by guest
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`on July 19, 2019.
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`
`Recombinant Soluble Interleukin-11 (IL-11) Receptor a-Chain Can Act as
`an IL-11 Antagonist
`
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`䉷 (cid:49)(cid:57)(cid:57)(cid:55)(cid:98)(cid:121)(cid:84)(cid:104)(cid:101)(cid:65)(cid:109)(cid:101)(cid:114)(cid:105)(cid:99)(cid:97)(cid:110)(cid:83)(cid:111)(cid:99)(cid:105)(cid:101)(cid:116)(cid:121)(cid:111)(cid:102)(cid:72)(cid:101)(cid:109)(cid:97)(cid:116)(cid:111)(cid:108)(cid:111)(cid:103)(cid:121)(cid:46)
`
`I
`
`NTERLEUKIN-11 (IL-11) was first identified because of
`its ability to support the proliferation of an IL-6–depen-
`dent plasma cell line.1 Its biological actions include the abil-
`ity to stimulate proliferation of multipotential hemopoietic
`progenitor cells,2 the enhancement of megakaryocyte and
`platelet formation,3-5 and the stimulation of acute phase pro-
`tein synthesis.6 Many of these actions are shared with
`IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM),
`cardiotrophin-1 (CT-1), and ciliary neurotrophic factor
`(CNTF).7-11 The overlapping functions of these cytokines is
`in part due to the sharing of the cell surface receptor compo-
`nent, gp1309 with specificity being determined by a specific
`low-affinity receptor a-chain.12
`The IL-11 receptor a-chain (IL-11R) has recently been
`cloned and shown to share many structural and functional
`similarities with the IL-6 receptor a-chain (IL-6R).13 Firstly,
`the extracellular domain shows 24% amino acid identity
`including the characteristic conserved Trp-Ser-X-Trp-Ser
`(WSXWS) motif. The short cytoplasmic domain (34 amino
`acids) lacks the Box 1 and 2 regions that are required for
`activation of the JAK/STAT signaling pathway. Secondly,
`the IL-11R binds its ligand with a low affinity (kd (cid:130)10
`nmol/L) and alone is insufficient to transduce a biological
`signal. The generation of a high affinity receptor (kd (cid:130)400
`to 800 pmol/L) capable of signal transduction requires coex-
`pression of the IL-11R and gp130.13,14 In contrast, the high-
`affinity receptors for LIF, CT-1, and CNTF signal through
`a heterodimer of the LIF receptor and gp130.10,15-17 Finally,
`the early signaling pathways of IL-6 and IL-11 appear similar
`and include phosphorylation of the JAKs, STAT1 and 3, and
`activation of the mitogen-activated protein (MAP) kinase
`pathway.18,19
`Naturally occurring soluble receptors, consisting of the
`extracellular domain, have been described for many cytokine
`receptors including the specific low-affinity receptor a-
`chains for IL-6 and LIF.20,21 The soluble forms bind their
`respective ligand with a similar low affinity (1 to 20 nmol/
`L) to their transmembrane a-chain counterparts in the ab-
`sence of gp130.20,22,23 Soluble IL-6R (sIL-6R) can associate
`with cell surface gp130 in the presence of IL-6 and transduce
`a signal.23 A soluble form of the mouse LIFR is generated
`by alternative splicing of mRNA transcripts.24 In contrast to
`the sIL-6R, soluble LIFR can bind LIF but is unable to
`transduce a signal and thus behaves as a LIF antagonist.20
`Recently, a transcript potentially encoding a soluble IL-11R
`
`(sIL-11R) has been identified25 although the naturally oc-
`curring protein has yet to be isolated. Recombinant forms
`of the sIL-11R, in the presence of IL-11, have been shown
`to mediate an IL-11–type signal in cells that express gp130,
`but not the transmembrane IL-11R. In cells expressing the
`transmembrane IL-11R, the sIL-11R augments the IL-11 re-
`sponse.26-28 These results suggest that IL-11R is very similar
`to the IL-6R. However, in vitro studies using tagged recom-
`binant forms of IL-11, soluble human IL-11 receptor, and
`gp130 have suggested that dimers of the IL-11/sIL-11R com-
`plex bind to a single gp130 molecule rather than two like
`that seen with the IL-6–signaling complex.28
`The experiments reported here were undertaken to exam-
`ine the action of a recombinant sIL-11R expressed in the
`Chinese hamster ovary (CHO) cell line. Confirming recent
`reports, we show that the sIL-11R, in the presence of IL-11
`mediated a proliferative or differentiative signal in appro-
`priate cell lines expressing gp130 alone. However, the sIL-
`11R was capable of antagonizing the IL-11 response of cells
`expressing gp130 and the transmembrane IL-11R. The an-
`tagonistic activity may have implications for the predicted
`in vivo effects of sIL-11R.
