Comprehensive gene expression profile of human activated Th1- and Th2-polarized cells

Shigenori Nagai, Shin-ichi Hashimoto, Taro Yamashita, Nobuaki Toyoda, Taku Satoh, Takuji Suzuki and Kouji Matsushima

Department of Molecular Preventive Medicine, School of Medicine and CREST, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Correspondence to: K. Matsushima


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
In response to antigen stimulation, Th cells differentiate into two types of effector cells, Th1 and Th2. Th1 cells predominantly mediate cellular immunity, whereas Th2 cells induce humoral allergic responses. We have conducted here serial analysis of gene expression (SAGE) in human activated Th1- and Th2-polarized cells from cord blood. SAGE analysis of 64,510 tags (32,219 and 32,291 tags from Th1 and Th2 cells respectively) allowed identification of 22,096 different transcripts. In activated Th1 cells, many of the known genes (12 genes, P < 0.01; 56 genes, P < 0.05), including genes encoding IFN-{gamma}, lymphotactin, osteopontin, MIP-1{alpha}, MIP-1ß, perforin, ß-catenin and CD55, are highly expressed. On the other hand, in activated Th2 cells rather limited numbers of known genes (four genes, P < 0.01; 10 genes; P < 0.05), such as genes encoding FUS, ILF-2, IL-13 and E2-EPF, are found to be selectively expressed. The comprehensive identification of genes selectively expressed in human activated Th1 or Th2 cells should contribute to our understanding of the molecular basis of Th1/Th2-dominated human diseases and may provide genetic information to diagnose these diseases.

Keywords: chemokine, cytokine, serial analysis of gene expression, Th, transcripts


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Th lymphocytes are classified into two subsets based on their cytokine production profile (1). Th1 cells produce a large amount of IFN-{gamma}, whereas Th2 cells produce IL-4, IL-5, IL-10 and IL-13. Development of Th1 cells is driven by IL-12 produced by macrophages and dendritic cells via the transcription factor Stat4 signaling. On the other hand, commitment to the Th2 lineage is induced by IL-4 via the Stat6 signaling pathway. Th1 cells mediate delayed-type hypersensitivity responses, and provide protection against intracellular pathogens and viruses, whereas Th2 cells promote B cells to produce IgE and contribute to the eradication of extracellular parasites, but also induce atopic reaction.

Significant progress has been made in identifying transcription factors that control the transition of naive T cells to the Th1 or Th2 lineage. c-Maf induces endogenous IL-4 production in non-T lineage cells (2) and GATA-3 promotes the expression of a broad spectrum of Th2-specific cytokines, even in developing and committed Th1 cells (3). In contrast, T-bet, which is one of the T-box family of transcription factors, has been recently identified as a key molecule in Th1 differentiation, which converts even committed Th2 cells into Th1 cells (4).

Furthermore, hematopoietic prostaglandin D synthase, which is well known as a key enzyme involved in prostanoid production by allergen-provoked mast cells, is preferentially produced in Th2 clones (5) and chemoattractant receptor CRTH2 is selectively expressed on the cell surface of Th2 cells (6). In contrast, the ligands of E-selectin and P-selectin are selectively expressed on the surface of Th1 cells (7).

With regard to chemokine receptors, CXCR3 and CCR5 are preferentially expressed on human Th1 cells. On the other hand, CCR4 is preferentially expressed on Th2 cells (8,9). The differential expression of chemokine receptors on each type of Th cell is thought to be critical to the selective cell migration of these cells into the particular immune/inflammatory sites. CCR5+ CD4+ T cells of the Th1 phenotype selectively accumulate in inflamed joints of rheumatoid arthritis (10), and CCR4-bearing Th2 cells are recruited by CC chemokines, thymus and activation-regulated chemokine (TARC), and macrophage-derived chemokine (MDC) (11), and the size of this population is much increased in human atopic diseases (K. Kurashima et al. and T. Miyawaki et al., unpublished observations). With regard to chemokines, preferential or high expression of macrophage inflammatory protein (MIP)-1{alpha}, MIP-1ß, and regulated upon activation, normal T cell expressed and presumably secreted (RANTES) in Th1 cells has been reported (12,13).

