antibody solution (mAbX 3.3 mg/mL) and was perfused continuously with ringer acetate buffer at different flow rates using a push-pull system (KDS Legato 272 push/pull pump) while maintained at 37°C. The recovery rate corresponds to the percent of mAbX diffusing in the perfusion fluid and was calculated as follows: (Cmicrodialysate/Creservoir) x100, where C corresponds to the concentration of mAbX in the dialysate (Cmicrodialysate) or reservoir (Creservoir) (Supplemental Figure S1). Results are representative of the recovery rate obtained with 3 probes and are expressed as median [IQR]. The recovery rate of an irrelevant murine IgG1 isotype was assessed for probe calibration in the in vivo experiments and measured by retrodialysis. In this case, the microdialysis probe was perfused with ringer acetate buffer containing the murine IgG1 (1μg/mL, Cperfusate). To ensure that the murine IgG1 mimicked mAbX, mAbX was added in the perfusion fluid and the probe was incubated in the buffer only in some experiments. The residual murine IgG1 or mAbX (Cresidual) concentrations were measured in the dialysate, and the recovery rates corresponded to the percentage of murine IgG1 or mAbX lost from the perfusion fluid (retrodialysis). The retrodialysis rate was calculated as follows: ((Cperfusate -Cresidual)/Cperfusate) x 100, where C corresponds to the concentration of the murine IgG1 or mAbX.

Animals and housing

Three healthy female cynomolgus macaques, weighing 3.6 to 4.5 kg and obtained from Bioprim®, were housed in accordance with the most recent European legislation (Directive 2010/63/UE). Animals were acclimatized to laboratory conditions and were trained to receive an aerosol before the first set of experiments. The experimental protocol was conducted according to NIH guidelines for the care and use of laboratory animals and was approved by the ethics committee of Val de Loire (n°04372.01).

Aerosol administration

Formulation and nebulization conditions as well post nebulization integrity of mAbX was previously characterized (data not shown). Macaques were placed on a restraint chair after being sedated via intramuscular (i.m.) injection of ketamine (10 mg/kg). Inhalation of mAbX (3 mL of 10 mg/mL solution) was done in conscious animals with a customized vibrating membrane eFlow® Technology nebulizer connected to the eFlow® PULSE controller at 50ms/50ms of ON/OFF associated with a baby face mask (Smartmask Baby), all from PARI Pharma GmbH. The volume mean diameter (VMD) was 3.3±0.1 µm as measured by laser diffraction, and the respirable fraction (0.5µm < particles < 3µm) was 34.2% ± 6.5 % with 0.9% NaCl solution. Aerosol administration lasted no longer than 15 min. In specific studies designed to evaluate pulmonary deposition in each animal with the device, a placebo solution traced with DTPA-Tc 99m (3 mL) was delivered in the same conditions, and animals were imaged with a gamma camera (Ecam, Siemens) immediately after the aerosol session, as previously described. [START_REF] Hervé | VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations[END_REF] Aerosol deposition in the lungs, stomach, ears, nose and throat was calculated taking into account the background, coefficient of tissue attenuation and radioactivity decay.

Implantation of the microdialysis probe and in vivo microdialysis

Immediately after aerosol delivery, animals were sedated with ketamine (10 mg/kg i.m.) then anesthetized with isoflurane. Animals were orotracheally intubated (tube internal diameter of 3.5 mm) and mechanically ventilated in a supine position with Datex Flexima ventilator.

Ventilator settings were: tidal volume 70 ml, respiratory rate: 30/min, air and oxygen flows between 0.5 and 1L/min, oxygen inspiratory fraction: 50%, Pi Max: 25 cmH20, FeCO2: 35-40 mmHg. Equipment for anesthesia monitoring was set up (pulse oximetry, end-tidal carbon dioxide, non-invasive blood pressure, and temperature monitoring), as was a urinary catheter with an hourly diuresis unit, an electric warming blanket and pressure ulcer-reducing devices.

After inserting two peripheral venous catheters, isoflurane was relayed by continuous injection of midazolam (0.9 to 5 mg.kg -1 .h -1 ), ketamine (9 to 40 mg.kg -1 .h -1 ) and buprenorphine (6 to 25 µg.kg -1 .h -1 ) for up to 54 hours. Animals were supplemented with dextrose in normal saline solution (Glucidion G10%, injected at 2 mL/h).

The microdialysis probe was inserted in the lung parenchyma through right mini-thoracotomy under direct vision and was sutured on the lung. An intercostal pleural drain was inserted before closing the surgical wound. Arterial catheterization of the femoral artery was then performed to facilitate blood sampling and for continuous hemodynamic monitoring. The probe was continuously perfused with ringer acetate buffer at 0.3 µL/min. According to in vitro results, in some experiments a murine IgG1 (1µg/mL, Mouse IgG1 Isotype Control #MAB002, R&D System) was added to the perfusate to evaluate the in vivo recovery rate.

