Netherlands

Rationale: In chronic obstructive pulmonary disease (COPD), the airways are characterized by goblet cell hyperplasia, decreased ciliated cell numbers and an altered turnover of the extracellular matrix (ECM). Current 2D (co-)culture systems lack the spatial in vivo environment that is formed by ECM. In this study we aimed to develop a lung ECM hydrogel-based co-culture model to investigate bidirectional interactions between fibroblasts and epithelial cells in relation to ECM changes. 

Methods: Porcine lungs were decellularized, lyophilized and ground to a powder. This powder was porcine pepsin-solubilized for 48h, pH neutralized, buffered with PBS and incubated at 37°C for 30 min to generate ECM hydrogels. Human primary lung fibroblasts (50,000/ml) from non-COPD donors were mixed with hydrogels prior to gelation in transwells. The next day, the hydrogels were coated with collagen type IV or left uncoated. After 24h, 100,000 primary bronchial epithelial cells (PBEC) from non-COPD donors were seeded on top of fibroblast seeded and unseeded gels. The cells were cultured submerged for 4 days and subsequently at air liquid interface for 14 days. The hydrogels were imaged using inverted light microscopy and by histology on paraffin-embedded sections

Results: PBECs attached and formed a mono layer on the collagen-IV coated and uncoated hydrogels. All gels contracted after 48 hours. The absence of fibroblasts appeared to reduce the degree of contraction. After 4 days of submerged culture the epithelial layer formed a stacked multicellular confluent layer on the hydrogels. After 14 days of air exposure the multicellular epithelial cells on the hydrogels further expanded and the fibroblasts remodeled their environment, as shown by a change in structure and staining intensity around the fibroblasts in the ECM hydrogel seen in the H&amp;E staining.

Conclusions: The combination of a lung ECM hydrogel with a basement membrane like coating made it possible to co-culture primary lung fibroblasts and PBECs in 3D at ALI, generating a morphology resembling the in vivo airway wall. 


Dutch Lung Congress

Airway epithelial cells, fibroblasts and the extracellular matrix: a ménage à trois resembling the airway wall in vivo


copd

extracellular matrix

model

[![](https://assets.underline.io/uploads/markdown_image/1/image/261902fdf187a88bfa74da226ff77186.png)](https://drive.google.com/file/d/1gUViQJJHCSgM38OhwpDoTdN89evCsZ-7/view?usp=sharing)
[![](https://assets.underline.io/uploads/markdown_image/1/image/1605292c232e6f185c7f87ee38e8502a.png)](https://drive.google.com/file/d/1kPkznVX_6cx-n846z_pUtyVDZql9KImc/view?usp=sharing)


### **Welcome Message**

Welcome to the inaugural **Dutch Lung Congress.**  We have all missed our opportunities to network and interact with our colleagues in the last twelve months or more.  While our desire was to meet face to face once again, when it became evident that this would not be possible until much later in 2021 (or even into 2022) the hard working committee organizing the Dutch Lung Congress found, what we hope will be, an excellent alternative to facilitate our annual meeting.

The Dutch Lung Congress aims to bring together everyone interested in research related to pulmonary diseases in the Netherlands.  We hope this event will provide opportunities for multidisciplinary interactions to enhance our approaches for addressing these challenging diseases.  Each session will include input from patients, clinical and basic and/or translational scientists discussing the latest information around the central themes chosen by the organizing committee.  One advantage of holding our congress as a virtual event this year is that we will have the opportunity to hear from world leading experts from all over the globe.  We hope you enjoy the programme the organizing committee have put together and will engage in dynamic discussions with all the speakers.

One highlight of our congress will be the Young Investigator Session.  Remember to join us for this exciting session when we will hear from the presenters who submitted the top scoring abstracts.  This promises to be a session full of exciting science that will display the strengths that constitute the future talent in our field.

When we are not listening to these great presentations we can all meet in Gathertown.  Within this interactive platform you will be able to meet colleagues and friends for networking. Enjoy a private chat over a virtual coffee, visit the booths of our sponsors and other parties, chat with the Young Investigator Board members or take some timeout in the relaxation spaces.  While in Gathertown take the opportunity to visit all the diverse posters on display. Chat live with the presenters or leave them a question on the discussion board for them to engage with you.  Make sure you check for any messages each time you enter Gathertown to make the most of your networking opportunities.