`
`(cid:77)(cid:65)(cid:84)(cid:69)(cid:82)(cid:73)(cid:65)(cid:76)(cid:83) (cid:65)(cid:78)(cid:68) (cid:77)(cid:69)(cid:84)(cid:72)(cid:79)(cid:68)(cid:83)
`
`Construction of a soluble murine IL-11R cDNA. The extracellu-
`lar domain of the murine IL-11R was amplified using Taq polymer-
`ase from a full length, previously described cDNA clone.13 The 5ⴕ
`
`From the Walter and Eliza Hall Institute of Medical Research,
`and The Co-operative Research Centre for Cellular Growth Factors,
`and Rotary Bone Marrow Research Laboratory, Royal Melbourne
`Hospital, Victoria, Australia.
`Submitted December 16, 1996; accepted August 2, 1997.
`Supported in part by grants from the National Health and Medical
`Research Council (Canberra, Australia); The Anti-Cancer Council
`of Victoria (Melbourne); and the Cooperative Research Centre for
`Cellular Growth Factors (Melbourne, Australia).
`Address reprint requests to David J. Curtis, MD, Walter and Eliza
`Hall Institute of Medical Research, Post Office, Royal Melbourne
`Hospital, Victoria, 3050, Australia.
`The publication costs of this article were defrayed in part by page
`charge payment. This article must
`therefore be hereby marked
`‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
`indicate this fact.
`䉷 1997 by The American Society of Hematology.
`0006-4971/97/9011-0030$3.00/0
`
`(cid:66)(cid:108)(cid:111)(cid:111)(cid:100)(cid:44)(cid:86)(cid:111)(cid:108) (cid:57)(cid:48)(cid:44) (cid:78)(cid:111) (cid:49)(cid:49) (cid:40)(cid:68)(cid:101)(cid:99)(cid:101)(cid:109)(cid:98)(cid:101)(cid:114) (cid:49)(cid:41)(cid:44) (cid:49)(cid:57)(cid:57)(cid:55)(cid:58) (cid:112)(cid:112) (cid:52)(cid:52)(cid:48)(cid:51)(cid:45)(cid:52)(cid:52)(cid:49)(cid:50)
`
`(cid:52)(cid:52)(cid:48)(cid:51)
`
`AID Blood 0020
`
`/
`
`5h43$$$381
`
`10-28-97 19:13:05
`
`blda
`
`WBS: Blood
`
`Lassen - Exhibit 1017, p. 1
`
`

`

`by guest
`
`
`
`www.bloodjournal.orgFrom
`
`on July 19, 2019.
`
`For personal use only.
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`(cid:67)(cid:85)(cid:82)(cid:84)(cid:73)(cid:83) (cid:69)(cid:84) (cid:65)(cid:76)
`
`and 3ⴕ ends of the cDNA were modified to include Xba I sites. The
`predicted protein began at amino acid residue Ser-24 in the sequence
`described by Hilton et al.13 A stop codon was inserted immediately
`5ⴕ of the transmembrane domain after Gln-367. The cDNA was then
`cloned into the Xba I site of a modified pEFBOS vector containing
`the IL-3–signal sequence followed by an inframe FLAG sequence
`(DYKDDDDK) so that the N-terminus of the predicted secreted
`protein was DYKDDDDKSPCPQA. The nucleotide sequence of the
`plasmid pEFBOS/sIL-11R was confirmed by dideoxy sequencing
`using a PRISM (Applied Biosystems, Inc, Foster City, CA) Ready
`Reaction DyeDeoxy Terminator Cycle Sequencing kit on an Applied
`Biosystems 373 DNA sequencer (Foster City, CA).
