Phenotypic Characterization of Macrophages in the Endometrium of the Pregnant Cow Lilian J

ORIGINAL ARTICLE

Phenotypic Characterization of Macrophages in the Endometrium of the Pregnant Cow Lilian J. Oliveira, Peter J. Hansen

Department of Animal Sciences, University of Florida, Gainesville, FL, USA

Keywords Problem Cow, endometrium, macrophage, placentome, Macrophages are recruited in large number to the interplacentomal pregnancy endometrium of the cow during pregnancy. We evaluated whether endometrial macrophages also accumulate in placentomal regions of Correspondence Peter J. Hansen, Department of Animal endometrium during pregnancy and whether endometrial macrophages Sciences, University of Florida, PO Box are regionally differentiated. 110910, Gainesville FL 32611-0910 USA. E-mail: [email protected]ﬂ.edu Method of study Interplacentomal endometrium and placentomes were subjected to Submitted June 18, 2009; dual-color immunoﬂuorescence using CD68 as a pan-macrophage accepted September 8, 2009. marker.

Citation Results Oliveira L J, Hansen PJ. Phenotypic CD68+ cells were abundant in stroma of the interplacentomal endome- Characterization of macrophages in the trium and caruncular septa of the placentomes. CD68+ cells were not endometrium of the pregnant cow. Am J Reprod Immunol 2009; 62: 418–426 present in fetal villi of the placentomes or in the interplacentomal chorion. Regardless of location, the majority of CD68+ cells also expressed + + doi:10.1111/j.1600-0897.2009.00761.x CD14. In interplacentomal endometrium, CD68 CD11b cells were present in deeper areas of the stroma but not in shallow endometrial stroma. In caruncular septa of the placentome, CD68+ cells were negative for CD11b. CD68+ cells in the interplacentomal endometrium were negative for MHC class II while most CD68+ cells in caruncular septa were positive for MHC class II.

Conclusion CD68+CD14+ macrophages present in the stroma of the interplacentomal endometrium and caruncular septa of the placentome are regionally differentiated with regard to expression of CD11b and MHC class II.

in number in caruncular septa after experimental Introduction infection with Neospora caninum in the pregnant Recruitment of macrophages to the endometrium is cow.8 Endometrial macrophages could also be key a characteristic of pregnancy in many species includ- regulators of the immunological interaction between ing the mouse,1 human,2–4 cynomologus and vervet mother and conceptus. Human decidual CD14+ cells monkeys,5 sheep6 and cow.7 One function of endo- express low levels of the costimulatory molecule metrial macrophages may be to serve as sentinels to CD86 when compared with cells in blood2 and prevent pathogens from establishing infection or to spontaneously express alternative activation markers clear dead cells. Endometrial macrophages increase interleukin (IL)-10, stabilin-1 and coagulation factor

