University of British Columbia Pulmonary Research Laboratory, St. Paul's Hospital, Vancouver, British Columbia, Canada V6Z 1Y6
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ABSTRACT |
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We examined the bone marrow response and the sequestration of polymorphonuclear leukocytes (PMNs) in lung using a bacteremic infection model in rabbits. PMNs were labeled with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) in the bone marrow, and the bone marrow release and the sequestration of BrdU-labeled PMNs were measured using immunohistochemistry. A focal subcutaneous infection (S) was induced, and the bacteremia (B) was produced 4 h later with Streptococcus pneumoniae (S+B). This S+B group was compared with other groups with only subcutaneous infection or only bacteremia. The S+B group developed a profound leukopenia after the bacteremia that was associated with an increase in circulating BrdU-labeled PMNs. Morphometric studies showed more PMN sequestration in the lung of the S+B group compared with the others (P < 0.05). Compared with unlabeled PMNs, BrdU-labeled PMNs, which represent newly released PMNs, preferentially sequestered in lung (P < 0.05) and were slow to migrate into the infected tissues (P < 0.05). We conclude that bacteremic infection is associated with an accelerated release of PMNs from the bone marrow and that these newly released PMNs preferentially sequester in lung and are slow to migrate into infected tissues.
neutrophil; granulocytopenia; Streptococcus pneumoniae
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INTRODUCTION |
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POLYMORPHONUCLEAR LEUKOCYTES (PMNs) have been implicated in the pathogenesis of acute lung injury observed in bacteremic infection (2, 36). These PMNs are recruited to the lung from both the circulating pool of cells and the bone marrow. Several studies have shown that younger PMNs in the bone marrow have different structural qualities (30) and functional capabilities (1, 5, 18) compared with mature PMNs in the peripheral blood. We postulate that these functionally immature PMNs could play an important role in PMN-induced lung injury. Pneumococcal pneumonia shortens the transit time of PMNs through both the mitotic and the postmitotic pools in the bone marrow and releases younger immature cells into the circulation (35). A recent study (33) from our laboratory showed that the bacteremia after a focal pneumococcal pneumonia accelerates this bone marrow response, possibly because the preceding focal pneumonia primed the bone marrow for a more intense response.
The mechanisms for the sequestration and migration of PMNs in the pulmonary vascular bed are different from the mechanisms in the systemic vascular bed. First, the longer transit time of PMNs through the lung capillaries than through systemic capillaries (21-23) increases the exposure of PMNs to inflammatory mediators and cytokines on activated capillary endothelium in infected lung tissue. Furthermore, mechanical deformation of PMNs in pulmonary capillaries causes F-actin assembly and upregulation of CD11b, which are factors involved in PMN sequestration and recruitment (27). Second, the molecular mechanisms for PMN recruitment in pulmonary circulation are different from the mechanisms in the systemic vascular bed (15). Third, the abundant alveolar macrophages present in the lung are capable of releasing cytokines that stimulate the bone marrow and activate both circulating PMNs and capillary endothelium (19). A recent study from our laboratory (33) has shown that a focal infection in the lung primed the bone marrow response with a subsequent bacteremia. This study was designed to determine the bone marrow response and the sequestration of PMNs in lung capillaries using a focal subcutaneous infection model with or without bacteremia, with focus on the behavior of newly released PMNs.
The thymidine analog 5-bromo-2'-deoxyuridine (BrdU) was used to label the dividing PMNs in the bone marrow, which allowed us to follow these labeled PMNs through the bone marrow, measure their release into the circulation, and determine their sequestration in the lung and their migration into the infected tissue (6, 29, 35).
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MATERIALS AND METHODS |
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Animals. Adult female New Zealand White rabbits (n = 19, weight = 2.5 ± 0.2 kg) were used in this study, and all of the experimental procedures were approved by the Experimentation Committee of the University of British Columbia. Three groups were studied: 1) rabbits with a focal subcutaneous infection followed by bacteremia (S+B; n = 7), 2) rabbits with only a focal subcutaneous infection (S; n = 6), and 3) rabbits with only bacteremia (B; n = 6).