`Expression and purification of sIL-11R. CHO cells (4 1 106)
`were washed twice in ice cold phosphate-buffered saline (PBS) and
`resuspended in PBS at 5 1 106 cells/mL. Cells were aliquoted into
`a 0.4-cm electroporation cuvette with 20 mg of pEFBOS/sIL-11R
`and 2 mg pPGKneo and then electroporated at 270 V and 960 mF
`in a Bio-Rad Gene-Pulser (Bio-Rad Laboratories, Hercules, CA).
`Cells were centrifuged through 1 mL fetal calf serum (FCS), resus-
`pended in RPMI-1640 medium containing 5% (vol/vol) FCS, and
`plated onto 10-cm diameter petri dishes. After 48 hours, geneticin
`(1 mg/mL) was added and 10 to 14 days later, resistant clones were
`picked. The highest sIL-11R expressing clone was identified by
`Western blot analysis.
`Conditioned medium was concentrated 101 using a YM-10 mem-
`brane (Amicon, Beverly, MA) and applied to an M2 anti-FLAG
`antibody affinity column (Eastman Kodak, New Haven, CT). After
`washing with 10 mmol/L Tris in PBS (pH 8.0) containing 0.02%
`(wt/vol) Tween-20 and 0.02% (wt/vol) sodium azide, bound sIL-
`11R was eluted from the column with 8 mL of 62.5 mg/mL FLAG
`peptide (Eastman Kodak). sIL-11R containing fractions were deter-
`mined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
`(SDS-PAGE) and Western blotting with M2 anti-FLAG antibody
`and exchanged into 150 mmol/L NaCl containing 0.02% Tween 20
`using gel filtration PD-10 columns (Pharmacia, Uppsala, Sweden).
`Fractions containing immunoreactive material were pooled and puri-
`fied by gel filtration using a Superdex 75 column (Pharmacia). Pro-
`tein estimation was performed by measuring the optical density (590
`nm) using Coomassie stain (Pierce, Rockford, IL) and compared
`with a bovine serum albumin standard. N- terminal sequencing anal-
`ysis was performed using a Beckman 6300 high performance amino
`acid analyzer (Irvine, CA) as previously described.29
`Immunoprecipitations. For antiphosphotyrosine assays, cell ly-
`sates from 1 1 107 cells were prepared using 1% Triton X-100
`(Sigma, St Louis, MO), 150 mmol/L NaCl, 50 mmol/L Tris HCl, 2
`mmol/L EDTA, 1 mmol/L sodium vanadate, 2 mg/mL aprotinin, and
`100 mg/mL phenylmethylsulfonyl fluoride. Five microliters of the
`antiphosphotyrosine antibody (4G10, UBI) or anti-LIF receptor anti-
`body (Regeneron Pharmaceuticals, Tarrytown, NY) was added and
`the antibody immobilized using Protein A sepharose. After incuba-
`tion at 4⬚C overnight, pellets were washed with 1 mL of lysis buffer
`containing 1% Nonidet P-40 (BDH Laboratory Supplies, Poole, UK),
`2 mg/mL aprotinin, and 1 mmol/L sodium vanadate and then resus-
`pended in 30 mL of reducing or nonreducing SDS sample buffer.
`Western blotting. Samples were run on 10% SDS-PAGE and
`electrophoretically transferred onto prewetted polyvinylidene diflu-
`oride (PVDF)-plus (Micron Separations Inc, Westborough, MA).