Blood and microdialysate were collected on a predefined schedule (every 6 and 3 hours, respectively) to analyze murine IgG1 (retrodialysis), mAbX and urea (used as an additional control of probe permeability). The clinical parameters (body temperature, mean arterial pressure, arterial blood oxygen saturation, heart rate and respiratory rate) of the cynomolgus macaques were monitored continuously during this period (n=54 for each animal). At the end of the experiment, animals were sacrificed by exsanguination, lungs were excised, and a part of each lobe was kept frozen at -80°C to measure mAbX in protein lysates.

Protein extraction and quantitative measurements of biological markers and Abs

Briefly, tissues (∼30 to 60 mg) were dissociated in RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 25 mM Tri-HCl pH 7.6) with a gentleMACS Dissociator (Miltenyi M tubes, program Protein_01), using 1 mL RIPA/20 mg tissue. After 5 minutes of centrifugation at 4,000xg, supernatants were collected, and proteins were quantitated using a BCA Protein Assay Kit (Thermo Fisher Scientific). Urea was quantified by a quantitative colorimetric assay (Bioassay System) according to the manufacturer's instructions. Murine IgG1 and mAbX were measured in the serum and/or the microdialysate by ELISA. The murine IgG1 was measured with the Mouse IgG1 Ready-SET-Go!® (affymetrix eBioscience) kit according to the manufacturer's instructions. For mAbX detection, the home-made ELISA allowed a range of quantification from 100 ng/mL to 410 pg/mL, with intra-assay CV% (1.13 [1.10]%) and inter-assay CV% (5.81 [6.10]%).

Immunohistochemistry of mAbX in the lung tissue

For the detection of residual mAbX in the lung tissue (at the end of the experiment), the lungs were sampled and fixed in 10% neutral formalin for 24 hours. Tissue from an animal that did not receive Ab with Fc-human was used as a control. Inhaled mAbX was detected on lung sections (5 μm) with a secondary anti-human IgG (horseradish peroxidase-conjugated antihuman IgG , #2049-05, Southern Biotech) , which do not cross-react with macaque IgGs (as confirmed by dot blot, data shown). The antigen was recovered by boiling the sections in a citrate-based buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). Endogen peroxidase activity was quenched by incubating the slides for 15 minutes in 3% H2O2 in methanol. For

  blocking, sections were incubated in diluted normal blocking serum (0.3% normal goat serum (#G9023, Sigma-Aldrich)) and 2.5% bovine serum albumin (#A7906, Sigma-Aldrich) in phosphate-buffered saline (PBS) 1X for 1 hour and washed twice. Next, sections were incubated with the diluted secondary peroxidase-conjugated anti-human IgG at 10 µg/mL in PBS-T (PBS 1X, 0.1% Tween 20) overnight at 4°C. They were then washed twice, and mAbX was then detected by incubation with DAB-peroxidase solution substrate (Dako, Les Ulis, France) until a satisfactory signal was obtained. The sections were counterstained with hematoxylin solution (Gill N°1, Sigma-Aldrich), cleared and mounted. Statistical analysis Results are expressed as median [IQR] for the indicated number of experiments performed independently. For the time-concentration profiles, results are expressed as mean +/-SEM. Statistical significance between the different values was analyzed by the Kruskal-Wallis test for multi-group comparisons or by the Friedman test for non-parametric repeated measures. Statistical analyses were performed using GraphPad Prism5 software. A p value of <0.05 was considered to be significant. FcRn: neonatal Fc receptor FeCO2: fraction of expired of CO2 IQR: interquartile range i.v.: intravenous i.m.: intramuscular NHP: non-human primates Pi Max: maximal inspiratory pressure PBS: phosphate-buffered saline PK: pharmacokinetic RIPA buffer: radioimmunoprecipitation assay buffer SDS: sodium dodecyl sulfate SEM: mean standard error Tc 99m : technetium-99m TMDD: target-mediated drug disposition VMD: volume mean diameter

Figure 1 :

 1 Figure 1: Setup for mAbX antibody microdialysis. A) The recovery rate of mAbX using a

Figure 2 :

 2 Figure 2: In vivo lung microdialysis of inhaled mAbX. A) Scheme of the experimental

Figure 3 :

 3 Figure 3: Fate of mAbX after inhalation in macaques. (A) Time-concentration of mAbX

Figure 4 :

 4 Figure 4: Immunodetection of residual mAbX in the lung tissue. Animals were sacrificed

Table 1 : Clinical parameters of the macaques during the microdialysis experiment.

 1 Parameters were measured each hour. Results are expressed as the median [IQR] for each macaque over a period of up to 54 hours.