BrdU labeling of PMNs. All of the animals received BrdU (100 mg/kg; Sigma, St. Louis, MO) injected into the marginal ear vein at a concentration of 10 mg/ml in pyrogen-free saline over 15 min.
Experimental protocol. Twenty hours after the infusion of BrdU, a focal subcutaneous infection was induced in the S+B and S groups using the method previously described (15) by our laboratory (time 0). Briefly, all rabbits were anesthetized with ketamine hydrochloride (35-50 mg/kg im) and xylazine (5 mg/kg im). Streptococcus pneumoniae (1.5 × 109 organisms) were mixed in 0.5 ml of sterile saline and 1% colloidal carbon (used to mark its distribution of the infection). This suspension was instilled into a sponge, and the sponge was inserted in the subcutaneous space of the lateral abdominal wall through an incision in the midline of the abdomen. Blood samples were taken for blood culture at 0, 2, 4, and 8 h after the induction of the subcutaneous infection. These blood cultures confirmed that the animals do not become bacteremic with subcutaneous infection over the 8-h period of study. Four hours after the induction of subcutaneous infection, pneumococcal bacteremia was induced in the S+B and B groups by mixing Streptococcus pneumoniae (3.0 × 109 organisms) in 20 ml of warm (37°C) sterile saline and infusing the mixture into the marginal ear vein. In the preliminary experiment, this dose of bacteria was established to be nonlethal but to induce a significant leukopenia and to result in significant positive blood cultures (mean 8.1 × 106 colony-forming units/ml; n = 8) 15 min after the infusion. Rabbit sedation was maintained with additional anesthetic as required and an infusion of normal saline (10 ml/h) with the rabbit in a prone position throughout the experiment. Blood samples (3 ml each) were taken at 0 and 2 h after the treatment with BrdU and hourly between 4 and 8 h. Bone marrow samples were aspirated from the iliac bone at 0, 4, and 8 h. After the last sample, the rabbits were killed with an overdose of pentobarbital sodium, the chest was opened rapidly, and the base of the heart was ligated to maintain the pulmonary blood volume. The trachea and both lungs were separated from other organs, weighed, and inflated by intratracheal instillation of 10% Formalin at 25 cmH2O pressure for 2 h. The abdominal wall containing the subcutaneous infection was removed, fixed in 10% Formalin for 2 h, and sampled for histology.
Processing of tissue and blood. The lungs were fixed for 2 h and then cut into five slices in the plane of gravity, randomly sampled, and processed into paraffin for histological evaluation. The subcutaneous infection sites were sampled and processed into paraffin in a similar fashion. The blood samples were collected in standard tubes containing potassium EDTA (Vacutainer, Becton Dickinson, Rutherford, NJ). Blood cell counts were performed with a model SS80 Coulter Counter (Coulter Electronic, Hialeah, FL), and differential counts were made on Wright's stained blood smears. Blood used for the preparation of leukocyte-rich plasma was collected in acid citrate-dextrose. The erythrocytes were sedimented by adding 4% dextran (average mol wt 267,000; Sigma) in PMN buffer. The resulting leukocyte-rich plasma was cytospun to obtain a monolayer of cells on slides precoated with 3-aminopropyltriethoxysilane. This was air-dried and fixed in methanol before being stained. The bone marrow aspirates were smeared on coated slides, air-dried, and fixed in methanol before staining.