`Membranes were blocked with 5% (wt/vol) skim milk in PBS. Phos-
`photyrosine containing proteins were detected by incubation with
`gp130 (UBI), STAT3 (Santa Cruz Biotechnology, Inc, Santa Cruz,
`CA) or SHP-2 (Transduction Laboratories, Lexington, Kentucky)
`antibodies and bound antibodies detected using goat antirabbit IgG
`conjugated to horseradish peroxidase. FLAG containing proteins
`were detected by incubation with the mouse M2 anti-FLAG antibody
`and then horseradish peroxidase conjugated rabbit anti-mouse anti-
`
`body (DAKO, Glostrup, Denmark). Bound antibody was visualized
`using the ECL substrate kit (Amersham, Buckinghamshire, UK)
`followed by autoradiography.
`PAGE. Electrophoreses were performed using Bio-Rad 8% to
`25% SDS-PAGE precast Pharmacia Phast-Gels according to the
`manufacturer’s instructions and the gels were visualized by silver-
`staining.
`Deglycosylation. Four micrograms of lyophilized sIL-11R was
`deglycosylated using the enzymatic deglycosylation kit (GLYKO,
`Movato, CA). Samples were treated for 1 hour with O-glycosidase
`DS and NANaseII (GLYKO) and followed by N-glycanase F for 3
`hours at 37⬚C. One microliter of reaction samples were then run on
`8% to 25% SDS-PAGE Phast-Gel under nonreducing conditions.
`Binding assays. One to two micrograms sIL-11R was radio-
`iodinated using a modified iodine monochloride method as pre-
`viously described.30 Specific radioactivity was estimated to be 25,000
`counts per minute (cpm)/ng. 2 1 107 cells/mL in RPMI-1640 me-
`dium containing 10% (vol/vol) FCS and 20 mmo/L HEPES were
`incubated at room temperature for 4 hours with various combinations
`of 125I-sIL-11R and hIL-11 in the presence or absence of cold com-
`petitor sIL-11R (100-fold excess). Cell associated and free 125I-sIL-
`11R were separated by rapid centrifugation through 200 mL of FCS
`and quantitated in a g-counter. For estimation of the affinity of IL-
`11 for the sIL-11R, 2 mg/mL of sIL-11R was immobilized using
`anti-FLAG coated beads and a titration of unlabeled IL-11 performed
`in the presence of 125I-labeled IL-11 (2 1 106 cpm/mL). The bindabil-
`ity of the 125I-labeled IL-11 was only 10%. This was accounted for
`when estimation of affinities by the nonlinear curve fitting program
`(LIGAND) was performed.
`Cytokines and biological assays. Human IL-11 and oncostatin
`M were purchased from R & D Systems (Minneapolis, MN). Cytos-
`pins of M1 cells after 5 days in liquid culture were performed as
`previously described.14,31 3H-Thymidine assays were performed by
`adding M1 or M1/IL-11R cells (1 1 104 /mL) into 96-well plates
`and then adding cytokines as indicated. Plates were incubated in
`humidified air with 10% CO2 at 37⬚C. 3H-Thymidine (1 to 2 mCi/
`well) was added after 4 days and 16 hours later, cells were procured
`and cell associated radioactivity measured. The proliferation of Ba/
`F3 cells in response to cytokines was measured in LUX 60 microwell
`HL-A plate (Nunc Inc, Naperville, IL) and semisolid agar assays to
`measure differentiation of M1 cells were performed as described.13
`
`(cid:82)(cid:69)(cid:83)(cid:85)(cid:76)(cid:84)(cid:83)
`
`Production and purification of sIL-11R. A stable CHO
`cell line producing sIL-11R was established by coelectropor-
`ation of the sense sIL-11R cDNA with pPGK neo. The super-
`natant was purified using an anti-FLAG affinity column.
`Western blots of the FLAG-eluted CHO cell fractions
`showed immunoreactive material of approximately 50 kD
`and 200 kD. When fractions were Western blotted from gels
`run under reducing conditions, the 200-kD band was not seen
`(Fig 1A). This suggested that the 200-kD band represented
`disulphide-linked multimers of the sIL-11R. Purification of
`pooled fractions 4 to 6 by gel filtration revealed a peak at
`44 kD (Fig 1B). Silver staining of an SDS-PAGE revealed
`at least 4 distinct bands of between 40 and 55 kD (Fig 1C).