Acknowledgments:

We would like to thank the members of the animal facility "PST Animaleries" of Tours (C. Aubert, B. Plante, G. Roseau, V. Schubnel), and those from Safety Pharmacology & In vivo PK supports PreClinical Safety Global Operations at Sanofi (A. Dudzicki, C. Battut, R.H Blanchard, F. Perrot), from CEPR-INSERM U1100 (E. Dalloneau, M. Cabrera and D. Le Pennec) for their technical assistance. We would like to thank the Department of the Biotherapeutic Formulation at Sanofi for providing the mAbX formulation for nebulization (S. Carayon) and the unit of molecular histopathology and bio-Imaging translational sciences (A.M. Lefebvre, R. Gorski and M. Classe) for their technical support with IHC, and the Laboratory of Biochemistry and Molecular Biology" CHRU of Tours (H. Blasco, C. Andrès) for their help in measuring urea. We acknowledge PARI Pharma GmbH (Germany) represented by M. Hahn for providing the eFlow Technology delivery system. We would like to thank Dr. B. Faivre d'Arcier and Franck Patin for their technical assistance. We thank Kimberly A. Barker for copy-editing assistance. This work was supported by public grants overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (reference: ANR-10-LABX -53-01) and provided by Région Centre (MicroPulm).

This work was supported by Sanofi-Genzyme, by a public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (reference: ANR-10-LABX -53-01) and a grant provided by Region Centre (MicroPulm) Conflict of interest: In accordance with Taylor & Francis policy and our ethical obligation as a researcher, we are reporting that PJ, CB and EG have business interests in

Abbreviations:

Abs: antibodies BAL: broncho-alveolar lavage CV: coefficients of variability Da: dalton DTPA: diethylenetriamine penta-acetic acid ELISA: enzyme-linked immunosorbent assay


Guillaume Desoubeaux
email: guillaume.desoubeaux@univ-tours.fr

Adélaïde Chesnay

Health threat caused by fungi of medical interest: where are we in 2021?

When non-scientific people are interviewed about the most dangerous fungus in the world, most instinctively cite the-so famous-death cap amanita (Amanita phalloides). However, this macroscopic species related to the Basidiomycota division, largely widespread in Europe and subsequently imported to all other continents, actually causes only few dozens of deaths per year. The real threat is rather microscopic and is responsible for a heavy burden in worldwide public health. According to the GAFFI (Global Action Fund for Fungal Infection) website (https://gaffi.org/), 13,500,000 people are currently suffering from common life-threatening fungal infections and >1.6 million die annually of related complications (Figure 1).

Fungal infection is described in both high and low resources countries, but their relative representations are variable therein [START_REF] Brown | Hidden killers: human fungal infections[END_REF]. In addition to the influence of numerous factors, their respective prevalence notably depends on the local climate, demographics and healthcare situation and habits, e.g., prevalence of tuberculosis and human immunodeficiency virus (HIV) infection, easy access to cancer chemotherapy, practice of transplantation, distribution of novel immunosuppressive regimens like monoclonal antibodies… [START_REF] Chandenier | Epidemiological transition of mycosis diseases in sub-Saharan Africa: from surface to depth[END_REF]. For instance, tropical areas are mostly concerned by endemic infections, e.g., mycetoma in the Saharan band from Senegal to Sudan, Penicillium marneffei talaromycosis in Southeast Asia, or histoplasmosis in central and South America: all these entities are associated with poverty, underlying infectious diseases and certain temperatures. In contrast, Northern and Western countries are rather interested in opportunistic diseases induced by iatrogenic processes, e.g., candidiasis following solid organ transplantation or surgery, aspergillosis associated to hematopoietic stem-cell transplantation or intensive anticancer chemotherapy [START_REF] Brown | Hidden killers: human fungal infections[END_REF].