We look forward to seeing you during the DLC and the great scientific (and other) discussions we are anticipating during our 2021 meeting.

On behalf of the DLC organizing Committee
<br>**Janette Burgess
<br>Chair, DLC Organizing Committee**
![](https://assets.underline.io/uploads/markdown_image/1/image/f461fb275d9667fb2a76a30813388e90.png)



### Special Session: Publieksmiddag
![](https://assets.underline.io/uploads/markdown_image/1/image/77e768dd500e6205175e5cafba59abdc.png)






![](https://assets.underline.io/uploads/markdown_image/1/image/f461fb275d9667fb2a76a30813388e90.png)
**More about the committee team:**:
[Barbro N. Melgert, PhD] (https://drive.google.com/file/d/1q_IIH3JvTivumCUzzsZGX7BgD41HxQmk/view?usp=sharing)
// [Bas Holverda, PhD] (https://drive.google.com/file/d/1QKXHmTTQjgs4OZVuM8doXU055S-fu_uU/view?usp=sharing)
// [Frits Franssen, PhD] (https://drive.google.com/file/d/1d7HRUJaqGuRFzkPN3SVgkbBfqFkDkWq2/view?usp=sharing)
// [Janette Burgess, PhD] (https://drive.google.com/file/d/1VHFlrEN6uCQGeeW3Lj4I8aTw7HsB0GFr/view?usp=sharing)
// [Michel van den Heuvel, PhD] (https://drive.google.com/file/d/1JeOEP8MHxiK_4lONr8UM2HaxdxpkAmA2/view?usp=sharing)
// [Patrick Bhairosing] (https://drive.google.com/file/d/1xI8ORq-g1bbVcrlRDviZ9NhAwanc4dFl/view?usp=sharing)
// [Gimano Amatngalim, PhD] (https://drive.google.com/file/d/1fyVDDKARLNjRk_JZk8_GyhGMjmp6DWHp/view?usp=sharing)
// [Peter Kunst, PhD] (https://drive.google.com/file/d/1QhtB4nQezpAjbfUAcBnINc-Bm3Shy2Lf/view?usp=sharing)
// [Dr. Michel van den Heuvel, Prof., MD, PhD] (https://drive.google.com/file/d/1JBdNEBXZCEikEZhJ6MoD5DhQfXMz3bP6/view?usp=sharing)
![](https://assets.underline.io/uploads/markdown_image/1/image/f461fb275d9667fb2a76a30813388e90.png)

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Rationale: In chronic obstructive pulmonary disease (COPD), the airways are characterized by goblet cell hyperplasia, decreased ciliated cell numbers and an altered turnover of the extracellular matrix (ECM). Current 2D (co-)culture systems lack the spatial in vivo environment that is formed by ECM. In this study we aimed to develop a lung ECM hydrogel-based co-culture model to investigate bidirectional interactions between fibroblasts and epithelial cells in relation to ECM changes. 

Methods: Porcine lungs were decellularized, lyophilized and ground to a powder. This powder was porcine pepsin-solubilized for 48h, pH neutralized, buffered with PBS and incubated at 37°C for 30 min to generate ECM hydrogels. Human primary lung fibroblasts (50,000/ml) from non-COPD donors were mixed with hydrogels prior to gelation in transwells. The next day, the hydrogels were coated with collagen type IV or left uncoated. After 24h, 100,000 primary bronchial epithelial cells (PBEC) from non-COPD donors were seeded on top of fibroblast seeded and unseeded gels. The cells were cultured submerged for 4 days and subsequently at air liquid interface for 14 days. The hydrogels were imaged using inverted light microscopy and by histology on paraffin-embedded sections

Results: PBECs attached and formed a mono layer on the collagen-IV coated and uncoated hydrogels. All gels contracted after 48 hours. The absence of fibroblasts appeared to reduce the degree of contraction. After 4 days of submerged culture the epithelial layer formed a stacked multicellular confluent layer on the hydrogels. After 14 days of air exposure the multicellular epithelial cells on the hydrogels further expanded and the fibroblasts remodeled their environment, as shown by a change in structure and staining intensity around the fibroblasts in the ECM hydrogel seen in the H&E staining.