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XIIIa.2,4 The comparison of the global transcriptome pattern of differentiation than the endometrial of circulating CD14+ cells to decidual CD14+ cells in macrophage in the interplacentomal region. To human revealed that decidual CD14+ have a distinct identify macrophages, we used antibody to CD68, a phenotype and express genes for regulatory proteins lysosomal-associated protein that is expressed on such as DC-SIGN.9 A population of DC-SIGN positive monocytes, macrophages and dendritic cells.19 macrophages is also present near the site of placental Expression of three markers by CD68+ cells in the attachment in early pregnancy in cynomologous and endometrium was evaluated to determine whether vervet monkeys.5 It has also been suggested that regional differentiation of endometrial macrophages endometrial macrophages regulate the concentra- occurs. The first marker examined was CD14, a tions of placental lactogen at the uteroplacental co-receptor for bacterial lipopolysaccharide expressed interface by binding of placental lactogen to macro- on monocytes, macrophages and neutrophils.20,21 phage stabilin-1 and subsequent processing of the CD14 is expressed on most interplacentomal CD68+ protein.10 A role in parturition is also possible cells in the pregnant cow.7 The second was CD11b because macrophages accumulate in the uterus near (b2 integrin) that, with CD18, composes the parturition.11,12 macrophage complex-1 (Mac-1) that is involved in The signal or signals that drive the accumulation leukocyte-endothelial adhesion.22 The third mole- and differentiation of macrophages in the pregnant cule was MHC class II, which is involved in antigen endometrium remains unclear. Using the unilater- presentation, and can be upregulated in activated ally-pregnant sheep as a model, there was evidence macrophages and downregulated in inactivated for both local and systemic signals for macrophage macrophages.23 accumulation in the interplacentomal endometrium during pregnancy.6 There are a variety of cytokines Materials and methods produced by the uterus or placenta that can regulate monocyte or macrophage differentiation. In the Materials cow, these include transforming growth factor-b13, IL-10, IL-12, IL-18, tumor necrosis factor-a and Mouse anti-human CD68 (clone EBM11; ascites, interferon-c.8 2.3 lg ⁄ mL) was obtained from Dako (Carpinteria, In the ruminant, the nature of the relationship CA, USA)6, mouse anti-bovine CD14 (clone between mother and fetus varies regionally within MM61A, ascites, 10 lg ⁄ mL),24 mouse anti-bovine the placenta. Most gas and nutrient exchange occurs MHC class II [a mixture of and equal mass of anti- at specialized structures called placentomes where DR alpha (clone TH14B) and an anti-DQ (clone fetal villi are interdigitated with maternal caruncular TH81A5); ascites, 1 mg ⁄ mL]25 and mouse anti- septa. In the intervening interplacentomal areas, the human CD11b (clone MM12A; ascites, 1 mg ⁄ mL)26 chorioallantois is apposed to the luminal epithelium. were from VMRD (Pullman, WA, USA). IgG controls A synctium forms in the endometrial epithelium as a matching the isotype of each antibody used were result of migration of fetal binucleated cells and their obtained as control mouse ascites fluid (Sigma- fusion with maternal epithelium.14 The immuno- Aldrich, St Louis, MO, USA). The Zenon Alexa Fluor genicity of the placenta differs between placentomal labeling kit and the Prolong Antifade mounting and interplacentomal sites. Chorionic tissue in the medium were obtained from Invitrogen (Eugene, placentome is negative for MHC class I expression OR, USA). throughout pregnancy while chorionic tissue in the The antibodies, including the IgG control, were interplacentomal regions can express MHC class I in tagged with fluorescently-labeled Fab fragments the third trimester of pregnancy.15,16 MHC class I against mouse IgG conjugated using the Zenon proteins on the chorion are predominately encoded Mouse Labeling IgG kits as per manufacturer’s by non-classical MHC class I genes.17 These non- instructions. The anti-CD68 was labeled with Alexa classical MHC class I proteins are possibly analogous Fluor 488, the anti-CD14 was labeled with Alexa to human leukocyte antigen (HLA)-G in humans Fluor 488 or Alexa Fluor 594 for immunofluores- that can regulate natural killer cell and macrophage cence and with Alexa Fluor R-Phycoerythrin for function.18 flow cytometry, and anti-bovine MHC class II and In this study, we hypothesized that the endome- anti-CD11b were labeled with Alexa Fluor 594. trial macrophage in the placentome has a different

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Normal goat serum and fetal bovine serum were washed an additional three times for 5 min each, purchased from Sigma-Aldrich or Pel-Freez Biologi- cover slips were mounted using Prolong Antifade cals (Rogers, AR, USA). The tissue freezing medium reagent and slides were examined using a Zeiss Axi- (Tissue-Tek OCT) was purchased from Sakura Fine- oplan 2 epifluorescence microscope (Zeiss, Gottin- tek USA, Inc. (Torrance, CA, USA). gen, Germany) with a 40· objective and using Zeiss Tissue Culture Medium-199 (TCM-199), bovine filter set 02 (DAPI filter), Zeiss filter set 03 (FITC fil- serum albumin (BSA) Fraction-V, Dulbecco’s phos- ter) and Zeiss filter set 15 (rhodamine filter). Digital phate buffered saline (DPBS), collagenase type I and images were acquired using AxioVision software Hoescht 33342 were purchased from Sigma-Aldrich (Zeiss) and a high-resolution black and white Zeiss (St. Louis, MO, USA). AxioCam MRm digital camera.