`Although the primary amino acid sequence predicts a protein
`of 30 kD, the extracellular domain of the sIL-11R contains 2
`potential N-glycosylation sites. To determine whether these
`multiple bands were due either to differential glycosylation
`or contamination, the pooled sample was lyophilized and
`then treated with deglycosylation enzymes. Treatment with
`N-glycanase F, which removes N-linked carbohydrates, re-
`
`AID Blood 0020
`
`/
`
`5h43$$$381
`
`10-28-97 19:13:05
`
`blda
`
`WBS: Blood
`
`Lassen - Exhibit 1017, p. 2
`
`

`

`by guest
`
`
`
`www.bloodjournal.orgFrom
`
`on July 19, 2019.
`
`For personal use only.
`
`(cid:73)(cid:76)(cid:45)(cid:49)(cid:49) (cid:65)(cid:78)(cid:84)(cid:65)(cid:71)(cid:79)(cid:78)(cid:73)(cid:83)(cid:77) (cid:66)(cid:89) (cid:65) (cid:83)(cid:79)(cid:76)(cid:85)(cid:66)(cid:76)(cid:69) (cid:73)(cid:76)(cid:45)(cid:49)(cid:49) (cid:82)(cid:69)(cid:67)(cid:69)(cid:80)(cid:84)(cid:79)(cid:82)
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`(cid:52)(cid:52)(cid:48)(cid:53)
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`examined using the murine myeloid cell line, M1, which
`expresses gp130 and the a-chain receptors for LIF and
`IL-6 but not for IL-11. After 4 days in suspension culture, 79
`{ 25% of cells stimulated with IL-11 and sIL-11R showed
`morphological features of macrophage differentiation (mean
`{ SD of 3 experiments, Fig 2D). This change was associated
`with inhibition of cellular proliferation as evidenced by re-
`duced incorporation of 3H-thymidine (see below). In con-
`trast, cells stimulated with IL-11 or sIL-11R alone showed
`no significant differentiation (Fig 2A and B). As reported
`previously,13 the majority of cells (93 { 4%) stimulated with
`LIF showed a macrophage phenotype (Fig 2C) as did cells
`that expressed the transmembrane IL-11R when treated with
`IL-11 (92 { 5%, data not shown). Flourescence-activated
`cell sorter (FACS) analysis of these cells confirmed in-
`creased surface expression of Mac-1a to levels similar to
`those observed with LIF treatment (data not shown).
`Experiments were then performed to examine the early
`phosphorylation events induced by sIL-11R. Lysates from
`M1 cells stimulated with various cytokines were immunopre-
`cipitated with either an anti-LIF receptor or an antiphospho-
`tyrosine antibody. Immunoprecipitates were run on SDS-
`PAGE gels, Western blotted, and probed with either the
`antiphosphotyrosine antibody in the case of the anti-LIF im-
`munoprecipitate (Fig 3A) or specific anti-gp130, STAT 3,
`or SHP-2 antibodies in the case of the antiphosphotyrosine
`immunopricipitates (Fig 3B through D). As expected, stimu-
`lation with maximal doses LIF (1,000 U/mL) or OSM(10
`ng/mL) resulted in phosphorylation of a 210-kD protein,
`which was confirmed to be the LIF receptor a-chain by
`immunoprecipitation (Fig 3A). Like IL-6, IL-11 (5 nmol/L)
`in the presence of sIL-11R (40 nmol/L) did not result in
`phosphorylation of the LIF receptor a-chain (Fig 3A). How-
`ever, consistent with the biological effects above, cells stim-
`ulated with sIL-11R and IL-11 showed phosphorylation of
`gp130 and STAT 3 (Fig 3B and C). Probing for the tyrosine
`phosphatase SHP-2 also showed phosphorylation in response
`to sIL-11R in the presence of IL-11 (Fig 3D). The phosphor-
`ylation pattern in cells stimulated with IL-11 or sIL-11R
`alone was no different to saline controls. These experiments
`suggested that IL-11 plus sIL-11R induced a differentiative
`response in M1 cells via phosphorylation of gp130. Thus,
`analogous to the transmembrane IL-11R and similar to re-
`sults with IL-6 and the sIL-6R,23 the sIL-11R acted as an
`agonist to effectively transmit an IL-11 signal. This showed
`that the intracellular domain of IL-11R was not required for
`a biological response.