Serial analysis of gene expression (SAGE) allows for the establishment of both a representative and comprehensive different gene expression profile in various cell types and organs under physiological and pathological states (14). Since each template contains identifiable tags corresponding to many genes, this method allows global gene expression profiling including unknown genes. In this study, we have analyzed here the expression profiles in activated Th1- and Th2-polarized cells using SAGE, and newly identified numerous genes for which expression is selective in either population. We have also compared our SAGE data with a recently published gene chip analysis of human Th1 and Th2 cells (15).


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Medium
The culture medium used throughout was RPMI 1640 supplemented with 2 mM L-glutamine, 1% non-essential amino acids, 1% pyruvate, 5x10–5 M 2-mercaptoethanol (Gibco/BRL, Gaithersburg, MD) and 10% FBS (JRH Biosciences, Lenexa, KS).

Generation of Th1 and Th2 cells from cord blood leukocytes
Human neonatal leukocytes were isolated from freshly collected, heparinized, neonatal cord blood by density- gradient centrifugation using Lymphoprep (density 1.077; Nycomed, Oslo, Norway). Th1- and Th2-polarized cells were generated by stimulating cord blood leukocytes with 1 µg/ml phytohemagglutinin (Gibco/BRL) in the presence of 2 ng/ml IL-12 (Genzyme Techne, Minneapolis, MN) and 200 ng/ml neutralizing anti-IL-4 antibody (34019.111; R & D Systems; Minneapolis, MN) for Th1 cultures, and 200 U/ml IL-4 (provided by Ono Pharmaceutical, Osaka, Japan) and 2 µg/ml neutralizing anti-IL-12 antibody (C8.6; Genzyme, Cambridge, MA) for Th2 cultures. Cells were washed on day 3 and expanded in each medium containing 4 ng/ml IL-2. At days 12–14, leukocytes were incubated with anti-CD4 mAb-coated magnetic beads and CD4+ cells were isolated by passing the cultured cells through a MACS system (Miltenyi Biotec, Bergish Gladbach, Germany).

Single-cell analysis of cytokine production
Cord blood-derived Th1 and Th2 cells were collected after 3 days of CD4+ cell separation, washed and re-stimulated with 50 ng/ml of phorbol myristate acetate (PMA) and 1 µg/ml of ionomycin (Sigma, St Louis, MO) for 4 h; 10 µg/ml of Brefeldin A (Sigma) was added during the last 2 h of the culture. Then the cells were fixed and permeabilized with IntraPrep permeabilization reagent (Immunotech, Marseille, France) according to the manufacturer's protocol. Fixed cells were stained with FITC-labeled anti-IFN-{gamma} (4S.B3) and phycoerythrin (PE)-labeled anti-IL-4 (MP4-25D2) mAb (PharMingen, San Diego, CA), and analyzed by an Epics XL (Coulter Electronics, Hialeah, FL). The software program System II (Coulter) was used on an Epics XL.

FACS analysis
Th1- and Th2-polarized cells were incubated with optimal concentrations of FITC-labeled anti-CCR4 mAb (11) and PE-labeled anti-CD45RO (UCHL1) mAb (PharMingen). After staining and fixation, analysis was performed using an Epics XL (Coulter Electronics).

SAGE protocol
mRNAs of Th1 and Th2 cells were purified from a mixture of total RNA from four donors. Poly(A)+ mRNA was isolated using the µMACS mRNA isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. SAGE libraries were generated using 2.5 µg poly(A)+ mRNA and were converted to cDNA with a BRL synthesis kit (Gibco/BRL) following the manufacturer's protocol, with the inclusion of primer biotin–5'-T18-3'. The outline of the SAGE protocol has been described in a previous report (16). Briefly, the cDNA was cleaved with NlaIII and the 3'-terminal cDNA fragments were bound to Dynabeads M-280–streptavidin (Dynal, Oslo, Norway). After ligation of the oligonucleotides containing recognition sites for BsmF1, the bound cDNA was released from the beads by digestion with BsmF1. SAGE tag overhangs were filled in with Klenow, and tags from the two pools were combined and ligated to each other. The ligation product was amplified with PCR, concatemerized and cloned into the SphI site of pZero-1 (Invitrogen, Carlsbad, CA). Samples were sequenced with the BigDye terminator kit and analyzed using a 96-lane 377 ABI automated sequencer (Perkin-Elmer, Branchberg, NJ).