Nowadays, historic infections like Pneumocystis pneumonia or Cryptococcus meningitis in HIV-positive patients are becoming less incident (especially because of the advent of highly active antiretroviral therapies), as illustrated by the -14.3% decrease per year in France during the 2001-2010 period for the former [START_REF] Bitar | Population-based analysis of invasive fungal infections, France, 2001-2010[END_REF], and the 3 to 4-fold reduction in Africa over the last decade for the latter [START_REF] Rajasingham | Global burden of disease of HIVassociated cryptococcal meningitis: an updated analysis[END_REF]. They have been progressively replaced by other fungal diseases that have been newly emergent or just relatively more represented. For instance, numbers of candidemia and invasive aspergillosis were +89.5% and 63.6% increased in France, during the same aforementioned period [START_REF] Bitar | Population-based analysis of invasive fungal infections, France, 2001-2010[END_REF]. Several causes linked to the medical advances can explain these findings: the diversification of the medical procedures and the arrival of new immunosuppressive drugs on the market, but also the prolongation of the overall survival of patients (so that consequently the exposure risks are proportionally increased). Currently, around 400,000 new cases of candidemia occur annually worldwide, primarily in intensive care units (ICU), but not exclusively [START_REF] Brown | Hidden killers: human fungal infections[END_REF]. The incidence rose up by >50% over only five years at the beginning of the 21st century in the USA [START_REF] Zilberberg | Secular trends in candidemia-related hospitalization in the United States, 2000-2005[END_REF]. Death rate for C. glabrata infection is approximately 55-60%, and only a little bit less for C. albicans  40%. Of note, a progressive increase in nonalbicans species prevalence has been globally observed, although it is slight and quite variable between the continents [START_REF] Falagas | Relative frequency of albicans and the various non-albicans Candida spp among candidemia isolates from inpatients in various parts of the world: a systematic review[END_REF][START_REF] Giacobbe | Changes in the relative prevalence of candidaemia due to non-albicans Candida species in adult in-patients: a systematic review, meta-analysis and metaregression[END_REF]. This trend could have some therapeutic consequences over time. For instance, the high overrepresentation of C. krusei (>10%) in the article written by Martin et al. [START_REF] Martin | Candida species distribution in bloodstream cultures in Lyon, France, 1998-2001[END_REF] might lead to systematic failures when using fluconazole. Over 300,000 patients develop invasive aspergillosis yearly [START_REF] Brown | Hidden killers: human fungal infections[END_REF][START_REF] Bongomin | Global and multi-national prevalence of fungal diseases-estimate precision[END_REF]; the mortality still remains close to 30-50%, in spite of adequate antifungal therapies [START_REF] Bitar | Population-based analysis of invasive fungal infections, France, 2001-2010[END_REF]. Recently, new Aspergillus-related entities have emerged in patients hospitalized in ICU because of severe underlying virus pneumonia, like influenza or COVID-19 (Coronavirus disease 2019) infection, thus leading to IAPA (influenza-associated pulmonary aspergillosis) or CAPA (COVID-19-associated pulmonary aspergillosis). Respective occurrence rates in such contexts were estimated at 12-28% [10] and 14-20% [START_REF] Salmanton-García | COVID-19-Associated Pulmonary Aspergillosis, March-August 2020[END_REF], but the lack of standard definitions in ICU precludes a reliable incidence picture in non-classical populations at risk. Besides, mucormycosis was reported with an incidence elevating from 0.7/million in 1997 (40 cases) to 1.2 in 2006 (72 cases), at a national scale in France [START_REF] Bitar | Increasing incidence of zygomycosis (mucormycosis), France, 1997-2006[END_REF]. The role of previous exposure to antifungal drugs lacking activity against Mucorales species (e.g., prophylaxis based on voriconazole during hematologic malignancies or bone marrow transplantation) has been raised to explain this trend in patients at risk, but does not seem exclusive. The recent COVID-19 pandemic reminded the importance of the local prevalence of diabetes in the occurrence of mucormycosis (in addition to the aforementioned conditions conventionally related to the viral infection: intubation, corticosteroid treatment, ICU stay) [START_REF] Al-Tawfiq | COVID-19 and mucormycosis superinfection: the perfect storm[END_REF]. Most case reports and series were published in India, where a recent survey found 11.8% inhabitants affected by such endocrine pathology [START_REF] Nagarathna | Prevalence of diabetes and its determinants in the young adults Indian population-call for yoga intervention[END_REF].

Diversification of the panel of the underlying chronic diseases, as well as the increasing absolute number of concerned patients, also plays a major role in the global burden of fungal infections. Thus, it is thought that at least 3,000,000 subjects are affected by chronic aspergillosis among those with debilitated terrain, like chronic obstructive pulmonary disease [COPD], alcoholic cirrhosis, sarcoidosis, or corticosteroid treatments [START_REF] Denning | Global burden of chronic pulmonary aspergillosis complicating sarcoidosis[END_REF]. More specifically, its prevalence was evaluated at about 1.2 million cases in the context of ancient tuberculosis [START_REF] Denning | Global burden of chronic pulmonary aspergillosis as a sequel to pulmonary tuberculosis[END_REF]. Case fatality rate approaches 15-40%. All over the world, more than 23,500,000 individuals are currently concerned by allergic fungal diseases, whatever the pathophysiology, like severe asthma with fungal sensitization (SAFS) in 5-20% asthmatics [START_REF] Denning | Fungal allergy in asthma-state of the art and research needs[END_REF] and allergic bronchopulmonary aspergillosis (ABPA) in 15% adults with cystic fibrosis (teenagers and kids are usually less affected) [START_REF] Armstead | Multi-country estimate of different manifestations of aspergillosis in cystic fibrosis[END_REF].

