Murine Leukemia: Depressed Response to Interferon Induction Correlated with a Serum Hyporeactive Factor - American Society ...
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INFnCTION AND IMMUNITY, Feb. 1976, p. 392-398 Vol. 13, No. 2
Copyright © 1976 American Society for Microbiology Printed in U.S.A.
Murine Leukemia: Depressed Response to Interferon
Induction Correlated with a Serum Hyporeactive Factor
DALE A. STRINGFELLOW
The Upjohn Company, Department of Experimental Biology, Kalamazoo, Michigan 49001
Received for publication 29 July 1975
Mice injected by the intraperitoneal route with either L1210 or P388 leukemic
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cells progressively developed a state of hyporeactivity to interferon induction
that was dependent upon inducer and time of administration. A circulating
factor was detected in the serum of both L1210 and P388 leukemic mice that
could transfer hyporeactivity to normal murine cells in vitro. A direct relation-
ship existed between the concentration of serum factor and development of
hyporeactivity to interferon induction in vivo. Characterization of the serum
hyporeactive factor from P388 or L1210 leukemic mice indicated that both were
similar to a hyporeactive factor previously detected in serum from virus-infected
mice. These results suggest a cause-effect relationship between the serum
hyporeactive factor and development of hyporeactivity in vivo.
Viral interference, interferon, and interferon either 105 L1210 or 10" P388 leukemic cells, which
inducers can inhibit the replication of onco- were generously furnished by G. L. Neil, Cancer
genic viruses, block in vitro transformation by Research, The Upjohn Co. Survival time of mice
such viruses, and alter the course of leukemia injected with L1210 or P388 cells was 8 to 11 days or 9
and tumors in animals (1, 5, 6, 10, 11, 15, 17, 21, to 12 days, respectively.
22). These observations suggest that interferon Cells. Murine interferon assays were carried out
in a cloned continuous line of mouse L-cell fibro-
and its inducers may have value in the treat- blasts (L929) originally obtained from the American
ment of malignant diseases as well as virus Type Culture Collection Cell Repository (Rockville,
infections that arise during malignancy. A Md.).
well-documented immunosuppression due to Secondary mouse embryo fibroblasts (MEF) were
malignancy has, however, been observed in ex- obtained from ICR strain mice (Upj:TUC-ICR; Up-
perimental animals and man (2, 7, 12, 14, 16), john Rearing and Procurement) as previously de-
and mice infected with Friend leukemia virus scribed (18).
(4) as well as other viruses (8, 13, 19) progres- Media. Eagle minimum essential medium (Micro-
sively develop a depressed ability to respond to biological Associates) containing 10% fetal calf se-
rum (Reheis Chemical Co., Phoenix, Ariz.), 100 U of
interferon induction. Therefore, agents de- penicillin per ml, and 50 ,tg of streptomycin per ml
signed specifically to bolster naturally occur- was used to propagate and maintain all tissue cul-
ring host defense mechanisms, such as inter- tures.
feron inducers, may be limited by the develop- Viruses. The Herts strain of Newcastle disease
ment of such a condition. virus (NDV) was originally obtained from S. Baron
The studies in this report were initiated to (National Institutes of Health). The stock NDV
determine whether an impaired ability to re- preparation used in these experiments was propa-
spond to interferon induction developed in mice gated in embryonated hen eggs which had been
bearing L1210 (9) or P388 (3) leukemia (both injected by the allantoic route and had a titer of 2.0
x 109 plaque-forming units (PFU) per ml in primary
originally induced by methylcholanthrene), chicken embryo cells.
and, after such a hypoactive condition was ob- Vesicular stomatitis virus (VSV), Indiana strain,
served, to elucidate mechanisms responsible for was obtained from the American Type Culture Col-
the hyporeactivity. lection. Pools of VSV were propagated in primary
embryonic chicken kidney cell monolayers. The
MATERIALS AND METHODS stock preparation of VSV used in these studies had a
titer of 6 x 10" PFU/ml when assayed in L929 mouse
Mice. BDF1 male mice (16 to 18 g) were obtained cells.
