M2 macrophages play an important role in tissue repair and regeneration. They have also been found to modulate β-cell replication in mouse models of pancreatic injury and disease. We previously reported that β-cell replication is strongly increased in a subgroup of human organ donors characterized by prolonged duration of stay in an intensive care unit (ICU) and increased number of leukocytes in the pancreatic tissue. In the present study we investigated the relationship between duration of stay in the ICU, M2 macrophages, vascularization, and pancreatic cell replication. Pancreatic organs from 50 donors without diabetes with different durations of stay in the ICU were analyzed by immunostaining and digital image analysis. The number of CD68+CD206+ M2 macrophages increased three- to sixfold from ≥6 days’ duration of stay in the ICU onwards. This was accompanied by a threefold increased vascular density and a four- to ninefold increase in pancreatic cells positive for the replication marker Ki67. A strong correlation was observed between the number of M2 macrophages and β-cell replication. These results show that a prolonged duration of stay in the ICU is associated with an increased M2 macrophage number, increased vascular density, and an overall increase in replication of all pancreatic cell types. Our data show evidence of marked levels of tissue repair in the human donor pancreas.

Diabetes is a heterogenous group of disorders characterized by a decreased functional β-cell mass, chronic hyperglycemia, and debilitating chronic complications. Type 1 diabetes has an autoimmune etiology with a T-cell–mediated destruction of most of the pancreatic β-cell mass, while type 2 diabetes is characterized by a more limited loss of β-cell mass in combination with insulin resistance and deficient β-cell function (13). Although β-cells show replication in fetal and neonatal life (4,5), they are generally considered to be long-living cells that rarely divide in adults (57). The relatively low capacity for growth and regeneration of adult human β-cells is thought to limit their capacity to compensate under conditions of decreased insulin availability. Interventions aimed at restoring a functional β-cell mass by stimulating β-cell replication would be of considerable interest from a clinical point of view. Although β-cell replication is rare in the adult human pancreas, we previously reported that some individuals display high levels of replication (8). Staining for the replication marker Ki67 in a large cohort of donor pancreata and statistical testing for a series of clinical parameters showed that in addition to young donor age, several factors related to the duration of stay in an intensive care unit (ICU) were significantly correlated with increased β-cell replication. Histopathological parameters that correlated with increased replication consisted of an increased number of leukocytes in the pancreatic parenchyma (8). As the alternatively activated (M2) macrophage subtype is thought to be of particular importance in regeneration, tissue repair, and angiogenesis (9,10) and as emerging data suggest a possible role for these M2 macrophages in the protection and replication of β-cells in experimental animals (11,12), we investigated the relationship between M2 macrophages, vascularization, and β-cell replication in a large series of human donor pancreata with increasing duration of stay in an ICU.

Collection of Pancreatic Tissue

Pancreatic biopsies of the body of the pancreas were taken as part of a quality control procedure in the framework of an islet transplantation program, approved by the local ethics committee (approval number BUN143201732095), and stored in a registered Biobank (Diabetes Biobank Brussels, local ethics committee approval number BUN143201524128). A single biopsy sample was taken from the body part of the gland, fixed in phosphate-buffered 4% formaldehyde, and embedded in paraffin. The study included 50 organ donors without diabetes and with different durations of stay in the ICU (0, 3, 6, 9, ≥12 days). The groups were matched for age, BMI, and sex (Table 1).