`Signaling by sIL-11R requires higher concentrations of
`IL-11 than by the transmembrane IL-11R. To quantitate
`the biological activity of the sIL-11R, we examined its ability
`to induce differentiation to postmitotic macrophages and
`thereby inhibit thymidine incorporation of M1 cells.31,32 Cells
`were stimulated for 5 days with sIL-11R plus IL-11. Titration
`of sIL-11R in the presence of IL-11 (500 pmol/L) showed
`a maximal biological response in the presence of 40 nmol/
`L sIL-11R (Fig 4A). In the presence of 40 nmol/L sIL-11R,
`an IL-11 titration was performed and compared with the
`dose-response for M1 cells expressing the transmembrane
`IL-11R in addition to gp130 (M1/IL-11R). The sIL-11R pro-
`
`(cid:70)(cid:105)(cid:103) (cid:49)(cid:46) (cid:80)(cid:117)(cid:114)(cid:105)(cid:174)(cid:99)(cid:97)(cid:116)(cid:105)(cid:111)(cid:110) (cid:111)(cid:102) (cid:115)(cid:73)(cid:76)(cid:45)(cid:49)(cid:49)(cid:82)(cid:46) (cid:40)(cid:65)(cid:41) (cid:87)(cid:101)(cid:115)(cid:116)(cid:101)(cid:114)(cid:110) (cid:98)(cid:108)(cid:111)(cid:116) (cid:111)(cid:102) (cid:67)(cid:72)(cid:79) (cid:115)(cid:73)(cid:76)(cid:45)(cid:49)(cid:49)(cid:82)
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`(cid:115)(cid:116)(cid:97)(cid:114)(cid:116)(cid:105)(cid:110)(cid:103) (cid:109)(cid:97)(cid:116)(cid:101)(cid:114)(cid:105)(cid:97)(cid:108)(cid:59) (cid:66)(cid:84)(cid:44) (cid:98)(cid:114)(cid:101)(cid:97)(cid:107)(cid:116)(cid:104)(cid:114)(cid:111)(cid:117)(cid:103)(cid:104) (cid:119)(cid:104)(cid:101)(cid:110) (cid:108)(cid:111)(cid:97)(cid:100)(cid:101)(cid:100) (cid:111)(cid:110)(cid:116)(cid:111) (cid:97)(cid:102)(cid:174)(cid:110)(cid:105)(cid:116)(cid:121) (cid:99)(cid:111)(cid:108)(cid:45)
`(cid:117)(cid:109)(cid:110)(cid:46) (cid:66)(cid:108)(cid:111)(cid:116)(cid:115) (cid:112)(cid:114)(cid:111)(cid:98)(cid:101)(cid:100) (cid:119)(cid:105)(cid:116)(cid:104) (cid:97)(cid:110)(cid:116)(cid:105)(cid:45)(cid:70)(cid:76)(cid:65)(cid:71) (cid:97)(cid:110)(cid:116)(cid:105)(cid:98)(cid:111)(cid:100)(cid:121)(cid:46) (cid:83)(cid:97)(cid:109)(cid:112)(cid:108)(cid:101)(cid:115) (cid:112)(cid:114)(cid:101)(cid:112)(cid:97)(cid:114)(cid:101)(cid:100) (cid:98)(cid:121)