In tropical countries, the actual burden of endemic fungal diseases is tough to assess, primarily because the access to efficient diagnostics still lacks in rural areas. Incidence rate of histoplasmosis is likely ~100,000 cases per year [START_REF] Denning | Minimizing fungal disease deaths will allow the UNAIDS target of reducing annual AIDS deaths below 500 000 by 2020 to be realized[END_REF], and the cumulated prevalence close to 50 million over time, although the infection remains most often asymptomatic [START_REF] Brown | Hidden killers: human fungal infections[END_REF]. According to a recent study, the number of deaths related to histoplasmosis in Latin America was estimated at range of 671 to 9394 during one unique year, so that they were somewhat equivalent to the 5062 deceases related to tuberculosis meantime [START_REF] Adenis | Burden of HIV-associated histoplasmosis compared with tuberculosis in Latin America: a modelling study[END_REF]. Like chromoblastomycosis (in all, 7740 cases have been reported worldwide, but this fungal entity is mostly present in Madagascar with prevalence up to 14/100,000 inhabitants, and in Brazil) [START_REF] Queiroz-Telles | Chromoblastomycosis[END_REF], mycetoma have been recently listed among the neglected tropical diseases by the World Health Organization (WHO). In the world, almost 20,000 cases of mycetoma have been reported in the literature in more than 100 countries. Notably, mycetoma is now described far outside the tropics, including for example Italy, China, Australia and USA [START_REF] Emery | The global distribution of actinomycetoma and eumycetoma[END_REF]. Sporotrichosis is mainly found in central and South America: a new epidemic wave has spread from Rio de Janeiro to the North through cat transmission [START_REF] Chakrabarti | Global epidemiology of sporotrichosis[END_REF]. Only in China, talaromycosis is supposed to concern 20% HIV-positive subjects geographically exposed, resulting in 0.35 cases per 100,000 inhabitants, whereas its worldwide incidence is supposed to be close to 10,000 new cases per year [START_REF] Zhou | Risk-Based Estimate of Human Fungal Disease Burden, China[END_REF]. Coccidioidomycosis due to Coccidioides immitis is largely underestimated: the actual incidence is likely closer to 140,000-150,000 cases per year, most occurring asymptomatically in America [START_REF] Brown | Hidden killers: human fungal infections[END_REF]. In California, 7466 workers were contaminated in 2017 subsequently to digging agricultural works and creation of embankments in the ground [START_REF] Laws | Coccidioidomycosis Outbreak among Workers Constructing a Solar Power Farm -Monterey County, California, 2016-2017[END_REF].

Finally, we can also cite the burden represented by superficial fungal infection, like tinea corporis, capitis, pedis or unguium that altogether probably count for a prevalence of one billion persons [START_REF] Panackal | Cutaneous fungal infections in the United States: Analysis of the National Ambulatory Medical Care Survey (NAMCS) and National Hospital Ambulatory Medical Care Survey (NHAMCS), 1995-2004[END_REF]. All people categories can be concerned, but tinea pedis, or athlete's foot, is more common in young sportsmen (-women), while tinea unguigum, or onychomycosis, is increasingly more prevalent in elderly, up to 50% individuals >70 years old [START_REF] Thomas | Toenail onychomycosis: an important global disease burden[END_REF]. Recurrent vulvovaginal candidiasis affects 138 million women per year [START_REF] Denning | Global burden of recurrent vulvovaginal candidiasis: a systematic review[END_REF].

In conclusion in the light of all the aforementioned rates (although they are probably significantly underestimated), one could legitimately acknowledge that mycologists and specialists of infectious diseases still have a lot of efforts to provide in order to better diagnose and treat the fungal diseases. The direct costs related to the care for managing fungal infections were estimated at ≥7 billion US$ per year in the United States, i.e., 0.22% of total health expenses [START_REF] Benedict | Estimation of Direct Healthcare Costs of Fungal Diseases in the United States[END_REF]. Recent outbreaks of Candida auris infection/colonization inside healthcare facilities are obvious examples of the emergence risk of new species that are highly resistant to conventional antifungals [START_REF] Schelenz | First hospital outbreak of the globally emerging Candida auris in a European hospital[END_REF][START_REF] Desoubeaux | Candida auris in contemporary mycology labs: a few practical tricks to identify it reliably according to one recent French experience[END_REF][START_REF] Ruiz-Gaitán | An outbreak due to Candida auris with prolonged colonisation and candidaemia in a tertiary care European hospital[END_REF], with up to 90% strains not susceptible to fluconazole, and around 10% to amphotericin B [START_REF] Chowdhary | A multicentre study of antifungal susceptibility patterns among 350 Candida auris isolates (2009-17) in India: role of the ERG11 and FKS1 genes in azole and echinocandin resistance[END_REF]. Focusing only in the USA, 948 cases of proven or probable C. auris infection have been notified so far to the Center for Diseases Control (CDC) in Atlanta.

Fig. 1 .

 1 Fig. 1. Summarizing representation of the relative burden for the most common fungal infections (according to the data of the GAFFI website (https://gaffi.org/). For each one, the global current prevalence and the annual related deaths are showed by the means of a radar graph, and compared to those caused by tuberculosis and human immunodeficiency virus (HIV) infection, according to a logarithmic scale.

Acknowledgment

Not applicable.

Funding

This research received no external funding.

Author contributions

GD was at the origin of the text and wrote it; AC reviewed and edited the document

Ethics approval and consent to participate

Not applicable.

Conflict of interest

GD was specifically invited by the editorial board to write this manuscript for the special issue dedicated to fungal diseases.


Victor Mercier
email: victor.mercier@gmail.com

Milène Sasso

Pascal Kouyoumdjian

Damien Sizaret

Simon Benzimra

Samia Gonzalez

Guillaume Desoubeaux

Some undesirable traps which can mislead the pathologist

Keywords: rice-bodies, ciliocytophthoria, starch grains

In clinical laboratories, the diagnosis of parasite diseases can sometimes be challenging for non-expert microbiologists. Indeed in spite of the advent of the molecular biology, macro-and microscopic examination still remains essential. Nonetheless, it is usually not automated and requires great skills to complete the correct diagnosis. It is not infrequent that inert elements mislead to erroneous diagnoses.