Immunostaining

Paraffin-embedded tissue blocks (4-µm sections) were immunostained as follows: 1) For characterization and quantification of the number of leukocytes in pancreatic tissue, sections were double stained using guinea pig anti-insulin (Diabetes Research Center [DRC], Vrije Universiteit Brussel) in combination with mouse anti-CD68 (clone KP1; Agilent Technologies, Heverlee, Belgium), mouse anti-CD45 (clone 2B11+PD7/26; Agilent Technologies), mouse anti-CD8 (clone 1A5; Novocastra Reagents, Leica Microsystems, Wetzlar, Germany), and mouse anti-CD20 (clone L26; Agilent Technologies). Specificity of leukocyte markers was determined on sections of human tonsils. 2) Macrophage subtypes were identified using triple staining for guinea pig anti-insulin, rabbit anti–macrophage mannose receptor CD206 (Abcam, Cambridge, U.K.), and mouse anti-CD68 or mouse anti-CD163 (clone 10D6; Novocastra Reagents). 3) Immunoperoxidase staining for mouse anti-Ki67 (clone MIB-1; Agilent Technologies) was performed for the quantification of replication in pancreatic tissue. Additional replication markers included mouse anti-MCM7 (clone 47DC141; Abcam) or rabbit anti–phosphorylated (phospho)–histone H3 (Sigma-Aldrich, Overijse, Belgium), stained in combination with mouse anti-Ki67. 4) For the quantification of the different replicating islet cell types, triple immunofluorescent staining for mouse anti-Ki67, guinea pig anti-insulin, and rabbit anti-glucagon (DRC) or double immunofluorescent staining for rabbit anti-Ki67 (clone SP6; Acris, Herford, Germany) was used in combination with mouse anti-amylase (clone G10; Santa Cruz, Heidelberg, Germany). 5) Demonstrating the different replicating cell types in pancreas tissue was performed by double immunofluorescent staining for rabbit anti-Ki67 in combination with mouse anti-CD68, mouse anti-CK19 (clone RCK108; Agilent Technologies), and mouse anti-vimentin (clone V9; Biogenex, The Hague, the Netherlands). 6) To verify the presence of replicating leukocytes inside the islets, triple immunofluorescent staining for rabbit anti-Ki67 and guinea pig anti-insulin in combination with mouse anti-CD45 and mouse anti-CD68 was performed. 7) For the analysis of apoptosis, immunoperoxidase staining using rabbit anti–cleaved caspase-3 (Cell Signaling Technology, Leiden, the Netherlands) was performed. 8) Double-color immunoperoxidase staining for mouse anti-CD31 (clone JC70; Ventana Medical Systems, Oro Valley, AZ) and guinea pig anti-insulin was performed for the analysis of the vascular density in pancreatic tissue and in islets of Langerhans. Antigen retrieval was performed in a 2100 retriever steam cooker (Aptum, Southampton, U.K.) with citrate buffer (pH 6.0) (ScyTek Laboratories, Logan, UT) for anti-CD68, anti-CD206, anti-CD163, anti-CD20, anti-Ki67, anti–phospho–histone H3, anti-amylase, anti-CK19, anti-vimentin, anti–cleaved caspase-3, and anti-CD31, and with Tris EDTA Buffer Solution (Klinipath, Olen, Belgium) for anti-CD8 and anti-MCM7.

Binding of primary antibodies was detected with 1) immunofluorescence: DyLight 488 anti–mouse IgG (H+L), DyLight 488 anti-guinea pig IgG (H+L), Alexa Fluor 488 anti-rabbit IgG (H+L), Cy3 anti-mouse IgG (H+L), Cy3 anti-rabbit IgG (H+L), and Alexa Fluor 647 anti–guinea pig IgG (H+L) (all from Jackson ImmunoResearch Laboratories, West Grove, PA), or with 2) immunohistochemistry: anti-mouse, anti-rabbit, and anti–guinea pig biotinylated IgG (Vector Laboratories, Burlingame, CA) in combination with the Vectastain Elite ABC kit and Vectastain ABC-AP kit (Vector Laboratories) using the Liquid DAB+ Substrate Chromogen System and Fuchsin+ Substrate Chromogen System (Agilent Technologies) as substrate.

Sections were mounted with fluorescent mounting medium (Agilent Technologies) containing DAPI (10 µg/mL) (Sigma-Aldrich) or with PERTEX mounting medium (Histolab, Göteborg, Sweden).

Image Acquisition and Morphometric Analysis of Leukocytic Cells

For the quantification of the number of leukocytes in pancreatic tissue, sections were digitally imaged using a Pathway 435 inverted fluorescence microscope (Becton Dickinson, San Jose, CA). Per section, 35 adjoining images were analyzed by automatic image analysis, corresponding to a total area of 1.13 cm2. AttoVision imaging software (Beckton Dickinson) was used for segmentation of individual DAPI-stained nuclei based on their DAPI positivity and nuclear size. The analysis was performed on all DAPI-stained nuclei in a given image and thus included both connective tissue and exocrine/endocrine tissue. Positivity for CD68, CD45, CD8, and CD20 was measured in a region of interest (ROI) of 3 pixels (1.8 µm) around the nucleus. A user-defined threshold for the mean intensity of the expression of the marker in the ROI was set for each marker and applied to all cases analyzed for that marker. A total of 2.07 ± 0.04 × 105 (mean ± SEM) nucleated cells were analyzed for each donor. Results per donor were expressed as the amount of nucleated inflammatory cells over the total of nucleated cells. Quantification was validated by a manual recount of selected sections.