`(cid:109)(cid:105)(cid:120)(cid:105)(cid:110)(cid:103) (cid:119)(cid:105)(cid:116)(cid:104) (cid:101)(cid:113)(cid:117)(cid:97)(cid:108) (cid:118)(cid:111)(cid:108)(cid:117)(cid:109)(cid:101) (cid:111)(cid:102) (cid:50)Ì (cid:110)(cid:111)(cid:110)(cid:114)(cid:101)(cid:100)(cid:117)(cid:99)(cid:105)(cid:110)(cid:103) (cid:40)(cid:108)(cid:101)(cid:102)(cid:116) (cid:115)(cid:105)(cid:100)(cid:101) (cid:111)(cid:102) (cid:103)(cid:101)(cid:108)(cid:41) (cid:111)(cid:114)
`(cid:114)(cid:101)(cid:100)(cid:117)(cid:99)(cid:105)(cid:110)(cid:103) (cid:40)(cid:114)(cid:105)(cid:103)(cid:104)(cid:116) (cid:115)(cid:105)(cid:100)(cid:101) (cid:111)(cid:102) (cid:103)(cid:101)(cid:108)(cid:41) (cid:115)(cid:97)(cid:109)(cid:112)(cid:108)(cid:101) (cid:98)(cid:117)(cid:102)(cid:102)(cid:101)(cid:114) (cid:97)(cid:110)(cid:100) (cid:114)(cid:117)(cid:110) (cid:111)(cid:110) (cid:56)(cid:37) (cid:116)(cid:111) (cid:50)(cid:53)(cid:37)
`(cid:83)(cid:68)(cid:83)(cid:45)(cid:80)(cid:65)(cid:71)(cid:69)(cid:46) (cid:77)(cid:111)(cid:108)(cid:101)(cid:99)(cid:117)(cid:108)(cid:97)(cid:114) (cid:119)(cid:101)(cid:105)(cid:103)(cid:104)(cid:116)(cid:115) (cid:111)(cid:110) (cid:108)(cid:101)(cid:102)(cid:116) (cid:115)(cid:105)(cid:100)(cid:101) (cid:40)(cid:107)(cid:68)(cid:41)(cid:46) (cid:40)(cid:66)(cid:41) (cid:67)(cid:104)(cid:114)(cid:111)(cid:109)(cid:97)(cid:116)(cid:111)(cid:103)(cid:114)(cid:97)(cid:109)
`(cid:111)(cid:102) (cid:83)(cid:117)(cid:112)(cid:101)(cid:114)(cid:100)(cid:101)(cid:120) (cid:55)(cid:53) (cid:103)(cid:101)(cid:108) (cid:174)(cid:108)(cid:116)(cid:114)(cid:97)(cid:116)(cid:105)(cid:111)(cid:110) (cid:40)(cid:80)(cid:104)(cid:97)(cid:114)(cid:109)(cid:97)(cid:99)(cid:105)(cid:97)(cid:41) (cid:111)(cid:102) (cid:112)(cid:111)(cid:111)(cid:108)(cid:101)(cid:100) (cid:102)(cid:114)(cid:97)(cid:99)(cid:116)(cid:105)(cid:111)(cid:110)(cid:115) (cid:52) (cid:116)(cid:111) (cid:54)(cid:46)
`(cid:40)(cid:67)(cid:41) (cid:83)(cid:105)(cid:108)(cid:118)(cid:101)(cid:114) (cid:115)(cid:116)(cid:97)(cid:105)(cid:110) (cid:111)(cid:102) (cid:110)(cid:111)(cid:110)(cid:114)(cid:101)(cid:100)(cid:117)(cid:99)(cid:105)(cid:110)(cid:103) (cid:83)(cid:68)(cid:83)(cid:45)(cid:80)(cid:65)(cid:71)(cid:69) (cid:111)(cid:102) (cid:102)(cid:114)(cid:97)(cid:99)(cid:116)(cid:105)(cid:111)(cid:110)(cid:115) (cid:52)(cid:48) (cid:116)(cid:104)(cid:114)(cid:111)(cid:117)(cid:103)(cid:104) (cid:52)(cid:52)
`(cid:102)(cid:114)(cid:111)(cid:109) (cid:103)(cid:101)(cid:108) (cid:174)(cid:108)(cid:116)(cid:114)(cid:97)(cid:1