Detection of BrdU-labeled PMNs. A mouse monoclonal antibody to BrdU and the alkaline phosphatase anti-alkaline phosphatase (APAAP) method were used to detect the presence of BrdU in PMNs in cytospins made from leukocyte-rich plasma, bone marrow smears, lung sections, and subcutaneous infection site sections (6). The cytospins and the bone marrow smears were fixed in methanol and subjected to digestion in 0.04% pepsin for 15 min. The lung sections and the subcutaneous infection site sections were deparaffinized, rehydrated, and digested in 0.4% pepsin for 10 min. All slides were incubated in 2 N HCl for 1 h to denature the DNA. Nonspecific binding sites were blocked with 5% normal rabbit serum, and the specimens were incubated for 1 h with the mouse monoclonal anti-BrdU antibody (2 mg/ml; Dako Laboratories, Copenhagen, Denmark). The specimens were then incubated with the secondary antibody, rabbit anti-mouse IgG (Dako Laboratories), for 30 min. Specific bindings were detected by incubation with mouse monoclonal APAAP complex (Dako Laboratories) followed by a new fuschin-based red substrate solution. The slides were counterstained with Mayer's hematoxylin, dehydrated through a graded series of alcohols and xylene, mounted, and covered with a coverslip.
Evaluation of BrdU-labeled PMNs. PMNs with any nuclei stained with BrdU were counted as positive. BrdU-labeled PMNs were evaluated on a Zeiss universal light microscope in random fields of view. On cytospins, 100 PMNs were counted, and the percentage of BrdU-labeled PMNs was determined; if <10% were positive, 500 cells were counted. Results are expressed as the percentage of PMNs that are BrdU labeled. On bone marrow smears, segmented PMNs and band cells were evaluated in a similar manner as the cytospins.
Morphological studies of lung tissue. The number of PMNs in tissue sections was determined with a modification of the sequential level stereological analysis described by Cruz-Orive and Weibel (11). A point-counting grid was placed over the cut surface of the lung slices, which were examined at ×4 magnification. The number of points falling on lung parenchyma, large blood vessels, and airways (>1 mm diameter) was determined. The volume fraction (VV) of each component was estimated as VV (object) = (sum of the points on object)/(sum of the total points). Paraffin-embedded sections stained for BrdU were point counted at ×400 magnification with a Nikon Microphot-fx light microscope (Nikon, Tokyo, Japan), a point-counting grid of 175 points that was superimposed onto the microscope image, and image-analysis software obtained from the National Institutes of Health (NIH Image). Twelve random fields of view of the lung and ten random fields of view of the sponge were evaluated. At this level, the number of points falling on air space, tissue in the lung, BrdU-labeled PMNs, and unlabeled PMNs in the tissue were counted, and the VV of each component was estimated. "Tissue" in this context consists largely of alveolar walls and includes capillaries and interstitial spaces. Results are expressed as the percentage of labeled and unlabeled PMNs in air space or tissue. The fixation and paraffin embedding reduce BrdU labeling by 53%, and this factor was used to correct values in the blood before comparison (29). The number of PMNs was also calculated. The number of PMNs sequestered in the lung was calculated as [lung volume × VV in tissue (excluding air space) × VV of PMNs in tissue]/143 fl, where 143 fl is the assumed volume of a rabbit PMN (13). Results are expressed as the number of PMNs per milliliter of tissue.
Morphological studies of the subcutaneous infection site. BrdU-labeled PMNs were evaluated in random fields of view on a section of subcutaneous infection sample. One hundred PMNs were counted, and the percentage of BrdU-labeled PMNs was determined. The total number of PMNs in sponges was evaluated using the same method as for the lung tissue.
Statistical analysis. Leukocyte counts and percentage in circulating blood and bone marrow were analyzed using a two-way ANOVA with time as a repeating factor and as a grouping factor. Total PMN number in tissue and the percentage of BrdU-labeled PMNs in tissue were analyzed using two-way and one-way ANOVAs, respectively, with blocking on animals. The sequential rejective Bonferroni test procedure was used to correct for multiple comparisons (24). A corrected P < 0.05 was considered significant. All values are expressed as means ± SE.