Sequence files were analyzed by means of the SAGE program group and DNAsis software (Takara, Osaka, Japan). After correcting sequencing mistakes, a total of 64,510 tags representing 32,219 and 32,291 tags from Th1 and Th2 cells respectively were analyzed.

RT-PCR
The RNA was reverse transcribed in 50 µl of 10 mM Tris–HCl (pH 8.3), 6.5 mM MgCl2, 50 mM KCl, 10 mM DTT, 1 mM of each dNTP, 2 µM random hexamer and 2.4 U/µl of Molony murine leukemia virus reverse transcriptase for 1 h at 42°C. The conditions for PCR were as follows: in a 50 µl reaction, 15 µM of each primer, 125 µM each of dNTP mixture (Toyobo, Osaka, Japan), 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2 and AmplyTaq (Perkin-Elmer). Primers used were as follows. Osteopontin: sense 5'-TGGCTAAACCC- TGACCCATCT-3', antisense 5'-TGGATGTCAGGTCTGCG- AAA-3'; GAPDH: sense 5'-CCTTCATTGACCTCAACTAC-3', antisense 5'-ACCACAGTCCATGCCATCACT-3'; FUS: sense 5'-AACGGGACAGCCCATGATT-3', antisense 5'-GGGCCTTACACTGGTTGCATT-3'; IFN-{gamma}: sense 5'-CTGTTACTGCCAGGACCCATATGTAAAAG-3', antisense 5'-CAACCATTACTG- GGATGCTCTTCGACCTTG-3'; MIP-1ß: sense 5'-CCGCCT- GCTGCTTTTCTTAC-3', antisense 5'-TGACAGTGGACCATC- CATAGGG-3'; IL-13: sense 5'-TCAATCCTCTCCTGTTGGC-AC-3', antisense 5'-CGTCCCTCGCGAAAAAGTTT-3'; lympho-tactin: sense 5'-AGACTTCTCATCCTGGCCCT-3', antisense: 5'-GCCAGAGACTACTAGCCAGTCA-3'; IL enhancer binding factor (ILF)-2: sense 5'-TTCCTTCAGTGAGGCCTTGCT-3', antisense 5'-GAAGATTGGGTGGCACTGTTG-3'.

Reaction mixtures were incubated in a Perkin-Elmer DNA thermal cycler for 25–35 cycles (denaturation for 30 s at 94°C, annealing for 60 s at 59°C and extension for 60 s at 72°C).

Statistical analysis
Statistical significance between samples was calculated using the equation:

where N1 and N2 represent the larger and smaller of the two numbers respectively, and k is the degree of confidence; P = 0.05 (k = 1.96) and P = 0.01 (k = 2.58). Positive values derived from the equation were deemed statistically significant at the respective confidence intervals (17).