from Jackson Laboratories (Bar Harbor, Me.) and Rochester mouse virus (RMV) is a strain of a
housed under conditions of constant temperature large-plaque mutant of encephalomyocarditis
and a 12-h light cycle, with food and water provided (EMC) virus which was isolated in MEF cell cul-
ad libitum. Mice were injected by the intraperito- tures from paralyzed and dying 2-week-old suckling
neal (i.p.) route with a 100% lethal inoculum of mice through which the virus had been blind pas-
392VOL. 13, 1976 MURINE LEUKEMIA 393 saged. This virus was identified as belonging to the inducers were administered 24 h after L1210 EMC group of murine picornavirus (D. A. Baker, cell injection (Fig. la). By 48 h, L1210 leukemic Master's thesis, Univ. of Rochester, Rochester, mice had a suppressed serum interferon re- N.Y.) and was obtained from L. A. Glasgow (Uni- sponse to RMV (250 U/ml compared to 640 U/ml versity of Utah, Salt Lake City). Stock preparations of RMV were obtained by injecting suckling mice in controls, 39%), NDV (4,500 versus 6,300 U/ intracerebrally with virus. At the height of disease, ml, 71%), tilorone (400 versus 1,600 U/ml, 25%), brains were removed, homogenized as a 10% suspen- and poly(I:C) (450 versus 1,000 U/ml, 45%), but sion in minimal essential medium plus 10% fetal calf appeared to regain responsiveness on days 3 serum, and stored at -70 C. The pool had a titer of and 4 of disease. From that point on the re- 2.5 x 101 PFU/ml in secondary MEF cell cultures. sponse to each inducer steadily declined, with Interferon assay. Confluent monolayers of L929 extremely low levels of serum interferon being cells grown in 35-mm plastic petri dishes (Falcon stimulated on day 8; RMV (
394 STRINGFELLOW INFECT. IMMUN.
a
780
T 630 b
500 T
340i 1330
200 21!00 r El Poly 1 C
--
X NOV
180 180 * Tilorone
O RMV
160 160
I-
z
0 140
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I-
2
0
120 _
ci
100 _
z z
0
80
,L
60
40
201-
I J-
3 4 5 6 1 2 3 4 5 6 8
DAYS POST L121o CELL INJECTION DAYS POST P3g8 CELL INJECTION
FIG. 1. Serum interferon levels induced by poly(I:C), NDV, tilorone hydrochloride, and RMV in L1210
(a) and P388 (b) leukemic mice. Interferon titers are expressed as a percentage of levels induced in matched
groups of normal uninfected control animals at each time period. L1210, P388, and uninfected control mice
received a single injection of inducers either 1, 2, 3, 4, 5, 6, or 8 days after leukemic cell injection. Blood
and subsequent serum samples were collected at the time of maximum serum interferon response [4 h after
poly(I:C), 6 h after NDV or RMV and 18 h after tilorone]. Each bar represents the mean of results from three
separate experiments, whereas the vertical lines designate range.
pendent state of hyporeactivity and that the tration of hyporeactive factor is expressed in
development of hyporesponsiveness could not units per milliliter, which represents the recip-
be explained solely on the basis of viral destruc- rocal of the highest dilution of serum inhibiting
tion of specific cell populations necessary for by 50% the interferon response induced by NDV
interferon production. Rather, a circulating in control plates treated with comparable dilu-
factor was detected in the serum of EMC virus- tions of serum from normal uninfected mice.
infected mice which could transfer hyporeactiv- A circulating hyporeactive factor was de-
ity to normal murine cells in vitro. It was there- tected in serum from L1210 leukemic mice (Fig.