To quantify the amount of CD68+ and CD206+ cells, 10 random images of each section were taken with a Nikon Eclipse 80i fluorescent microscope with a 20× objective. The amount of CD68+ and CD206+ cells was quantified manually and expressed as means ± SEM of the cell number/mm2. In sections stained for CD163 in combination with CD206, 10 random images were taken with a Nikon Eclipse 80i fluorescent microscope with a 20× objective and analyzed manually. The copositivity for CD206 and CD163 was analyzed and expressed as the percentage of CD206+ cells that were CD163+ and vice versa. The amount of CD68+ and CD206+ cells inside the islets was quantified manually in all islets and expressed as the average amount per islet.

Quantification of Replication and Apoptosis

The replication in the pancreatic tissue was quantified in sections stained for Ki67. The sections were digitally imaged with an Aperio CS2 slide scanner. The amount of Ki67+ cells was evaluated manually in 10 random image fields and expressed as means ± SEM of cell number/mm2. Replication was confirmed in sections from donors with 0 and ≥12 days in the ICU by additional staining for MCM7 or phospho–histone H3 in combination with Ki67. The amount of MCM7+ and phospho–histone H3+ cells was evaluated in 10 random image fields and expressed as mean ± SEM of cell number/mm2.

Slides triple stained for Ki67, insulin, and glucagon were scanned with an Eclipse Ti fluorescent microscope (Nikon) with a 20× objective. If possible, at least 1,000 insulin- and glucagon-positive cells were evaluated manually, and the results were expressed as the percentage ± SEM of insulin- or glucagon-positive cells that were positive for Ki67. Per section, 1,000 amylase-positive cells were analyzed manually for Ki67 positivity on slides double stained for Ki67 and amylase. The results were expressed as percent ± SEM of amylase-positive cells positive for Ki67.

The presence of Ki67+ leukocytes inside the islets was verified manually on selected slides stained for Ki67 and insulin in combination with CD45 or CD68.

Apoptotic cells in pancreatic tissue were quantified in sections stained for cleaved caspase-3. Sections were digitally imaged with an Aperio CS2 slide scanner and evaluated manually. Human lymph node tissue, duodenum, and pancreas obtained from a patient with acute pancreatitis were used as positive controls.

Image Acquisition and Morphometric Analysis of Vascular Density

Sections stained for CD31 and insulin were digitally imaged with an Aperio CS2 slide scanner. Pancreatic and islet vessel area was measured semiautomatically by ImageJ software. Image processing was performed with Fiji, an open-source distribution of ImageJ (version 1.48d; National Institutes of Health, Bethesda, MD), allowing user extensibility via Java plugins. In a first step, an ImageJ macro was created to automatically select the hematoxylin-positive area on each section as a representation of total tissue ROI. These tissue boundary ROIs were subsequently corrected for false-positive signals. Second, fixed thresholds were applied to select the insulin+ and CD31+ signal within the boundary ROIs. The fixed threshold was chosen as the median threshold that correlated most accurately with the specific signal in 3 random fields of 3 random sections in 10 samples. In manually verified analyses, the insulin and CD31 ROIs were corrected for false-positive and false-negative signals, which were manually deselected or included, respectively. Manual verification was performed by checking the conformity of each separate ROI with the corresponding insulin+ or CD31+ area. Third, for the quantification of the pancreatic areas, islet areas, and vessel areas, the plane areas of the manually verified and automatically determined boundary ROIs were measured. Finally, an output file was generated and saved for peer evaluation of each analysis. This output file can easily be overlaid on the source image to confirm vessel and islet outlines and trace the individual particles. Vascular density in individual donors was expressed as the total CD31+ area over the total pancreatic or total insulin+ islet area per tissue section that was analyzed.