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RESULTS |
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Leukocyte and PMN counts. The leukocyte counts in the three groups over the study period are shown in Fig. 1. In the S+B group, the total leukocyte and PMN counts decreased after bacteremia compared with the baseline (8.9 ± 0.6 ×109 leukocytes/ml and 3.7 ± 0.4 ×109 PMNs/ml at 0 h; 3.5 ± 0.2 ×109 WBCs/ml and 1.9 ± 0.3 ×109 PMNs/ml at 8 h; P < 0.05), whereas in the S and B groups the values did not change significantly. The percentage of band cells in the S+B group increased from 7 to 8 h (P < 0.05).
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Release of BrdU-labeled PMNs into the circulation. The release of BrdU-labeled PMNs from the bone marrow into the circulation is shown in Fig. 2. The percentage of BrdU-labeled PMNs in the circulation increased between 5 and 8 h compared with baseline values in both the S+B group (0 h, 1.1 ± 0.1%; 8 h, 27.9 ± 4.2%; P < 0.05) and the S group (0 h, 0.8 ± 0.3%; 8 h, 11.2 ± 1.6%; P < 0.05). In the B group, the increase was not significant. The percentage of BrdU-labeled PMNs in the S+B group was significantly higher than in the other groups from 5 to 8 h (P < 0.005). The PMN release from the bone marrow was accelerated most in the S+B group.
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BrdU-labeled PMNs in the bone marrow. The percentages of segmented PMNs and BrdU-labeled PMNs in the bone marrow are shown in Fig. 3, A and B, respectively. Only in the S+B group did the percentage of segmented PMNs decrease at 8 h (from 51.0 ± 2.2% at 0 h to 35.5 ± 3.0% at 8 h; P < 0.05). The percentage of segmented BrdU-labeled PMNs in the bone marrow increased in the S+B group at 8 h (20.3 ± 2.4 to 46.9 ± 3.7%; P < 0.005). The percentage of segmented BrdU-labeled PMNs in the S+B group at 8 h was higher than in the other groups (S, 23.0 ± 3.6%; B, 18.2 ± 4.1%; P < 0.005). The percentage of band cells and BrdU-labeled band cells in the bone marrow did not change significantly over the study period in all three groups (data not shown). The data show that the PMN maturation in postmitotic pool of the bone marrow was accelerated in S+B group.
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PMNs sequestrated in the lung. The total number of sequestrated PMNs in the lung of each group is shown in Fig. 4, which includes historical data of the group of normal rabbits without any infection (14). More PMNs sequestered in untreated tissues of the S+B group (16 ± 0.3 × 108/ml tissue) than in both the S and B groups (S, 11 ± 0.3 × 108/ml tissue; B, 12 ± 0.6 × 108/ml tissue; P < 0.05). These values were all more than the control group of normal rabbits (1.2 ± 0.5 × 108/ml tissue; P < 0.001) (12). Very few PMNs migrated into air spaces (<1% of sequestered PMNs), with no difference in migration of PMNs among groups.
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PMNs migrated into subcutaneous infection site. The numbers of PMNs that migrated into the subcutaneous infection site of the S and S+B groups are shown in Fig. 5. There was no difference between the two groups. The sequential bacteremia did not change the migration of PMNs into the sponge.
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BrdU-labeled PMNs in the lung and infected sponge. The percentages of BrdU-labeled PMNs in the blood, lung capillaries, and infected subcutaneous tissues at 8 h are shown in Fig. 6. In all groups, lung tissues have higher percentages of BrdU-labeled PMNs than blood (S+B group: lung capillaries, 21.1 ± 1.3%, and blood, 14.8 ± 0.8%; S group: lung capillaries, 13.6 ± 0.6%, and blood, 5.9 ± 0.9%; B group: lung capillaries, 15.6 ± 0.9%, and blood, 6.2 ± 1.6%; P < 0.05). The data demonstrate preferential sequestration of BrdUlabeled PMNs compared with unlabeled PMNs in the lung capillaries in all groups. In S+B and S groups, the percentages of BrdU-labeled PMNs of infected subcutaneous tissue were lower than those of blood at 8 h (S+B, 1.8 ± 0.5%; S, 1.6 ± 0.7%; P < 0.05). The data demonstrate slow migration of BrdU-labeled PMNs in the subcutaneous infection site compared with unlabeled PMNs.