Conclusions: The combination of a lung ECM hydrogel with a basement membrane like coating made it possible to co-culture primary lung fibroblasts and PBECs in 3D at ALI, generating a morphology resembling the in vivo airway wall. 


poster

Introduction: 
Nowadays, identifying disease progression in an early stage has direct treatment implications in patients with fibrotic interstitial lung diseases (F-ILDs). eHealth could be a solution for close monitoring and detecting disease progression, at low burden for patients and clinicians. The main aims of this study are to assess long-term FVC change in patients with F-ILD measured with home spirometry and to evaluate the feasibility of a multinational patient-led registry in F-ILD. In addition, we aim to better validate patient-reported outcomes (PROMs) in patients with F-ILD.
Methods: 
The I-FILE is a prospective multinational observational study, conducted in seven European countries. We aim to include 700 treatment naive patients with newly diagnosed F-ILD. Patients perform daily home spirometry in the first three months followed by once weekly measurements for a period of 2 years. In addition, PROMs will be completed online every 6 months, including the King’s brief Interstitial Lung Disease Health Status, EQ5D-5L, Visual Analogue Scale on cough, dyspnea, fatigue and general complaints, Leicester Cough Questionnaire, Fatigue Assessment Scale , Work Productivity and Activity Impairment Questionnaire, Global Rating of Change Scale and Living with Pulmonary Fibrosis questionnaire. Results are encrypted and directly sent to a secured server through the online I-FILE application.
Outcomes: 
The primary endpoint is change in FVC after 12 months assessed by home monitoring. Measurements will be compared to in-hospital FVC obtained during regular follow up visits. Other endpoints are adherence to daily and weekly home spirometry and validity, consistency and responsiveness of PROMs in patients with F-ILD. 
Conclusions: 
The I-FILE study will be the ultimate collaboration between patients and doctors to provide much needed insights in disease behavior and progression in a large and diverse patient group with F-ILD. Results can be used to optimize monitoring of dieasese course to enable tailored treatment for patients with F-ILD

Study design of an international patient-led registry in fibrotic interstitial lung diseases using eHealth technology: I-FILE

Objective: To determine the cause of work-related asthma.
Method: A city transit bus driver (57 yrs.) of a public transport company with non-allergic, non-eosinophilic asthma developed progressive work-related symptoms of productive cough, wheezing and dyspnea when driving in buses with a crude polyurethane filter (Bus A). Symptoms did not worsen when driving on the only bus with a different type of climate control, including a filter for particles > 0,1 μm (Bus B).
Lung function was monitored during and after passenger transport services with Bus A, on regular routes in a city (Breda) and suburban areas. Subsequently, air quality was measured in 2 buses of type A and in Bus B, on the same day and under standardized conditions during rides without passengers, but following regular routes.
Results: Increased symptoms without significant changes in FEV1 were observed during 3 successive regular rides with Bus A. However, two days afterwards, FEV1 dropped from 2,7 l to 1,9 l (30%). Real time measurements showed constantly lower levels of soot in Bus B. Mean soot concentrations in Buses A were 2,4 and 2,1 μg/m3 and in Bus B 1,2 μg/m3 (Fig. 1). Levels of PM 2,5 did not differ between buses A and B (28-32 μg/m3). In all buses, levels of ultrafine particles depended on outdoor air pollution.
Conclusion: Soot concentrations in Bus A were comparable with levels present in busy streets and urban centres and may be responsible for work-related asthma. 
(Guarnieri M, Balmes JR. Lancet 2014; 383:1581-92).


Work-related meets environmental asthma in a bus driver

Background
Approximately 8% of all births occur between 30-36 weeks of pregnancy. Respiratory tract infections (RTI) and wheezing illnesses disproportionally affect such preterms in early life. Next to the relatively underdeveloped lungs at birth, an unfavourable microbiome associated with an aberrant immune maturation might underlie the susceptibility to RTI. Early- and severe RTI increase the likelihood of subsequent poor respiratory health. Oral bacterial lysates have shown to be effective in the prevention of RTI in children through the interaction with the gut immune system and stimulation of non-specific immune responses. Our systematic review showed reduction of pre-school wheezing episodes and asthma exacerbations after bacterial lysate use (mean difference of -1.40 episodes (95% CI -2.01--0.80 ;figure 1). Also, primary prevention of RTI and wheezing episodes in very young infants is described and currently being studied (Orbex study, USA). However, studies so far excluded preterms despite their extra-ordinary disease burden.