Statistical Analysis

A permutation test was used to avoid making strong distributional assumptions and to deal with a limited sample size. Permutation tests construct the null distribution assuming that grouping (days in the ICU) is irrelevant in order to evaluate how likely the actual observations are. This type of test also provides CIs that allow for testing differences between groups or, in other words, makes it possible to verify that scores on different days differ. Such analysis is performed to establish pairwise differences between the observations obtained at 0 days in the ICU and each of the conditions with 3, 6, 9, or ≥12 days in the ICU. Significance of differences was calculated at the 50th percentile (median) level with multiple testing adjustment of the P values using the method of Hommel (13). The distribution of scores is visualized using boxplots enhanced with data points. The pancreas weight per unit BMI was regressed on the duration of stay in the ICU, both linearly and curvilinearly, using polynomials of first and second order. Spearman correlation was used to examine the relationship between the different parameters. All statistical tests were performed using the R Companion package (R Companion: Functions to Support Extension Education Program Evaluation. R package version 2.0.0. https://CRAN.R-project.org/package=rcompanion; Mangiafico, 2018).

Data and Resource Availability

The detailed data sets generated during and/or analyzed during the current study are available from the corresponding author upon request.

Prolonged Duration of Stay in the ICU Is Accompanied by an Increased Number of Pancreatic M2 Macrophages

Pancreas tissue of organ donors with a short duration of stay in the ICU (0 or 3 days) showed low levels of CD68+, CD45+, CD8+, and CD20+ cells. In contrast, a significant increase was observed in pancreata of donors with 6, 9, or ≥12 days of stay in the ICU, with a marked elevation of CD68+ and CD45+ cells and a lesser rise of CD8+ and CD20+ cells (Figs. 1 and 2).

The M2 macrophage subtype was assessed by the presence of CD206 and CD163. The number of CD68+CD206+ macrophages showed a significant three- to sixfold increase in donors with 6, 9, and ≥12 days of stay in the ICU (Fig. 3A and B). Overall, 73.4 ± 1.8% (mean ± SEM) of CD68+ cells expressed CD206 and virtually all CD206+ cells were CD163+ (data not shown). Within the islets of Langerhans, the number of CD68+ and CD68+CD206+ macrophages was twofold higher in donors with 9 days of stay in the ICU compared with donors with a short duration of stay in the ICU (Fig. 3C and D). CD45+, CD8+, and CD20+ cells were present in low numbers inside the islets, without significant changes between donor groups (data not shown).

Replication and Apoptosis of Pancreas Cells

Pancreas sections from organ donors with different durations of stay in the ICU were immunostained for the replication markers Ki67 (Fig. 4A–I), MCM7 (Fig. 4J), and phospho–histone H3 in combination with Ki67 (Fig. 4K and L). Ki67+ cells were found throughout the tissue, including connective tissue, acinar cells, ductal cells, macrophages, and endocrine β- and α-cells. The identity of these cells was ascertained by double immunostaining for cell type–specific markers vimentin, amylase, CK19, CD68, insulin, and glucagon, respectively.

A significant fourfold increase in Ki67+ cells/mm2 pancreas was observed in donors with 6 days of stay in the ICU and an eight- to ninefold increase in donors with 9 and ≥12 days of stay in the ICU (Fig. 5A). Triple staining for Ki67, insulin, and glucagon showed a significant fivefold increase in Ki67 positivity in both β- and α-cells at 9 and ≥12 days of stay in the ICU (Fig. 5B and C). Double staining for Ki67 and amylase showed a significant 2.5- to 5-fold increase in Ki67 positivity in acinar cells at 6, 9, and ≥12 days of stay in the ICU (Fig. 5D). Immunostaining for MCM7 or phospho–histone H3 and Ki67 in donors with 0 and ≥12 days of stay in the ICU confirmed replication in these cells, with a significant ninefold increase in positivity between 0 and ≥12 days of stay in the ICU (Fig. 5E and F). No replicating leukocytic cells were found inside the islets.

Apoptosis was evaluated using staining for cleaved caspase-3 on pancreas sections from all 50 donors. Less than 0.005% of pancreas cells showed positivity for cleaved caspase-3, and no significant differences were found between groups (data not shown).