fore of interest to determine whether a similar 2a). The concentration of serum hyporeactive
hyporeactive factor could be detected in serum factor (SHF) reached peak levels of 57 U/ml 2
from leukemic mice. days after L1210 cell injection and then steadily
Serum was collected from L1210, P388 leu- declined. During the early stages of disease an
kemic, or normal uninfected mice daily for the indirect relationship existed between the con-
first 8 days after cell injection and assayed for centration of circulating SHF and the ability to
hyporeactive factor as previously described respond to interferon induction. On day 2,
(18). Confluent secondary MEF cell monolayers when SHF concentrations were at a maximum,
grown in 35-mm petri dishes (Falcon Plastics) the average interferon response of L1210 leu-
were rinsed with PBS and in triplicate received kemic mice to the four inducers was depressed,
1 ml of a serum dilution per plate. After 18 to 24 and as the level of SHF decreased, the ability to
h of incubation at 37 C each plate received 4 x respond to interferon induction increased. SHF
107 PFU of NDV and was returned to 37 C for 24 was also detected in the serum of P388 leukemic
h. Growth medium was then harvested and mice (Fig. 2b). High circulating levels of SHF
frozen until assayed for interferon. The concen- (75 U/ml) were detected 48 h after leukemic cellVOL. 13, 1976 MURINE LEUKEMIA 395
injection and remained relatively high (45 to 70 ing the later stages of P388 and L1210 leukemia
U/ml) through day 8 of disease. A correspond- may at least in part be attributed to major
ing initial and continuous suppression of the changes in various cell populations. They do
interferon response was also observed. These not, however, explain the hyporeactivity ob-
results suggest a direct relationship between served during the early stages of disease and
the presence of SHF and a depressed interferon further support the possible role of SHF in mod-
response in leukemic mice. ulating hyporeactivity in vivo.
Daily total and differential peripheral leuko- Properties of hyporeactive factor in the
cyte (WBC) counts and spleen weights were sera of leukemic mice. The physicochemical
also determined for both L1210 and P388 leu- properties of hyporeactive factor in serum col-
kemic mice (Table 1). Significant increases in lected 72 h after P388 or 48 h after L1210 cell
total WBC numbers and spleen weights began injection were determined by using a previously
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to appear 3 to 4 days after injection of P388 described procedure (18). Fundamental proper-
leukemic cells. In L1210 leukemic mice a tran- ties of SHF from either L1210 or P388 leukemic
sient increase in WBC and lymphocyte counts mice were similar to those of a previously char-
appeared on days 3 and 4 followed by a gradual acterized hyporeactive factor detected in the
decline. Significant increases in total WBC serum of EMC virus-infected mice (18, 20; Table
numbers again appeared 7 to 8 days after L1210 2). Each was protein in nature and was de-
cell injection. Low numbers of blast and juve- stroyed by incubation at 37 C for 30 min with 50
nile cells appeared in the circulation of L1210 ,ug of trypsin per ml (3 x crystallized; Worthing-
and P388 leukemic mice as early as 48 h and ton Biochemical Corp., Freehold, N.J.) but was
progressively increased in numbers, reaching not affected by ribonuclease or deoxyribonucle-
levels of up to 30% by day 8. These results ase (50 ,ug/ml; Worthington Biochemical Corp.).
suggest that the hyporeactivity observed dur- No loss in biological activity was observed when
a
530 INDUCERS
320
o Pit 1: C
220b
a Tilarese INDUCERS
200 O NOV 200 O Ply I C
O RMV A Tirerme
130 ISO O Nov
O RMV
160 160
S140
70
120 !S120 J# I
100~~~~~~~~~0 us100 ' -
-
m
go I 0~~~~~~~~~~0
60~~~~~~~~~0
/ a3 cc 40
"I I~~~
20
20 0
1 2 3 4 5 6 7 3 - 1 2 3 4 5 6 7 3
DAYS POST L1212 CELL INJECTION DAYS POST P388 CELL INJECTION
FIG. 2. Serum interferon and SHF levels in L1210 (a) or P388 (b) leukemic mice. Interferon titers are
expressed as a percentage of the levels induced in matched groups of normal uninfected control mice at each
time period. L1210, P388, and uninfected control animals received a single injection of poly(I:C), tilorone
hydrochloride, NDV, or RMV either 1, 2, 3, 4, 5, 6, or 8 days after leukemic cell injection. Blood and
subsequent serum samples were collected 4 h after poly(I:C), 6 h after NDV or RMV, or 18 h after tilorone.
Each point represents the mean of results from three separate experiments, whereas the solid line designates
the average response to all four inducers. The broken line designates the average level of SHF in serum from
leukemic mice estimated by the ability of dilutions of serum to inhibit the interferon response induced by NDV
in MEF cells. SHF levels are expressed in units per milliliter, which represents the reciprocal of the highest
serum dilution inhibiting by 50% the interferon response induced by NDV in control plates.396 STRINGFELLOW INFECT. IMMUN.