Aim To reduce RTI and wheezing in moderate-late preterms in the first year of life by bacterial lysate administration and to determine the correlation of biological markers with respiratory symptoms, immune protection and treatment effect. 

Methods
This randomised placebo-controlled trial will include 500 otherwise healthy preterm infants (30+0-35+6 weeks). OM-85 or placebo powder will be given from 6-10 weeks after birth until 12 months of age. Clinical data will be continuously collected by e-Health and study visits; with optional biosampling (blood; nasal fluid, brush and swab; faeces) and sleep impedance pneumography at baseline, 6 and 12 months. 

Outcome
Lower RTI and wheezing episodes in the first year of life. Immune maturation, as well as microbiome development in the respiratory tract and gut. Biomarkers for risk-group selection and/or treatment success. 

Impact
We envision finding an affordable therapy for a large patient group thereby improving life-long respiratory health and (family) quality of life. 


Protecting late-moderate preterm infants from respiratory tract infections and wheeze in their first year of life by using bacterial lysates: a study protocol


Chronic Obstructive Pulmonary Disease (COPD) patients frequently develop osteoporosis, severely limiting life quality and increasing hospitalisation. Accelerated bone degradation is traditionally explained by elevated Receptor Agonist of NFKB signalling Ligand (RANKL) levels in the bone, resulting in osteoclast activity, however, COPD patients with dominant emphysema also presented with elevated serological levels of RANKL (Bai et al, 2011). Recent data from Woldhuis et al shows that RANKL is produced by lung fibroblasts, and that in COPD, compared to a plethora of secreted cytokines, RANKL secretion has the highest fold increase. Also, local production of the inflammatory cytokine IL1β is known to be a potent inducer of RANKL in osteoblasts, which in the inflamed COPD lung could translate to increased RANKL production. However, it is currently unclear whether IL1β also induces local RANKL production in the lung. We hypothesize that IL1β induces RANKL expression in lung fibroblasts, resulting in higher production and secretion of RANKL. To test this, we first explored a single-cell sequencing dataset generated by Lee et al. comprising IL1β treated murine lung organoids. Re-analysis showed that stromal cells in this model strongly upregulated RANKL in response to IL1β. To further explore the effects of IL1b, primary fibroblasts from COPD lung were treated with il1b for 48 hours. Although free RANKL was not detected in culture supernatants in either condition, levels of the RANKL/osteoprotegerin (OPG) complex were increased under influence of IL1β, whereas OPG levels remained constant, indicating that RANKL production indeed increased. Combined, these results show that RANKL production is regulated by IL1β in lung fibroblasts. Also, these data mark the importance of measuring both free- and OPG-bound RANKL, as OPG might obscure any effects of RANKL. Building on this, future research should focus on exploring whether this regulatory effect is altered in COPD by checking both free- and OPG-bound RANKL in lung tissue and serum and examining the possible role of RANKL in lung tissue.

IL1β induces RANKL secretion in human lung fibroblasts

DHA but not EPA reduces IL-8 (KC) cytokine release from lung alveolar epithelial cells
Dan Li, Danique Lobee, Melissa Van der Veen, Hoeke.A. Baarsma, Reinoud Gosens, Martina Schmidt 