Vascular Density

Immunostaining for the endothelial cell marker CD31 (Fig. 6A) showed a significant two- to threefold increase in vascular density in pancreata of donors with 6, 9, or ≥12 days of stay in the ICU versus day 0 (Fig. 6B). Within islets, no significant differences in vascular density were observed between the different conditions under study (data not shown). As a prolonged duration of stay in the ICU could potentially affect pancreas size, and as this would influence the relative vessel area (CD31+ area/total pancreas area), we investigated whether prolonged stay in the ICU was associated with a change in pancreatic weight. We obtained whole pancreas weights from 394 organ donors from the Diabetes Biobank Brussels (age 15–50 years; stay in the ICU ≤15 days) and normalized pancreas weight to BMI for each individual donor. The normalized weight is referred to as “relative pancreatic weight.” We performed regression analysis of the relative pancreatic weights versus the duration of stay in the ICU, but no statistically significant relations were found (Fig. 7). Donor characteristics, including administration of vasopressors and insulin, are given in Supplementary Table 1.

M2 Macrophage Number Correlates With Replication Level and Vascular Density

A strong correlation was found between the number of CD68+CD206+ macrophages and the number of Ki67+ cells/mm2 of pancreatic tissue (r = 0.70) (Fig. 8A). Similarly, the number of CD68+CD206+ macrophages/mm2 strongly correlated with the percentage of Ki67+ β-cells (r = 0.63) (Fig. 8B), Ki67+ α-cells (r = 0.59) (Fig. 8C), and CD31+ vascular density (r = 0.61) (Fig. 8D). The number of CD68+CD206+ macrophages per islet only weakly correlated with the percentage of Ki67+ β-cells (r = 0.34) (Fig. 8E) and Ki67+ α-cells (r = 0.27) (Fig. 8F).

In this study we investigated the number of M2 macrophages present in human pancreas donors with prolonged duration of stay in the ICU. We observed markedly elevated levels of M2 macrophages from 6 days of ICU stay onward and found a strong correlation between increased M2 macrophages, vascular density, and overall cellular replication. Our results indicate that pancreas donors with prolonged duration of stay in the ICU show marked histopathological signs of tissue repair.

We previously reported that β-cell replication is rare in human donor pancreas, with a virtual absence of replicating β-cells in most of the 363 organ donors investigated (8). However, levels of up to 7% replicating Ki67+ β-cells were found in a small subset of donors. Univariate analysis showed that prolonged duration of stay in the ICU, kidney dysfunction, relatively young donor age, increased numbers of CD45+ leukocytes and CD68+ macrophages, prolonged brain death, and use of steroids were all found to be significantly associated with an increased number of replicating β-cells (8).

In the present study we investigated the potential role of M2 macrophages in the pancreas of patients with a prolonged duration of stay in the ICU. Such analysis is limited by the static nature of the fixed pancreas samples available, and these limitations should be taken into consideration when discussing potential mechanisms and processes involved. M2 macrophages play an important role in tissue repair and regeneration in mice (1417). Several studies suggest that M2 macrophages can also play a role in β-cell protection and replication. Macrophage-deficient mice showed defects in pancreatic islet development and a decreased β-cell mass (18). Other studies showed macrophage-promoted angiogenesis in mouse islets, protection against islet loss, and stimulation of islet cell replication during chronic pancreatitis (19). In addition, M2 macrophages were shown to stimulate β-cell regeneration and replication in different rodent models of pancreas injury, including partial duct ligation (2026). It should be noted that macrophages are extremely plastic cells and that the classification into proinflammatory M1 macrophages and regulatory M2 macrophages is an oversimplified description of the heterogeneity of macrophages (9,11).

Our study confirms that an increased duration of stay in intensive care is associated with an increased number of leukocytes in the pancreatic tissue, especially CD68+ macrophages. We showed that most of the CD68+ macrophages expressed M2 macrophage–specific markers, possibly indicating an ongoing tissue repair process in the pancreatic tissue. We therefore investigated whether the increased number of M2 macrophages, observed from 6 days of ICU stay onward, was accompanied by histopathological changes indicative of tissue repair. We observed that a prolonged duration of stay in the ICU and an increased number of M2 macrophages were accompanied by markedly increased levels of replication in many cell types present in the pancreas and by an increased vascular density. Both endocrine and acinar cell replication were increased in donors with prolonged duration of stay in the ICU, while replication was also present in connective tissue components and ductal structures. Furthermore, the vascular density was found to be increased in donors with 6, 9, or ≥12 days of stay in the ICU. The strong statistical correlation found between the number of M2 macrophages, the number of replicating cells (including β- and α-cells), and vascular density suggests that M2 macrophages are involved in the stimulation of both replication and angiogenesis.