Tissues Dual-Color Flow Cytometric Analysis for CD68 and CD14 in Dispersed Endometrial Cells Uteri were obtained from pregnant cows of various breeds at a local abattoir. The uterus was transported Two-color immunofluorescence was performed using to the lab on ice within 1.5 hr after slaughter. Fetal preparations of dispersed endometrial cells from the crown-rump length was measured to estimate fetal interplacentomal endometrium of a pregnant cow at age.27 Tissues from a total of 20 pregnant cows an estimated Day 166 of pregnancy. The uterus was (estimated fetal age 137–242 days; mean ± S.D. = obtained at a local abattoir and transported to the lab 182.0 ± 37 days) were collected. Samples of inter- on ice. Interplacentomal endometrium was obtained placentomal endometrium and placentome ipsilateral by dissection. Tissue fragments were collected into a to the corpus luteum were snap-frozen in Tissue-Tek 50 mL sterile culture tube containing 30 mL Tissue OCT embedding compound (see Noden and de Culture Medium (TCM)-199 supplemented with type Lahunta27 and Davies et al.28 for representation of I collagenase at 150 U ⁄ mL and 10% (v ⁄ v) fetal bovine the anatomy of the bovine placenta highlighting fea- serum. Cells were incubated at 37C for 1 hr under tures of interplacentomal and placentomal endo- gentle rotation. Dispersed cells were then filtered metrium). Samples of lymph node, thymus and through a sterile 200 lm cell strainer into 50 mL ster- spleen were also collected from cows at Day 17 of ile culture tubes and centrifuged at 110 · g for 5 min. pregnancy for use as positive controls. The cell pellet was resuspended with 5 mL TCM-199 supplemented with 10% (v ⁄ v) fetal bovine serum and cell number was determined using a hemacytometer. Two-Color Immunofluorescence A total of 5 · 106 cells were placed into Tissue was processed for dual-color immunofluores- 13 · 100 mm polyethylene tubes in staining buffer cence by preparing 4 lm sections with a cryostat [Dulbecco’s PBS supplemented with 0.1% (w ⁄ v) microtome. Sections were placed onto precleaned BSA and 0.1% (w ⁄ v) sodium azide], washed twice poly-L-lysine coated glass slides, fixed in ice-cold with 2 mL staining buffer and resuspended in the acetone for 10 min and air dried. The sections were smallest volume possible with staining buffer. Cells rehydrated in phosphate buffered solution (PBS) at were stained for dual-color flow cytometry analysis

4C [0.01 m Na2HPO4, pH 7.4 containing 0.85% using Zenon-labeled anti-CD68 (2.3 lg ⁄ mL; tagged (w ⁄ v) NaCl]. with Alexa Fluor 488) and anti-CD14 (10 lg ⁄ mL; Sections were incubated with blocking buffer [PBS tagged with Alexa Fluor R-phycoerythrin) diluted in containing 10% (v ⁄ v) goat serum] for 1 hr followed antibody staining buffer. After incubation, samples by incubation overnight at 4C with labeled anti- were washed with 2 mL staining buffer, and resus- CD68 (2.3 lg ⁄ mL) as a pan macrophage marker con- pended with Dulbecco’s PBS containing 4% (w ⁄ v) comitant with either labeled anti-CD14 (10 lg ⁄ mL), paraformaldehyde for fixation. Before analysis, cells anti-CD11b (10 lg ⁄ mL) or anti-MHC class II were washed once with 1 mL of staining buffer and (10 lg ⁄ mL). Other sections were incubated with resuspended in 300 lL staining buffer. The flow Zenon-labeled isotype controls (10 lg ⁄ mL). Sections cytometry profiles were obtained on Fluorescent were then washed three times for 5 min using PBS Analysis Cell Sorter FACSCaliburTM using CELLQuest and incubated with Hoescht 33342 reagent flow cytometry software (Becton-Dickinson, Franklin (2.3 lg ⁄ mL in PBS) for 15 min. Sections were Lakes, NJ, USA).

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Gates for identiﬁcation of positive cells were estab- in deep stroma but most CD68+ cells closer to the lished by analyzing other aliquots of cells that were luminal epithelium did not express CD11b (Fig. 3c). incubated with control IgG (tagged with Alexa Fluor In the placentome, CD11b+ cells were rare (Fig. 3e) 488 and Alexa Fluor R-phycoerythrin) at the same and none of the CD68+ cells were positive for dilution as the primary antibodies described above. CD11b (Fig. 3g,h).