TABLE 1. Daily peripheral, WBC profile and spleen weights of L1210 and P388 leukemic mice
Characteristics of Days post L1210 or P388 cell injection
mice 1 2 3 4 5 6 7 8
L1210
Total WBC/mm3 10,2000 9,200 12,500 11,000 9,900 9,700 12,200 29,900
Lymphocytes/mm3 7,200 7,100 11,000 9,600 7,900 6,600 3,200 5, 100
Monocytes/mm3 1,300 1,000 500 400 400 800 1,700 4,500
Granulocytes/mm3 1,700 1,100 1,000 1,000 1,600 2,300 7,300 20,300
Avg spleen wt (g) 0.1 0.08 0.08 0.1 0.08 0.1 0.1 0.11
P388
Total WBC/mm3 9,200 10,600 15,200 15,400 13,600 19,900 15,300 15,000
Lymphocytes/mm3 6,500 8,300 12,300 11,000 8,400 12,700 6,900 4,500
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Monocytes/mm3 1,700 800 800 1,400 1,500 2,000 3,400 3,800
Granulocytes/mm3 2,000 1,500 2,100 3,000 3,700 5,200 5,000 6,700
Avg spleen wt (g) 0.07 0.06 0.11 0.17 0.24 0.23 0.24 0.24
Normal controls
Total WBC/mm3 9,800 9,800 9,400 10,200 9,600 9,800 10,500 9,200
Lymphocytes/mm3 6,900 7,800 7,600 8,400 7,900 7,500 8,400 7,400
Monocytes/mm3 1,300 900 940 900 800 1,100 900 860
Granulocytes/mm3 1,600 1,100 860 900 900 1,200 1,200 940
Avg spleen wt (g) 0.061 0.07 0.06 0.05 0.07 0.06 0.06 0.07
a
Each value represents a mean of 18 animals, six animals per experiment in three separate experiments.
TABLE 2. Physicochemical properties of SHF from tween 20,000 and 40,000, using G-200 Sephadex
EMC virus-infected, L1210, or P388 leukemic mice (Pharmacia Fine Chemicals, Uppsala, Sweden)
SHF sourceb
column chromatography (18). Using an identi-
PropertiePa cal system, the molecular weight of SHF from
L1210 P388
__
___ EMC L1210 or P388 leukemic mice was estimated to
be between 100,000 and 150,000. Also, the SHF
Trypsin sensitivity + + + from virus-infected or leukemic mice had no
RNase sensitivity - - -
DNase sensitivity - - - effect on the sensitivity of L929 cells to the
Dialyzability - - - antiviral action of interferon (but effectively
Sedimentation at 100,000 - - - impaired their ability to produce interferon),
x g
Species specific + + +
nor could any interferon be detected in serum
Heat stability: from L1210 or P388 leukemic mice.
56C
37C + + + DISCUSSION
pH stability (2.0-11.0) + + + Results presented in this report indicate that
Mol wt (Sephadex G-200 i100,000- 100,000- 20,000-
chromatography) 150,000 150,000 40,000 both L1210 and P388 leukemic mice progres-
I sively develop a state of hyporeactivity to inter-
a
RNase, ribonuclease; DNase, deoxyribonuclease. feron induction. If such a condition develops in
b Serum was collected 96 h after EMC virus infection, 72 man it may limit the use of interferon inducers
h after P388 or 48 h after L1210 cell injection. as effective therapeutic agents in the treatment
of neoplasia or secondary virus infections that
sera from leukemic or EMC virus-infected mice arise during malignancy. Reports that man de-
were dialyzed for 48 h at 4 C against PBS or velops an immunosuppressive state during ma-
when centrifuged at 100,000 x g for 2 h. The lignancy (7) suggest that such a depression of
biological activity of SHF from all three sources interferon responsiveness may occur. The hypo-
was species specific in that each depressed the reactivity created in mice by L1210 and P388
NDV-induced interferon response of murine leukemia, however, was inducer dependent.