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DISCUSSION |
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This study shows the bone marrow response and the behavior of newly released PMNs during focal and bacteremic subcutaneous infections. Pulse labeling of dividing PMNs in the bone marrow with the thymidine analog BrdU allows us to quantitate the bone marrow response and evaluate the behavior of newly released PMNs both in the lung and at the site of infection (6, 29, 33).
A subcutaneous infection or bacteremia alone did not change the different cell populations (metamyelocytes, band cells, and segmented PMNs) in the maturation pool of the bone marrow, but bacteremia after a subcutaneous infection decreased the percentage of segmented PMNs (Fig. 3A). The number of PMNs in the postmitotic pool of human bone marrow is 20 times that in the circulating blood, and this pool provides a source of PMNs that are available for immediate release into the circulation during an acute infection (3, 4). The fact that this large storage pool was depleted in our study indicates that the magnitude of the bone marrow response elicited by subcutaneous infection associated with bacteremia is much greater than each of these stimuli alone. Interestingly, the increase in the percentage of segmented BrdU-labeled PMNs in the bone marrow observed with bacteremic subcutaneous infection indicates an accelerated PMN maturation in the bone marrow (Fig. 3B). These findings suggest that PMNs are released from the bone marrow faster than they mature.
Granulocytopenia is the hallmark of a diffuse intravascular activation of PMNs associated with bacteremic infection (8, 10, 20). This granulocytopenia may result from 1) an inadequate bone marrow response to the stimulus, 2) excess sequestration of leukocytes within microvessels, or 3) excess migration of PMNs out of the vascular space into the tissues. In our study, granulocytopenia was seen only during bacteremic subcutaneous infection (Fig. 1, A and B). This granulocytopenia occurred in conjunction with the increase in band-form PMNs in the circulation (Fig. 1C) as well as the marked increase in the BrdU-labeled PMNs in the circulation (Fig. 2). These observations verify an accelerated bone marrow response during bacteremic subcutaneous infection. The development of granulocytopenia together with an accelerated bone marrow response indicates that the PMNs are removed from the circulation at a faster rate than their release from the bone marrow. This was supported by the quantitative morphometric studies, which showed an increased sequestration of PMNs in the lung during bacteremic subcutaneous infection compared with the other groups (Fig. 4). A small fraction (1-2%) of PMNs delivered to a focus of infection actually migrates into tissues (14). Furthermore, sequential bacteremia did not increase the PMN migration in this study (Fig. 5). Therefore, we conclude that the major mechanism for the granulocytopenia in the bacteremic subcutaneous infection group was increased sequestration of PMNs in capillaries rather than excessive migration into tissues.
Our data clearly show that the presence of a focal subcutaneous infection resulted in sequestration of PMNs in lung capillaries compared with noninfected animals (Fig. 4). This sequestration is in concordance with the previous report from our laboratory (14) that showed increased sequestration of PMNs in normal noninfected lung during a focal pneumococcal pneumonia, although there was a small but significant difference in the magnitude of sequestration between a focal subcutaneous infection and a focal pneumonia in our comparison (Table 1). Noninfectious inflammation such as ischemia-reperfusion injury and thermal injury models also cause the sequestration of PMNs in normal lung tissue (7, 31, 32). Although the mechanisms responsible for the sequestration of PMNs in normal lung tissue during a focal inflammatory response are unclear, our findings suggest that these mechanisms are similar. Release of inflammatory mediators and cytokines from cells participating in the focal inflammatory response could influence PMN sequestration in the lung. Inflammatory mediators also release PMNs from the bone marrow, activate circulating PMNs or vascular endothelium, and change PMN-endothelial interactions, all factors that may contribute to sequestration of PMNs in the lung.