Results Localization of Cells Positive for MHC Class II In the interplacentomal endometrium, there were Localization of Cells Positive for CD68 and CD14 three major populations of MHC class II+ cells. The In interplacentomal regions, CD68+ cells were abun- first two were in the stroma immediately adjacent to dant in the endometrial stroma (Fig. 1b). Density of the basement membrane of the luminal epithelium CD68+ cells was lowest in stroma closer to the lumi- (Fig. 4a,e). These cells were negative or weakly posi- nal epithelium and in the deepest regions of the tive for CD68 (Fig. 4c,d,g,h). One population of stroma near glands (Fig. 3b). Regardless of location, MHC class II+ cells near the luminal epithelium were the majority of CD68+ cells also expressed CD14 also positive for CD14 (Fig. 5c,d) whereas the other, (Fig. 1c,d). There were also many cells in the stroma, more abundant, population, were negative for CD14 most prominently in the areas closest to luminal epi- (Fig. 5c,d). The third population was in the stroma thelium, which were positive for CD14 but either immediately adjacent to the basement membrane of negative or weakly staining for CD68 (Fig. 1c,d). the endometrial glands. These cells were negative for In the placentomal areas, CD68+ cells and CD14+ CD68 (Fig. 4c,d) and CD14 (not shown). cells were present in the caruncular septa (maternal In the caruncular septa of placentomes, there tissue) but were absent in fetal villi (Fig. 1e,f). The were numerous MHC class II+ cells (Fig. 4i,m). Some majority of CD68+ cells also expressed CD14 (Fig. 1g,h). were CD68+ and others were CD68), Most CD68+ Analysis by flow cytometry confirmed that the cells were also positive for MHC class II (Fig. 4k,l,o,p). majority of CD68+ cells in interplacentomal endome- No MHC class II+ cells were found in fetal villi trium expressed CD14 and vice versa (Fig. 2). (Fig. 4i,k,l,m,o,p).

Expression of CD11b on Cells Positive for CD68 Discussion In the stroma of the interplacentomal endometrium, Results indicate that the endometrial accumulation cells dual-labeled for CD68 and CD11b were present of CD68+ cells during pregnancy in the cow seen

CD14 CD68 CD14/CD68 CD14/CD68 (a) (b) (c) (d)

Str

50 µm 50 µm 50 µm 50 µm

Fig. 1 Immunoﬂuorescent labeling of cells for (e) (f) (g) (h) CD68 (green) and CD14 (red) in endometrium from a cow at Day 145 of pregnancy. The top panels represent sections of interplacentomal endometrium (a–d) and the bottom panels represent sections of placentome (e–h). FV Nuclei were labeled with Hoescht 33342. Merged images are in panels c, d, g and h. CS

CS, caruncular septum; FV, fetal villi; LE, lumi- 50 µm 50 µm 50 µm 50 µm nal epithelium; Str, endometrial stroma.

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4 (a) 1024 (b) 104 (c) 10 1.46% 66.67% Fig. 2 Acquisition dot-plots from ﬂow cyto- 3 3 768 10 10 metric analysis of the proportion of CD68+ cells in dispersed endometrial cells from the inter- 2 512 102 10 FL2 SSC

CD14 placentomal endometrium of a cow at Day + 1 166 of pregnancy that were CD14 . Plots 256 101 10 represent: (a) analysis of cells on the basis of

Gate 1 95.75% 11.60% 0 0 0 size (FSC, forward scatter characteristics; 10 10 0 1 2 3 4 0 256 512 768 1024 100 101 102 103 104 10 10 10 10 10 FSC FL1 CD68 x axis) and granularity (SSC, side scatter characteristics; y axis), with the gate subjected (d) to further analysis (Gate 1) shown as the red rectangle, (b) ﬂuorescence for cells incubated with isotype controls, (c) dual-labeling of cells in Gate 1 for CD14 (y axis) and CD68 (x axis) and (d) representative histogram showing cells labeled with isotope control in FL1 (black), 0 1 2 3 4 10 10 10 10 10 anti-CD68 (green) and anti-CD14 (red).