Background:
The acute inflammatory response to injury or pathogens protects the host from systemic infection and initiates tissues repair. Recent research showed that resolution of acute inflammation is an active process which is initiated by the omega 3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Here we aim to investigate in the role of DHA and EPA in models of inflammation in the lung epithelium. 
Method:
Human bronchial epithelial (BEAS-2B) cells and human alveolar epithelial (A549) cells were exposed to different concentrations and the indicated time periods (up to 24h) to DHA and EPA (up to 100 μM), followed by stimulation with lipopolysaccharide (LPS) (0.01-100 μg/ml). Cell viability was measured by MTT. The inflammatory markers interleukin-8 (IL-8, IL-6) were measured by ELISA. Expression of IkkB, IkB-a, p65 expression has been detected by western blot and immunofluorescence. To translate the findings from the 2D cell cultrues to the 3D cell cultures, a mouse organoid model comprised of a co-culture of primary mouse CD31-CD45-Epcam+ cells with CCL206 fibroblasts was used. 
Results:
DHA and EPA alone and in combination with LPS did not affect the cell viability in both BEAS-2B and A549 cells. In BEAS-2B cells, DHA and EPA did not reduce the LPS-stimulated IL-8 and IL-6 release. On the contrary, in A549 cells DHA (60μM) but not EPA significantly reduced the LPS-induced IL-8 release, leaving the IL-6 release unchanged. Total IKKB and p65 expression was not changed by DHA, EPA, measured alone or in combination with LPS. The p-IKKa/b, IkB-a expression and p-65 nuclear translocation are being researched now by Western blot and immunofluorescence. Importantly, we found in the organoid model that LPS induces mouse KC release, a process significantly reduced by DHA but not EPA. 
Conclusion: 
DHA but not EPA may reduce the inflammatory response in lung alveolar epithelial cells by suppressing IL-8 (KC) release. The mechanism involved still needs to be studied further. 


DHA but not EPA reduces IL-8 (KC) cytokine release from lung alveolar epithelial cells

Allergen-specific subcutaneous immunotherapy (SCIT) is a treatment for allergic airway disease that depends on regulatory T cell (Tregs) induction by dendritic cells (DCs) to achieve long-term tolerance. Linkage of specific sialic acid residues to antigens (sialylation) enhances their binding to immune inhibitory Siglec receptors on DCs, instructing a tolerogenic phenotype. Here, we test whether use of sialylated rPhleum pratense 5a (Sia-rPhl p5a), or Sia-Phl p5a- and Phl p1-derived peptides, can enhance efficacy of SCIT in a grass pollen (GP)-driven mouse model of allergic asthma.
CFSE labeled CD4+ T-cell activation by (un)modified peptide-loaded DCs was measured in vitro to select responsive peptides. Next, BALB/cByJ mice were GP sensitized, treated with injections of saline, (un)sialylated rPhl p5a, Phl p1 or Phl p5a-peptides, or GP extract, followed by GP-challenges. We measured immunoglobulins, ear swelling responses (ESR), inflammatory cells and cytokines in broncho-alveolar lavage (BAL) and lung tissue and airway hyperresponsiveness (AHR).
T-cells isolated from GP-sensitized mice showed increased proliferation and higher production of TGF-β1 in response to sia-peptide-loaded DCs in vitro, as compared to their unsialylated controls. In vivo, SCIT using GP and rPhl p5a effectively suppressed AHR and airway inflammation. Sia-rPhl p5a SCIT significantly enhanced suppression of ESR and AHR as well as IL-5 and IL-13 levels and eosinophilia in BALF compared to unmodified rPhl p5a. In addition, sialylation of Phl p5a-peptides, but not of Phl p1a-peptides, enhanced suppression of ESR and AHR.
Sialylation of rPhl p5a (full protein or peptides) leads to superior suppression of allergic inflammation, while Phl p1-peptides were ineffective, also after sialylation. Use of the recombinant full protein induces neutralizing antibodies, which was not enhanced by sialylation. SCIT with Sia-rPhl p5a and or peptides is safe in our experimental mouse model. Glycan-allergen conjugates have the potential to be developed as novel improved SCIT treatment.


Glycan modification of rPhl p5a leads to superior suppression of allergic inflammation in a SCIT mouse model of allergic asthma

Spirometry is essential in the diagnosis of airway disease and can be useful in monitoring patients. Despite the essential role, spirometry remains largely underused in primary care. Currently, due to COVID-19, the use of office spirometry is contraindicated in many countries. Furthermore, post COVID-19 spirometry in general practices can be optimalized. Spirometric devices are costly and personnel requires special training. Referral for spirometry increases the cost for patients and lowers the feasibility. Spirometry takes up significant amounts of time that could be used for extra information gathering via more extensive conversation and questionnaires. Spirometry in general practices can be a problem for airway disease that is presenting in specific situations, such as exercise-induced asthma. In recent years, handheld spirometry linked to phones/apps has been developed for study purposes and remote monitoring. 

In this implementation study, we will evaluate whether a spirometry test performed at home with NuvoAir would be a feasible and good alternative to spirometry performed at the general practice office.