Through three different concrete examples, this article aims at underscoring the actual risk of parasite misidentification and at highlighting the systematic approach to be conducted in order to enable reliable diagnosis.

Introduction

In clinical laboratories, the diagnosis establishment of parasite diseases can be challenging for pathologists or non-expert microbiologists. Thorough macro-and microscopic observation and histological examination of miscellaneous biological specimens, such as bronchial-alveolar lavage fluids (BALF), feces or biopsies of various natures, are the key-steps for reaching an accurate diagnosis. So far, these methods have been usually non-automated yet and both require extensive experience. Misidentification pitfalls are numerous, and pathologists or microbiologists should always be aware of them to avoid failure in the diagnosis process. Indeed, several inert or non-pathogenic elements can frequently mislead to erroneous diagnoses, and thus subsequently to inadequate curative therapies. Diagnostic difficulties are primarily emphasized when managing a patient who is used to live or has lived in tropical areas; the imported diseases are often neglected, and thus the pathologists and microbiologists are less seasoned with them and their diagnosis.

Therefore to reinforce the accuracy of their diagnostic conclusions, all laboratory staff should be informed of few very-practical tricks to enable better recognition of pathogenic elements and for distinguishing them easily from non-parasite structures. In this article, we will expose three concrete examples that focus on thorough observations of fake parasites in biological samples, and we will discuss more the differential diagnosis for each one.

Maggots or cestode vesicles?

A 58-year-old woman underwent a total left hip replacement due to coxarthrosis. In the following months, she began to feel chronical pain next to the joint, in spite of the painkiller medications. No clear etiology was found through the magnetic resonance imaging (MRI). Likewise, a punction was not contributive. Twenty-two months after the initial surgery, the hip prothesis was removed in order to avoid a septical issue. During this second intervention, numerous little white inert bodies, measuring seven to eight millimeters in length, were found around the soft tissues (Figure 1 A) and above the prothesis (Figure 1 B). The sampleswhich look like maggots or cestode vesicles at first glancewere sent to the hospital laboratory for parasite identification (Figure 1 C), but were finally characterized as rice-bodies. The in vitro bacterial culture of the tissue surrounding the rice-bodies eventually isolated Cutibacterium acnes colonies, i.e. natural saprophyte bacterium of the skin, whereas the mycological culture remained sterile. Based on periodic acid Schiff (PAS) and hematoxylin and eosin safron staining, the histopathological examination showed fibrin deposition with inflammatory cells, but no parasite. To definitively exclude cysticercosis, the research of Taenia solium antibodies was conducted, but was found negative. A second total hip replacement was programmed, four months later. No complication occurred, and the patient left the hospital with favourable outcome.

Ciliated protozoans?

A 65-year-old woman underwent surgical resection of womb fribroma that was evolving over one year. There was no known underlying infection at time of the surgery. Concomitantly to the intervention, aspirate of the pertioneal liquid was systematically addressed to the hospital laboratory for biological investigations. At the fresh mounting examination, several mobile structures of 10-25 µm were observed. At higher magnification, they appeared ciliated on their whole circonference (Figure 2 video 1), a little bit like a protozoan. However, the motions were quite unatural, depending mostly on the Brownian flow. Finally, these structures were identified as ciliocytophthoria artifacts coming from uterine tubes or endometrium. No in vitro cultures were positive. There was no complications for the patient and no occurrence of infection. The outcome was favourable.

Segmented helminth?

A 58-year-old male was found dead at home and subsequently autopsied by forensic scientists for suspicion of COVID-19 (coronavirus infectious disease -2019) infection. During necropsy, some lung lesions were clearly observed, and biopsies were then performed for further investigations.

Histopathology examination confirmed the presence of foci of acute pneumonitis. Moreover, several unusual round-or oval-shaped structures, with size ranging from 2-3 µm to 10-15 µm, were noticed within the lumen of alveoli and bronchioles (Figure 3). Due to the size and the regular section, helminth larvae or nematode remnants were first suspected. However at higher magnification, the appearance was rather suggestive of starch grains. The grains had been probably unintentionally aspirated into the lungs, and thus were not assumed to be responsible for the death. The latter was finally inferred to a stroke.

Discussion

Correct identification of parasites in clinical samples can be critically challenging, including for quiteskilled microbiologists. The latter have to conduct a systematic thorough approach during the macroand microscopic examination of all the specimens that are addressed to them. Indeed, the risk of confusion can be really detrimental in some circumstances.