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
SAGE technology can provide quantitative and simultaneous analysis of large numbers of transcripts. In this study, we investigated human activated Th1- and Th2-polarized cells. Surface CCR4 expression and the cytokine expression profile were analyzed on these two subsets (Fig. 1A and BGo). CCR4 has been reported to be selectively expressed on Th2 cells (11). Our results confirmed that the lymphocytes derived from cord blood cells were well differentiated into Th1 or Th2 cells. The mRNAs were prepared from these cells after stimulation with PMA and ionomycin for 6 h, and were processed to SAGE analysis. A total of 64,510 tags, including 32,219 and 32,291 tags from activated Th1 and Th2 cells respectively, allowed identification of 22,096 different transcripts. The expressed genes were searched for through the GenBank database to identify individual genes. The top 50 transcripts in two subsets are listed in Table 1Go. Twenty-four and 25 out of the top 50 transcripts were ribosomal proteins in activated Th1 and Th2 cells respectively. However, many genes identical to the cDNAs of secreted proteins were also identified. A Th1 cytokine, IFN-{gamma}, was greatly expressed in activated Th1 cells, whose expression frequency is 5.78%. High expression of genes encoding granulocyte macrophage colony stimulating factor (GM-CSF) (0.84%), MIP-1ß (0.83%), IL-2 (0.82%), IL-3 (0.74%), TNF-{alpha} (0.42%), lymphotactin (0.38%), granzyme B (0.36%) and MIP-1{alpha} (0.28%) were also observed in activated Th1 cells. On the other hand, the genes encoding IL-2 (0.37%), GM-CSF (0.36%), TNF-{alpha} (0.29%), IL-3 (0.28%), granzyme B (0.27%) and IL-13 (0.22%) were detected in activated Th2 cells. Unlike monocyte-derived macrophages and dendritic cells (18,19), the transcripts related to cytoskeleton or cell structure are not highly expressed. This may reflect minimal morphological changes of T cells even after activation.



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Fig. 1. Polarization of Th1 and Th2 cells from cord blood cells. (A) Surface CCR4 expression on Th1- and Th2-polarized cells. Both cells were memory (CD45RO+) subsets and Th2-polarized cells were preferentially expressed in CCR4. (B) Cytokine expression profile of Th1- and Th2-polarized cells stimulated with PMA and ionomycin.

 

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Table 1. Transcript profile in activated Th1 and Th2-polarized cellsa

 
Expressed genes between activated Th1 and Th2 cells were compared, and are shown in Fig. 2Go. Each dot represents a gene expressed in these two subsets and the expression levels of most of the transcripts between these two subsets were very similar. However, the expression profiles also showed significant difference in many transcripts of these cells.



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Fig. 2. Distribution of the different tags from Th1 and Th2 cells. The number of times each unique SAGE tag appeared was plotted on a logarithmic scale, using a total of 32,219 tags from activated Th1 cells (x-axis) versus 32,291 tags from activated Th2 cells (y-axis). To avoid division by 0, we used a tag value of 1 for any tag that was not detectable in one sample and the tag populations were normalized. The line with slope of a unity through the center predicts equal gene expression in the two subsets.

 
Tables 2 and 3GoGo show the genes selectively expressed in activated Th1 or Th2 cells (Th1 > Th2, 68 genes; P < 0.05; Th1 < Th2, 14 genes; P < 0.05). The genes in the EST database or unidentified in the GenBank database are excluded from the tables and are available at http://www.prevent.m.u-tokyo.ac.jp/SAGE.html. Among cytokine and chemokine genes, IFN-{gamma} was expressed 49.2-fold higher in activated Th1 cells than in activated Th2 cells. Furthermore, we identified numerous Th1-predominantly expressed genes such as osteopontin (19.0-fold), MIP-1ß (15.5-fold), lymphotactin (12.5-fold), perforin (11.0-fold), MIP-1{alpha} (6.6-fold), RANTES (6.0-fold), lymphotoxin {alpha} (5.0-fold), IL-3 (2.7-fold), GM-CSF (2.3-fold), NK enhancing factor (NKEF) (2.3-fold) and IL-2 (2.2-fold).


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Table 2. Differential tag abundance in activated Th1- and Th2-polarized cells (1)a

 

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Table 3. Differential tag abundance in activated Th1- and Th2-polarized cells (2)a

 
It has been reported that MIP-1 and RANTES, which are ligands of CCR5, chemoattract the cells related to Th1-dominated cellular immunity (8,20,21). Interestingly, lymphotactin, expressed in activated Th1 cells, has chemotactic activity to CD8+ and NK cells (22). Furthermore, NKEF has an ability to enhance NK cytotoxicity (23). Thus, it suggests that once Th1 cells are activated, effector cells in cellular immunity are recruited around Th1 cells and then accelerate immune response against invading microorganisms or neoplasm. Osteopontin is not only an adhesive matrix protein (24) but also a chemotactic molecule for smooth muscle cells and T cells (25,26). The granulomatous responses in sarcoidosis and tuberculosis have been reported to be associated with high expression of osteopontin (27,28).