Home spirometry for the diagnosis and monitoring of asthma and COPD in primary care: an implementation study

Aim
We aimed to identify facilitators and barriers of adherence to multimorbid exacerbation action plans in patients with COPD and comorbidities.

Methods
Individual semi-structured interviews on patients’ experiences with use of multimorbid symptom diaries and exacerbation action plans in a self-management trial (the COPE-III study) were conducted with patients with moderate to severe COPD and ≥1 comorbidity. Interview data were thematically analysed using the Capability, Opportunity and Motivation-B model to understand behaviour regarding patient adherence to multimorbid exacerbation action plans. 

Results
Ten patients (5 Australian, 5 Dutch; 6 men, age 59-83 years) were interviewed (Table 1). Patients indicated several distinctive perspectives on their own role and healthcare providers’ roles towards disease management (Table 2). Facilitators of adherence included the availability of social support, taking responsibility for disease management, and improved disease comprehension. Barriers to adherence included aversion against daily symptom monitoring and medication use, complexity of symptom diaries and action plans, and difficulties to recognise increased symptoms and to differentiate between COPD symptoms and comorbid symptoms.

Conclusion
To optimise adherence to multimorbid exacerbation action plans, self-management training should be tailored to patients’ level of understanding, directed towards comprehension of COPD, comorbidities and medication, symptom recognition and differentiation between COPD and comorbid symptoms, and focus on individual patients’ roles towards disease management to enhance patients’ responsibility. Our results also highlight the importance of implementation of effective social support strategies in COPD self-management interventions to further improve adherence.


Facilitators and barriers of adherence to exacerbation action plans in patients with COPD and comorbidities: a qualitative study

Background: IL11 was recently reported to induce the formation of myofibroblasts and their secretion of collagen, and its subcutaneous administration drives the development of pulmonary fibrosis in mice. While the role of IL11 on myofibroblast differentiation is described, less is known as to how IL11 affects epithelial-mesenchymal communication associated with epithelial repair. Furthermore, it is not yet well-established which cell types are the main producers and responders to IL11 in vivo.

Here we aim to investigate the role of IL11 in alveolar regeneration.

Methods: Epithelial progenitor cell behavior was studied using an organoid model, comprised of a co-culture of primary mouse CD31-CD45-Epcam+ cells with either CCL206 fibroblasts or primary human fibroblasts. After 14 days of growth, the number and size of the organoids were determined. Additionally, immunofluorescent staining for Surfactant protein C (SPC) and acetylated tubulin (ACT) was performed to define alveolar vs airway organoids. Immunohistochemical staining of IL11 and IL11 receptor was performed on human lung tissue to observe producer and responder cells of IL11 signaling.

Results: The presence of rhIL11 significantly decreased the number of both mouse and human organoids formed in a concentration-dependent manner. Additionally, the fraction of SPC expressing organoids was concentration-dependently reduced by rhIL11 whereas the fraction of SPC-ACT- organoids was increased. Organoid size was not affected by rhIL11. Finally, immunohistochemistry revealed that IL11R is found in the smooth muscle, airway epithelium, endothelium and macrophages, whereas IL11 was primarily observed in the airway epithelium, alveolar type 2 cells and macrophages.

Conclusions: IL11 may impair epithelial progenitor function by suppressing their activation and by suppressing the formation of mature alveolar cell types. IL11R localization suggests mesenchymal activation by IL11 may contribute to the observed effects on progenitor function, possibly through mesenchyme-epithelial crosstalk.

IL11 negatively impacts adult lung alveolar organoid formation

Nasal high-flow therapy (NHFT) appears to be a suitable treatment in COVID-19 patients, but healthcare professionals are reluctant to use the therapy on the ward. This observational study included 86 COVID-19 patients in whom NHFT was initiated on the ward. As a control group, 43 patients were retrospectively included. In patients with a restricted medical policy, 51% of the patients in the NHFT group recovered compared to 20% in the control group. In patients with a full medical policy, ICU admission was 71% in the NHFT group compared to 46% in the control group. Overall, NHFT did not lead to increased or delayed intubations. Furthermore, the overall mortality rate and the mortality rate after intubation is similar in both groups (control 15%, NHFT 17%). The study shows that NHFT on the ward is feasible and effective in both patients with a full medical policy and in patients with a restricted policy.

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