CD11b CD68 CD11b/CD68 CD11b/CD68 (a) (b) (c) (d) LE

Str

100 µm 100 µm 100 µm 100 µm Fig. 3 Immunoﬂuorescent labeling of cells for (e) (f) (g) (h) CD68 (green) and CD11b (red) in endometrium from a cow at Day 145 of pregnancy. The top panels represent sections of interplacentomal endometrium (a–d) and the bottom panels represent sections of placentome (e–h). Nuclei were labeled with Hoescht 33342. Merged images are in panels c, d, g and h. EG, endometrial gland; LE, luminal epithelium; 100 µm 100 µm 100 µm 100 µm Str, stroma.

previously for the interplacentomal endometrium7 negative for CD11b. This regional differentiation also occurs in the caruncular septa of the placen- likely reﬂects different trafﬁcking patterns and regu- tome. That these CD68+ cells are originated from the latory signals in the two placental regions. myeloid hematopoietic lineage is likely because they The most striking difference between CD68+ cells also express CD14. CD68 is expressed on monocytes, in interplacentomal and placentomal regions was in macrophages and dendritic cells19,29,30 and CD14 is the expression of MHC class II. Presence of MHC expressed on monocytes, macrophages and neu- class II on CD68+ cells in the caruncular septa of the trophils.20 It is also apparent from this study that placentomal endometrium is consistent with the endometrial macrophages are regionally differenti- activation of these cells by the M1 or M2 pathway.23 ated. CD68+ cells in the interplacentomal endome- By contrast, the absence of detectable MHC class II trium were negative for MHC class II and sometimes in interplacentomal CD68+ cells could indicate that positive for CD11b while CD68+ cells in the the cells are inactivated macrophages23 or monocytes placentomal endometrium were MHC class II+ and newly recruited to the endometrium that have

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MHC class II /CD68 MHC class II CD68 (a) (a) LE (b) (c) (d)

Str

EG 50 µm 50 µm 50 µm 50 µm

(e) (f) (g) (h)

Str

50 µm 50 µm 50 µm 50 µm

(i) (j) (k) (l)

CS Fig. 4 Immunoﬂuorescent labeling of cells for 50 µm 50 µm 50 µm 50 µm CD68 (green) and MHC class II (red) in endometrium from a cow at Day 216 of preg- (m) (n) (o) (p) nancy. The top panels represent sections of interplacentomal endometrium (a–h) and the bottom panels represent sections of placen- FV tome (i–p). Nuclei were labeled with Hoescht 33342. Merged images are in panels c, d, g, h, k, l, o and p. CS, caruncular septum; EG, CS endometrial gland, FV, fetal villi, LE, luminal 50 µm epithelium; Str, endometrial stroma. 50 µm 50 µm 50 µm

defective antigen-presentation function. Further evi- expression of CD11b by circulating blood cells was dence suggesting that at least some of the interplac- higher in pregnant women compared with non- entomal CD68+ cells are newly arrived in the pregnant women.31 endometrium is the staining pattern for CD11b. It is also possible that CD68+ cells in the interplac- This protein, which is involved in monocyte move- entomal endometrium are negative for MHC class II ment through the endothelium,22 was only because they are more distant from trophoblast- expressed on a population of CD68+ cells in the derived activation signals than CD68+ cells in the pla- deeper stromal areas of interplacentomal endome- centomes or that a molecule secreted by the trium. The lack of CD11b+ cells in shallow stroma interplacentomal trophoblast or endometrium inhibits and the placentome could reflect loss of CD11b macrophages. Non-classical MHC class I antigens are expression as macrophages migrate towards the pla- expressed on the interplacentomal trophoblast of the centa and differentiate in response to changes in cow.17 In the human, trophoblast non-classical MHC the endometrial microenvironment. Further evi- class I proteins can regulate endometrial macrophage dence for a role for CD11b in monocyte trafficking function.18 The interplacentomal endometrium pro- to the endometrium is the finding in humans that duces uterine serpin in glandular epithelium32 and, at

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MHC class II CD14 MHC class II /CD14