Only IL-13 and IL-9 were cytokines predominantly expressed in activated Th2 cells, which are related to asthma or atopic dermatitis (2931). The mRNA expression of IL-4 and IL-10 was not significantly different between Th1 and Th2 cells, and the tag of IL-5 was not detected in either library (Table 3Go). However, we could confirm the expression of mRNAs of these Th2-related cytokines in activated Th2 cells by RT-PCR (data not shown). This might be due to too low expression of these molecules to be analyzed by SAGE.

Th1/Th2 differentiation-regulatory genes, such as T-bet, GATA-3 and c-Maf, were not significantly different after activation. However, many immediate-early genes such as c-jun and c-fos, which propagate the cellular responses to growth stimuli, were highly induced in activated Th1 cells. NOT (TINUR), which was originally cloned from apoptotic human T lymphoid PEER cells stimulated with PMA and calcium ionophore (32), and TRAIL, which is a member of the TNF family and mediates activation-induced cell death of mature T lymphocytes (33), were highly expressed in Th1 cells as listed in the `Apoptosis' category of Table 3Go. These results may indicate that early-responsive genes in activated Th1 cells are up-regulated more than in activated Th2 cells; however, apoptosis-related genes are also induced quickly in activated Th1 cells in order to eliminate over-activated Th1 cells. ILF-2 (8.0-fold in activated Th2 cells) is one of the components of NF-AT (34) and the activation of NF-AT is reported to be involved in the effector function of Th2 cells (35,36). Thus, it suggests the possibility that ILF-2 is one of the regulators of NF-AT and affects the function of activated Th2 cells.

All genes categorized in apoptosis and proteolysis were predominantly expressed in activated Th1 cells. TIMP-1 is an inhibitor of matrix metalloproteinases, which play a crucial role in the infiltration of inflammatory cells and the induction of airway hyper-responsiveness (37). Thus, lower expression of TIMP-1 in the Th2-dominant condition may contribute to disease onset of some Th2 diseases, such as asthma.

With regard to enzymes and signaling molecules, the genes encoding kinases and phosphatases are categorized in Table 3Go. However, JAK and STAT families, which are related to the cytokine signaling pathway (33), were barely detected in either activated Th1 or Th2 cells. ß-Catenin (Th1; 7 tags, Th2; 0 tags) provides a link between cell-surface-expressed cadherins and represents a key molecule connecting cellular adhesion to signal transduction pathways (38). Cadherin expressed on T lymphocytes forms a complex with ß-catenin (39), and might be involved in the interactions between activated T cells, especially activated Th1 cells, and their cellular targets or the extracellular matrix.

Ca2+ controls various functions of the cells and is very important for signal transduction. Recently, it has been reported that the rate of Ca2+ clearance from the cytosol in Th2 cells was higher than that in Th1 cells and the expression of the Ca2+-activated K+ channel, which controls the membrane potential, is increased in Th1 cells, and these differences may affect the production of different cytokines between Th1 and Th2 cells (40). Thus, a gene encoding TASK, which is one of the potassium channels and preferentially expressed in activated Th1 cells, might be also related to preferential cytokine gene expression in Th1 and Th2 cells by controlling the membrane potential.

Furthermore, stress-induced transcripts such as some of the heat shock proteins were markedly expressed in activated Th1 cells. On the other hand, the gene encoding fusion, derived from t(12;16) malignant liposarcoma (FUS) is preferentially expressed in activated Th2 cells. FUS protein contains an RNA-recognition motif and is a component of nuclear riboprotein, which is related to not only cell proliferation but also cell differentiation (41). Interestingly, Hicks et al. reported that disruption of FUS had an effect on B cell development and activation in the cause of the defect of accessory cells (42). Thus, in type 2 immunity (humoral immunity), FUS in activated Th2 cells may have an important role in generating the specific antibodies in B lymphocytes.