cells (L929, MEF, or peritoneal exudate cells) That is, the degree of hyporeactivity and rate of
but had no effect on human foreskin fibroblast, onset varied with each inducer. For example,
rabbit kidney, or embryonic chicken kidney poly(I:C) and NDV generally induced higher
cells. At 56 C greater than 90% of the biological interferon levels later during L1210 leukemia
activity of each was lost within 10 min but each than did tilorone or RMV. These results sug-
was stable at 37 C for at least 4 h and over a pH gest that the ability of the diseased as well as
range of 2.0 to 11.0 for 48 h at 4 C. The molecu- the normal host to respond to a particular in-
lar weight of SHF from EMC virus-infected ducer may be a vital point to consider in selec-
mice has previously been estimated to be be- tion and development of these agents for thera-VOL. 13, 1976 MURINE LEUKEMIA 397
peutic use. ACKNOWLEDGMENTS
Several explanations might account for the The technical assistance of H. C. Vanderberg and F. T.
development of hyporeactivity. The data pre- Maida and statistical assistance of P. Good and T. Tesar are
sented in this report indicate that a circulatory appreciated.
factor was detected in the serum of L1210 and
P388 leukemic mice which could transfer hypo- LITERATURE CITED
reactivity to normal murine cells in vitro. Dur- 1. Atanasiu, P., and C. Chany. 1960. Action d'un inter-
ing the initial stages of disease a direct rela- feron provenant de cellules malignes sur l'infection
experimentale du hamster nouveau-ne par le virus du
tionship appeared to exist between the develop- polyome. C. R. Acad. Sci. 251:1687-1689.
ment of hyporeactivity and the concentration of 2. Ceglowski, W. S., and H. Friedman. 1968. Immunosup-
SHF of leukemic mice. When SHF levels were pressive effects of Friend and Rauscher leukemia dis-
high, interferon responsiveness was suppressed. ease virus on cellular and humoral antibody forma-
tion. J. Natl. Cancer Inst. 40:983-995.
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Correspondingly, on day 1 or 4 when no or low 3. Dawe, C. J., and M. Potter. 1957. Morphologic and
levels of SHF were detected in the sera of L1210 biologic properties of a lymphoid neoplasia of mice in
mice and on day 1 when low circulating SHF vivo and in vitro. Am. J. Pathol. 33:603.
levels were present in P388 mice, the in- 4. DeMaeyer-Guignard, J. 1972. Mouse leukemia: depres-
sion of serum interferon production. Science 177:797-
terferon response induced by poly(I:C), tilo- 799.
rone, or NDV was normal or enhanced. These 5. Gresser, I., L. Berman, G. De-The, D. Brouty-Boye, J.
data suggest that high circulating levels of SHF Choppey, and E. Falcoff. 1968. Interferon and murine
decreased interferon responsiveness and that in leukemia. V. Effect of interferon preparations on the
evolution of Rauscher disease in mice. J. Natl. Can-
some cases extremely low SHF levels may have cer Inst. 41:505-522.
potentiated the response to some inducers. 6. Gresser, I., J. Coppey, E. Falcoff, and D. Fontaine.
Physicochemical properties of the factor de- 1966. Action inhibitrice de l'interferon brut sur le
tected in the sera of leukemic mice were similar developpement de la leucemie de Friend chez la
souris. C. R. Acad. Sci. Ser. D 263:586-588.
to those of a factor previously detected in the 7. Hollinshead, A. C., G. Tarro, P. Ghretein, and W.
serum of virus-infected mice (except for molecu- Rawls. 1974. Characterization of herpesvirus non-
lar weight, which may be similar to interferon viral antigens: relation to squamous cell carcinomas,
where several molecular weight species occur) p. 301-317. In E. Kurstak and R. Morisset (ed.), Viral
immunodiagnostics. Academic Press
(18, 20), suggesting more than a coincidental 8. Hoterman, 0. A., and E. A. Havell.Inc.,
New York.
1970. Reduced
relationship between development of hyporeac- interferon response in mice congenitally infected
tivity in vivo and presence of circulating SHF. with lymphocytic choriomeningitis virus. J. Gen. Vi-
Hollinshead et al. (7) have reported that a cir- 9. Law, rol. 9:101-103.