Deformability of PMNs could explain increased sequestration of PMNs in normal pulmonary capillaries. PMNs must deform to negotiate the pulmonary capillary bed because of the discrepancy between their size and that of the pulmonary capillary segments (13, 21, 23). Granulocytes from the bone marrow maturation pool are larger and less deformable than their circulating counterparts (30), although it is unclear how long these immature cells maintain these functional properties after they are released into the circulation. The percentages of newly released PMNs (BrdU-labeled PMNs) in the lung were higher than the percentages in circulating blood at 8 h in all groups (Fig. 6). This suggests that younger PMNs released from the bone marrow are still less deformable and preferentially sequester in lung capillaries compared with the circulating mature counterparts. This suggests that the release of younger PMNs from the bone marrow contributes to the sequestration of PMNs in normal lung.
Decreased deformability of activated PMNs has been shown to result in the sequestration of PMNs in pulmonary capillaries (16, 25, 37). Bacteremia after a focal subcutaneous infection caused more sequestration than subcutaneous infection alone, which suggests that the sequential bacteremia further reduced deformability of PMNs and resulted in further increased sequestration of PMNs in the lung.
The influence of cell adhesion molecules on either the PMNs or the endothelium in the sequestration of PMNs in lung capillaries is still unclear. L-selectin and CD18 are not necessary in the immediate sequestration of PMNs after zymosan-activated plasma infusion but contribute to the prolonged sequestration of PMNs (12, 17). In a model of focal pneumococcal pneumonia, intercellular adhesion molecule-1 expression increased on endothelium in the pneumonic region but not in the adjacent normal lung (9). The recruitment of PMNs in lung tissue during a focal pneumococcal pneumonia is CD18 independent, whereas recruitment of PMNs in systemic capillaries is CD18 dependent (15). These reports suggest that the effect of adhesion molecules on PMN sequestration in normal lung during focal infection is small.
The observation that the newly released PMNs were slow to migrate into the infected subcutaneous tissues (Fig. 5) is consistent with previous studies (29, 33) showing that recently released PMNs are slow to migrate into the alveoli during a focal pneumococcal pneumonia. These findings support in vitro studies (1, 5, 18) comparing PMNs in the bone marrow with PMNs in peripheral blood, which show that bone marrow PMNs are less motile and do not migrate and phagocytose as well as PMNs obtained from the circulation. PMNs from the bone marrow express a lower baseline level of CD18 and less upregulation of CD18 with a stimulus than do PMNs in the peripheral blood (28). These findings are consistent with the slow migration of newly released PMNs (BrdU-labeled PMNs) into the infection site (Fig. 5). We speculate that the preferential sequestration and slow migration of newly released PMNs provide these cells with a greater opportunity to injure the capillary endothelium.
Several studies (8, 10, 20) have demonstrated PMN sequestration during bacteremia and endotoxemia, implicating PMN-mediated endothelial injury by neutrophil-derived mechanisms (26, 34). The population of PMNs that sequester and their phenotypic and functional characteristics are likely to be very important in determining the magnitude and extent of this injury. Our data show that newly released PMNs preferentially sequester in the lung capillaries compared with mature PMNs already in circulation (Fig. 6). We speculate that those newly released PMNs could play a major role in injuring the capillary endothelium if they were activated during their prolonged transit through the lung capillaries.
In summary, the data presented here show that the granulocytopenia observed during bacteremic subcutaneous infection is associated with an accelerated release of younger, less mature PMNs into the circulation. These younger PMNs preferentially sequester in the lung and are slow to migrate into the site of infection. This observation suggests that they still possess immature functional characteristics. These results underline the important additive effect of bacteremia after an initial infection on the bone marrow release and sequestration of PMNs in lung.
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ACKNOWLEDGEMENTS |
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We thank Lorri Verburgt for assistance with the statistical analysis.
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FOOTNOTES |
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This work was supported by Medical Research Council of Canada Grant 4219.
Address for reprint requests: S. F. van Eeden, Pulmonary Research Laboratory, St. Paul's Hospital, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6.
Received 24 December 1997; accepted in final form 14 April 1998.
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