It should be noted that increased vascular density can be caused by several processes, including vessel neoformation, atrophy of the acinar component, or dilatation of vessel diameter. No evidence was found of decreased relative pancreatic weight with time in the ICU, making it unlikely that atrophy plays a role in the observed changes.

Presently, the underlying events driving the increase in the number of M2 macrophages in pancreatic tissue are not clear. Hypoxia has been described to promote M2 macrophage infiltration and polarization (27,28) and was also reported to lead to increased vascularization (29). As discussed previously (8), ischemic tissue damage sustained as a result of the events leading to admission to the ICU, often due to head trauma or a (cerebro)vascular accident, could form one possible explanation. A transiently decreased perfusion of the pancreatic gland may lead to cell death, necrosis, infiltration of inflammatory cells, and finally repair, including cell replication and vascularization induced by M2 macrophages (30). Such a mechanism would be compatible with the results in experimental animals where inflammatory conditions were found to stimulate macrophage infiltration in addition to islet and β-cell replication (11,12). However, the lack of clear signs of necrosis or apoptosis in any of the donor organs that were investigated in the current study argues against such a mechanism.

Other pathways potentially leading to tissue damage and subsequent repair may include an increased expression of proinflammatory markers described in donor liver and kidney in organs from patients with a prolonged duration of stay in the ICU, traumatic death, and episodes of infection (3133). Prolonged brain death in combination with hemodynamic instability was found to lead to progressive inflammation, leukocyte infiltration, and organ dysfunction in rodent liver and kidney (34,35). The effect of such conditions on donor pancreas was not previously studied, although brain death in rodents was reported to lead to increased cytokine and chemokine expression, including interleukin-1β, interleukin-6, tumor necrosis factor-α, and MCP-1, in isolated islets (36) and to increased leukocyte adherence in postcapillary venules in rodent pancreas (37). Furthermore, a recent study demonstrated that β-cells isolated from brain-dead donors showed signs of stress even before islet isolation and culture (38). In this context, it would be important to investigate whether increased cytokine signaling can be demonstrated in donor organs prior to the increase in M2 macrophages and proliferation. The observations in the current study may not be limited to the pancreas, and analysis of other human donor organs, including kidney and liver, would be of interest.

A complicating factor in the interpretation of the present data is that patients in an intensive care setting often show altered metabolism and nutritional demands, with notable impact on pancreas function and potentially also on the macrophage population and vascular density.

There are conflicting reports in the literature about the influence of the clinical history of the donor on the extent and time course of histopathological changes in the human donor pancreas (39). Using a cohort of North European organ donors, we found major changes in the number of leukocytic cells, replication, and vascularization in donors with 6–12 days’ duration of stay in the ICU. Other studies, using a U.S. cohort of organ donors (7,40,41), were unable to find such changes, although these studies often focused on donors with a relatively short duration of stay in the ICU. In addition to differences in duration of stay in the ICU, different clinical treatment protocols and different medication around the time of organ donation may also play a role in explaining differences in results. Larger cohorts of pancreas donors with different clinical backgrounds should be investigated to study these differences in more detail.

In conclusion, our results show that prolonged duration of stay in the ICU is associated with an increased number of M2 macrophages, increased vascular density, and an overall increase in replication of all pancreatic cell types. Our data suggest that prolonged stay in the ICU is associated with markedly increased levels of tissue repair in the human donor pancreas.

Acknowledgments. The authors thank Sabrina D’Haese, Nicole Buelens, and Ann Demarré from the Vrije Universiteit Brussel and the staff at the Department of Pathology at the Universitair Ziekenhuis Brussel for expert technical assistance.

Funding. This study was supported by the Fonds Wetenschappelijk Onderzoek (Research Foundation Flanders) (G019211N).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. S.S. interpreted the results and wrote the manuscript. S.S. and L.R. performed the experiments and collected the data. S.S., D.L.D.P., N.D.L., and P.i.V. designed the study. G.S., G.L., and Z.L. designed the macros and script used for the automated images analysis. W.C. provided statistical expertise and was responsible for the statistical analyses. N.D.L. and P.i.V. reviewed and edited the manuscript. All of the authors read the manuscript and approved the version to be published. P.i.V. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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