Although we obtained cord blood from a minimum of four healthy volunteers to find the average gene expression, there could be differences in gene expression between individual donor-derived cells. To justify the SAGE results, we picked up seven genes of which expression is distinct between activated Th1 and Th2 cells, and analyzed their expression by RT-PCR (Fig. 3Go). The PCR results validated the SAGE data.



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Fig. 3. RT-PCR analysis of genes expressed differently in activated Th1 and Th2 cells. RT-PCR was performed on total RNA isolated from both cells. Genes preferentially expressed in (A) activated Th1 cells and (B) activated Th2 cells.

 
Very recently, transcript imaging of human Th1/Th2 cells using oligonucleotide arrays of ~6000 genes was reported (15). When our SAGE data was compared with the result of oligonucleotide arrays (part of their results is cited in Table 2Go), it was noticed that very limited numbers of genes were examined by an oligonucleotide array by the other group. The number of human genes has been estimated to be in the range from ~35,000 to 120,000 (4345). The SAGE data in this study includes >22,000 transcripts; however, comprehensive gene expression profile analysis using the DNA arrays lifted only 6000 genes, which is definitely too limited. Furthermore, they did not describe the abundance of these genes, either. Among the genes preferentially expressed in either cell type, the expression of genes encoding IFN-{gamma} [SAGE, 49.2-fold (Th1, 1770 tags; Th2, 36 tags); array, 61.5-fold], MIP-1ß [SAGE, 15.5-fold (Th1, 248 tags; Th2, 16 tags); array, 15.2-fold], MIP-1{alpha} [SAGE, 6.6-fold (Th1, 86 tags; Th2, 13 tags); array, 6.1-fold], perforin [SAGE, 11.0-fold (Th1, 11 tags; Th2, 0 tags); array, 7.2-fold], NOT [SAGE, 6.5-fold (Th1, 13 tags; Th2, 2 tags); array, 3.5-fold], TRAIL [SAGE, 6.0-fold (Th1, 12 tags; Th2, 2 tags); array, 5.8-fold] and IAP homolog C [SAGE, 5.0-fold (Th1, 5 tags; Th2, 0 tags); array, 2.3-fold] in activated Th1 cells, and CD6 [SAGE, 2.6-fold (Th1, 15 tags; Th2, 39 tags); array, 3.2-fold] in activated Th2 cells is more or less similar, but most of the genes shown to be differentially expressed are different from their results. The most probable reason for this discrepancy is that they generated Th1- and Th2-polarized cells for array hybridization analyses at an early stage (3 days) of the differentiation, whereas we prepared the RNAs from well-differentiated (2 weeks) Th cells.

In conclusion, thorough identification of the genes selectively expressed in human activated Th1 and Th2 cells should provide useful information to clarify the functions of these cells. In the future, by combining with a DNA microarray system, the data presented in this report should be very informative to diagnose various human Th1/Th2-dominated immune diseases. Furthermore, cloning of numerous unknown genes identified only in the EST database should provide further understanding of molecular pathogenesis of Th1/Th2 dominated human diseases and may provide novel therapeutic targets for the treatment of these diseases.


    Acknowledgments
 
We are very grateful to Drs V. Velculescu, L. Zhang, W. Zhou, B. Vogelstein and K.Kinzler for their help in SAGE analysis, and also to Mr Muto and Ms Ayabe for sequencing. We highly appreciate Dr H. A. Young (National Cancer Institute, Frederick, MD) for thoughtful comments on this work.


    Abbreviations
 
FUS fusion, derived from t(12; 16) malignant liposarcoma
GM-CSF granulocyte macrophage colony stimulating factor
ILF IL enhancer binding factor
MDC macrophage-derived chemokine
MIP macrophage inflammatory protein
NKEF NK enhancing factor
PE phycoerythrin
PMA phorbol myristate acetate
RANTES regulated upon activation, normal T cell expressed and presumably secreted
SAGE serial analysis of gene expression
TARC thymus and activation-regulated chemokine

    Notes
 
Transmitting editor: M. Miyasaka

Received 18 October 2000, accepted 6 December 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 

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