Regarding the first case, one could have first suggested vesicles due to any cestod infection, e.g. cysticercosis, when facing the oval white structures. However in the medical history of the patient, there were no clear clues that could have reinforced this diagnostic hypothesis. In contrast, the observation was finally more in adequation with rice-bodies description.[1, 2] Rice-bodies appear rubbery when cut into pieces, and they are theoreteically not hollow. However, there contain neither liquid nor parasite elements, wether living or dead, inside. Rice-bodies are rarely observed; they sometimes form during inflammatory processes of the synovial liquid, like infectious arthritis, rheumatoid arthritis, non-specific arthritis or osteoarthritis. [START_REF] Jeong | Candida septic arthritis with rice body formation: a case report and review of literature[END_REF] The pathogenesis may be explained by microinfarcted synovium and release of tissue into the joint, and subsequent encasement by fibrin deposition. [START_REF] Cheung | Synovial origins of Rice bodies in joint fluid[END_REF] Another hypothesis suggests a de novo formation in synovial fluid independently of any synovial elements, and progressive enlargement with aggregation of inflammatory nodules consisting of fibrin, collagen, inflammatory cells and blood vessels. [START_REF] Hung | Tuberculous Arthritis of the Knee with Rice Body Formation: A Report of a Rare Case[END_REF] In the present case, the rice-bodies probably developed after the first hip surgery. Usually, rice-bodies are isointense on T1-weighted MRI and slightly hyperintense on T2-weighted MRI relative to muscles; [START_REF] Chen | Distinguishing multiple rice body formation in chronic subacromial-subdeltoid bursitis from synovial chondromatosis[END_REF] but in the present case, ricebodies were unperceivable during medical imaging.

At first glance, the second observation reported herein could have suggested a vegetative stage of ciliated protozoans, [START_REF] Simões-Silva | Peritoneal Microbiome in End-Stage Renal Disease Patients and the Impact of Peritoneal Dialysis Therapy[END_REF] like trichomonids or paramecia. [START_REF] Zalonis | Rare case of trichomonal peritonitis[END_REF] Actually, the microscopic structures were rather consistent with ciliocytophthoria artefacts. [START_REF] Hilding | The common cold[END_REF] Ciliocytophthoria are usually the result of the degenerative process of ciliated cells consequently to various common infections, like miscellaneous virus diseases. [START_REF] Gelardi | Ciliocytophthoria of nasal epithelial cells after viral infection: a sign of suffering cell[END_REF] They are produced from the triangular top part of the bronchial or uterine cells, at the luminal border, which is separated from the cytoplasm bearing the decaying nucleus.

Ciliocytophthoria size is quite variable. The cilia are straight, sometimes only represented on a limited part of the outer perimeter, and have a uniformed short length. They express a coordinate and synchronous rhythmic beating which does not lead to any active movement. [START_REF] Alam-Eldin | Identification criteria of the rare multi-flagellate Lophomonas blattarum: comparison of different staining techniques[END_REF] All these features should theoretically allow a clear distinction with (non-)pathogenic flagellated or ciliated protozoans.

For example, the vegetative stage of Giardia duodenalis displays a flat pear-shape with two transverse, claw-shaped median bodies, and with four pairs of flagella. Lophomonas blattarum bears irregular long flagella arising over the anterior part, and which are longer at center and shorter at sides. [START_REF] Alam-Eldin | Identification criteria of the rare multi-flagellate Lophomonas blattarum: comparison of different staining techniques[END_REF] Furthermore, the motility of flagella is much more active. [START_REF] Martínez-Giron | From the 19th century to the 21st, an old dilemma: ciliocytophthoria, multiflagellated protozoa, or both?[END_REF] Among the ciliate phylum, Balantioides coli trophozoites are large, up to 150 μm in length, and their entire cell surface is covered by short cilia. [START_REF] Ponce-Gordo | Balantioides coli[END_REF] The third case about the observation of starch grains highlights how histopathology examination can be tricky for pathologists and parasitologists. Starch grains are quite common structures. Their morphologic presentation is the same for all vegetables; they are stained in dark pink color by the PAS method, which indicates high concentration in carbohydrates. Starch grains contain a lobulated and empty internal part with no clear well-defined structures inside, and there is a lack of inflammatory response surrounding these exogenous particles. [START_REF] Kradin | Diagnostic pathology of infectious disease[END_REF] The presence of such starch particles has already been described in inhalation pneumonia, [START_REF] Crome | Pulmonary nodular granulomatosis caused by inhaled vegetable particles[END_REF] but with no clear demonstration of any pathogenic role.

Likewise, starch grains may be sometimes confused with pinworm remnants. However, Enterobius vermicularis pinworm is quite different, regarding its morphology: the transversal section of its body displays a pair of cuticular crests. Usually, it also shows typical eggs in the uterus and the characteristic narrow meromyarian layers (two to three muscle layers per quarter section divided by four cords). [START_REF] Oertel | Be careful with lentils! About a forensic observation[END_REF] In conclusion, in case of any doubt about the parasite etiology, the clinical specimens have to be systematically addressed to the parasitology laboratory in parallel before the tissue fixation which is mandatory for histological examination, and in order to get a specialized advice on a fresh sample.

Although the multiplex real-time PCRs have been increasingly used as first-line diagnostics, [START_REF] Van Lieshout | Clinical consequences of new diagnostic tools for intestinal parasites[END_REF] the macro-and microscopic examination still play a major role in the diagnostic process. The three aforementioned cases show how numerous can be the pitfalls leading to erroneous identification. In such a context, one should remember that the microbiologists own a critical expertise added to an habit of systematic approach of biological analysis. Ciliocytophthoria artifacts are degenerated from bronchial cells and each one is constituted by the nucleus (nc) with small cytoplasm that bears the cilia (ci). The latter arise from the terminal bar (tb).