(a) (b) (c) (d)

Fig. 5 Immunofluorescent labeling of cells for MHC class II (red) and CD14 (green) in Str interplacentomal endometrium from a cow at Day 243 of pregnancy. Nuclei were labeled LE with Hoescht 33342. Merged images are in 50 µm 50 µm 50 µm 50 µm panels c and d. LE, luminal epithelium; Str, endometrial stroma. least in the sheep, this protein can inhibit proliferation References of peripheral mononuclear cells in vitro.32–34 1 Hunt JS, Manning LS, Mitchell D, Selanders JR, Another possibility is that some or all of the Wood GW: Localization and characterization of CD68+ cells in the interplacentomal endometrium macrophages in murine uterus. J Leukoc Biol 1985; are not derived from a myeloid mononuclear phago- 38:255–265. cyte lineage but instead represent stromal cells that 2 Heikkinen J, Mo¨ tto¨ nen M, Komi J, Alanen A, Lassila have differentiated in a manner associated with O: Phenotypic characterization of human decidual upregulation of certain genes characteristically macrophages. Clin Exp Immunol 2003; 131:498–505. expressed by macrophages. In species where endo- 3 Cupurdija K, Azzola D, Hainz U, Gratchev A, Heitger metrium undergoes a decidual response, decidual- A, Takikawa O, Goerdt S, Wintersteiger R, Dohr G, ized stroma express cytokines and chemokines Sedlmayr P: Macrophages of human first trimester characteristically secreted by immune cells.35,36 Per- decidua express markers associated to alternative haps similar differentiation pathways exist in species activation. Am J Reprod Immunol 2004; 51:117–122. like cattle that do not undergo decidualization. 4 Kim J-S, Romero R, Cushenberry E, Kim YM, Erez O, In the interplacentomal endometrium, there were Nien JK, Yoon BH, Espinoza J, Kim CJ: Distribution numerous MHC class II+ cells that were not positive of CD14+ and CD68+ macrophages in the placental for CD68. These included cells in stromal tissue bed and basal plate of women with preeclampsia and beneath the luminal epithelium, some of which preterm labor. Placenta 2007; 28:571–576. were CD14+, and cells in the glandular epithelium 5 Dambaeva SV, Breburda EE, Durning M, Garthwaite that were negative for CD14. A similar pattern of MA, Golos TG: Characterization of decidual leukocyte MHC class II+ cells exists in sheep.37,38 The CD14+ populations in cynomolgus and vervet monkeys. MHC class II+ cells could be mononuclear phagocytic J Reprod Immunol 2009; 80:57–69. cells derived from CD68+ macrophages in deep 6 Tekin S, Hansen PJ: Regulation of numbers of macrophages in the endometrium of the sheep by stroma or another other cell type. Presence of CD14+ systemic effects of pregnancy, local presence of the cells in the subepithelium stroma has been previ- conceptus, and progesterone. Am J Reprod Immunol ously reported at Day 16 after estrus in cyclic and 2004; 51:56–62. pregnant cattle.39 Some MHC class II+ cells in the 7 Oliveira LJ, Hansen PJ: Deviations in populations of subepithelial stroma could also be B cells because peripheral blood mononuclear cells and endometrial cells staining for the B cell marker CD21 were local- macrophages in the cow during pregnancy. 39 ized mostly to this region. Reproduction 2008; 136:481–490. As mentioned in the Introduction, recruitment of 8 Rosbottom A, Gibney EH, Guy CS, Kipar A, Smith macrophages to the endometrium is a characteristic RF, Kaiser P, Trees AJ, Williams DJL: Upregulation of 4 of pregnancy in many species. What sets the sheep cytokines is detected in the placentas of cattle infected and cow apart from the other species examined is with Neospora caninum and is more marked early in the magnitude of infiltration, particularly into the gestation when fetal death is observed. Infect Immun interplacentomal endometrium. The consequences of 2008; 76:2352–2361. this infiltration for the course of pregnancy and 9 Gustafsson C, Mjo¨ sberg J, Matussek A, Geffers R, immune function in the post-parturient period Matthiesen L, Berg G, Sharma S, Buer J, Ernerudh J: remain to be defined. Gene expression profiling of human decidual

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