L. W., T. B. Dunn, P. J. Boyle, and J. H. Miller.
culating factor was detected in the serum of 1949. Observations on the effect of a folic acid antago-
patients with immunosuppression due to ma- nist on transplantable lymphoid leukemias in mice.
lignancy and that the humoral factor may have 10. Levy, J. Natl. Cancer Inst. 10:179-193.
H. B. 1972. Interferon and interferon inducers in
mediated the immune defect. Whether the fac- the treatment of neoplastic diseases, p. 53-61. In I.
tor described in this report is similar is at pres- Bradsky, S. B. Kahn, and J. H. Meyer (ed.), Cancer
ent unknown, although studies are currently chemotherapy II. Grune & Stratton, New York.
under way to determine the effect of SHF from 11. Levy, H. B., Law, L. W., and A. S. Rabson. 1969.
Inhibition of tumor growth by polyinosinic-polycyti-
leukemic and virus-infected mice on various in dylic acid. Proc. Natl. Acad. Sci. U.S.A. 62:357-363.
vitro immune functions. SHF action does not, 12. Metcalf, D., and R. Moulds. 1967. Immune responses in
however, appear to be nonspecific nor due to preleukemic and leukemic AKR mice. Int. J. Cancer
2:53-58.
cellular toxicity since cells exposed to hyporeac- 13. Osborn,
antiviral J. E., and D. N. Medearis. 1967. Suppression of
tive factor are sensitive to the action interferon and antibody and multiplication of New-
of interferon and supported virus replication. castle disease virus in cytomegalovirus infected mice.
The data presented do not support an expla- Proc. Soc. Exp. Biol. Med. 124:347-353.
nation of the development of hyporeactivity 14. Peterson, R. D. A., R. Hendrickson, and R. A. Good.
destruc- 1963. Reduced antibody forming capacity during the
during the initial stages of disease on a incubation period of passage A leukemia in C3H mice.
tion or shift in cell populations necessary for Proc. Soc. Exp. Biol. Med. 114:517-520.
interferon induction or production. This may be 15. Rhim, J. S., C. Greenawalt, and R. J. Heubner. 1969.
particularly important since if the hyporeactiv- Synthetic double stranded RNA: inhibitory effect on
murine leukemia and sarcoma viruses in cell culture.
ity observed is mediated by a serum factor, Nature (London) 222:1166-1168.
studies directed toward elucidating the mecha- 16. Salaman, M. H., and N. Weddenburn. 1966. The immu-
nisms involved may also suggest means of cir- nosuppressive effect of Friend virus. Immunology
cumventing the development of hyporespon- 17. Sarma, 10:445-458.
P. S., G. Shiu, R. H. Neubauer, S. Baron, and
siveness which may concomitantly enhance the R. J. Huebner. 1969. Virus induced sarcoma of mice:
utility of interferon inducers potentially
as use- inhibitor by a synthetic polyribonucleotide complex.
ful antiviral agents. Proc. Natl. Acad. Sci. U.S.A. 62:1046-1054.398 STRINGFELLOW INFECT. IMMUN.
18. Stringfellow, D. A. 1975. Hyporeactivity to interferon ditis virus-infected mice. Infect. Immun. 10:1337-
induction: characterization of a hyporeactive factor in 1342.
the serum of encephalomyocarditis virus-infected 21. Wheelock, E. F., and R. P. B. Larke. 1968. Efficacy of
mice. Infect. Immun. 11:294-302. interferon in the treatment of mice with established
19. Stringfellow, D. A., and L. A. Glasgow. 1972. Hyporeac- Friend virus leukemia. Proc. Soc. Exp. Biol. Med.
tivity of infection: potential limitation to therapeutic 127:230-238.
use of interferon-inducing agents. Infect. Immun. 22. Zeleznick, L. D., and B. K. Bhuyan. 1969. Treatment of
6:743-747. leukemic (L1210) mice with double stranded polyri-
20. Stringfellow, D. A., and L. A. Glasgow. 1974. Hyporeac- bonucleotides. Proc. Soc. Exp. Biol. Med. 130:126-
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hyporeactive factor in the serum of encephalomyocar-
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