Figure legends

Figure 1 :

 1 Figure 1: Rice-body elements. A) Dissection of the left hip compartment showing multiple

Figure 2 :

 2 Figure 2: Observation of ciliocytophthoria particles at the fresh mounting examination x400.

Figure 3 :

 3 Figure 3: Histological observation of starch grains (sg) within the lumen of bronchioles at time of

Ethics

The patients were informed and did not oppose to the publication process. As the study is retrospective with no impact on the routine healthcare, no specific approval was required by the authorities.

Conflict of interest

None

Bibliographic references

[1] Faller G, Haagensen M, Barrow M. Juvenile idiopathic arthritis flare due to rice bodies in the knee of a 10-year-old girl. S Afr Med J. 2018;108 [START_REF] Gelardi | Ciliocytophthoria of nasal epithelial cells after viral infection: a sign of suffering cell[END_REF]:833-835.

Video 1: Observation of ciliocytophthoria particles at the fresh mounting examination x400. Note the 216 unilateral disposition of the cilia and the unnatural movements of their beating.


M.D Antoine Guillon

Karen Reckamp

Ph. D L Karen

Nathalie Reckamp

Heuzé-Vourc'

Immunotherapy improves the prognosis of lung cancer: do we have to change ICU admission and triage guidelines

Immunotherapy improves the prognosis of lung cancer: do we have to change intensive care unit admission and triage guidelines?

Title:

Immunotherapy improves the prognosis of lung cancer: do we have to change ICU admission and triage guidelines?

Authors:

Antoine Guillon 1,2,3 , antoine.guillon@univ-tours.fr Karen L. Reckamp 4 , KReckamp@coh.org Nathalie Heuzé-Vourc'h 2,3 nathalie.vourch@univ-tours.fr

Affiliations:

1. CHRU de Tours, Service de Réanimation Polyvalente, F-37000 Tours, France Soon, bald heads may not be a sign which identifies a patient with cancer receiving treatment. Indeed, therapies for cancer patients are improving dramatically, leading to increased survival rates and most are associated with a different toxicity profile. Recently, antibody-based therapy has transformed the therapeutic landscape and biology of non-small cell lung cancer (NSCLC) and other solid tumors. This may also reshuffle the playing cards for an ICU admission policy due to improved outcomes. In November 2016, the results of the KEYNOTE-024 trial showed for the first time the superiority of immunotherapy over chemotherapy as first-line treatment for NSCLC [START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF]. In this phase 3 trial, a humanized monoclonal antibody (mAb) against programmed death 1 (PD-1) was tested in patients who had previously untreated advanced NSCLC. The clinical trial was stopped by the safety monitoring committee on the basis of substantial clinical benefit of immunotherapy, and patients remaining in the chemotherapy group were switched to receive immunotherapy. Antibody-based therapy in NSCLC is a therapeutic breakthroughthe first mAb was approved only a decade ago [3]. It is difficult to predict lung cancer's mortality because new mAbs therapeutic options continue to emerge and improve survival for patients. When a clinical trial is completed in lung cancer, the overall survival may have change due to innovative immunotherapy, making previous results obsolete overnight. Indeed, the development of immunotherapy and combinations for lung cancer causes triage criteria for admission into the ICU to be in constant flux. We believe that we have entered into a time when triage criteria based on lung cancer prognosis will be almost impossible to define for ICU-clinician. Consequently, ICU admissions will have to be determined by high-quality consultation between the intensivist and thoracic-oncologist to define prognosis and appropriate treatment goals. Intensivists should note: the prognosis and survival for lung cancer will be transformed by this mAb immunotherapy revolution.

  Immunotherapy with immune checkpoint inhibitors has ushered in a new chapter in the treatment of solid tumors. In place of directly targeting cancer cells, these drugs stimulate immune cells to enhance the host immune response against cancer. Two classes of immune checkpoint inhibitors with blocking antibodies have demonstrated a high level of antitumor activity in a variety of malignancies: (i) anti-cytotoxic T-lymphocyte antigen-4 antibodies (not yet approved in NSCLC); (ii) inhibitors of PD-1 (nivolumab, atezolizumab and pembrolizumab) -all established second-line therapeutic options for patients with metastatic NSCLC [2].

  Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al. Paclitaxelcarboplatin alone or with bevacizumab for non-small-cell lung cancer. N. Engl. J. Med. 2006;355:2542-50.

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Acknowledgements

Not applicable

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Availability of supporting data

Not applicable

Abbreviations

ICU: intensive care unit NSCLC: non-small cell lung cancer mAb: monoclonal antibody PD-1: programmed death 1

Declarations section

Ethical Approval and Consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

No competitive interests to declare Authors' contributions AG, KLR, NHV contributed equally to the preparation of this manuscript.

Authors' information

AG is intensivist, specialized in respiratory disease; KLR is thoracic-oncologist, specialized in lung cancer; NHV is researcher in biology, specialized in immunotherapies.