- Email: [email protected]
Thiomersal preservative, pertussis vaccine and intussusceptions of the bowel in mice
ff~
of
~tan
1 ~ 8 6, 267-270
om
tolye, p
l
vaccine b m mtce
A.C. and I!_. C.
t N.B. t
o f the
t I . O.
mad p~rtussis to ~ e eeptior~ ~ a ~f ~ o f t~es, ma by the in I ~ u t e . ~ e acti~qry i~ labile at 75 *O ~nd ~ absent frc~rr~comb of tbd with b a e t e ~ from other g ~ e ~ , I~a ~ e ent of waccines i~4~ B. p from ~S~ ~ fro~a ~ho~e due tO B, toxiedty. ~
D
control t e s s o n v a c c i n ~ ~ g cells p r e ~ r ' ¢ ~ ~ we have e~'ac~untered e e t e d d e a t h s in g r o u p s o f otShe g m i c e . S u c h deatahs p r o v e d ~arly t r o u b in m o u s e t-g~ tats wbAe.h o n e w ~ e n ~ m g to o h ~ ~rne ~d of q a d v e corn he ~ ~ r a t h e r ~ a n j u s t a 'p~ss" o r "fail' ~ u l t . W e i r ~ f i t u t e d r o u t i n e au of d i e d w ~ t s t o n t ~ t . S u d a autopsies that of the ~~e~ed d~s w e r e d u e to in 6oa, generally in the ecaA area, with obstr~j~on and w i ~ Ltltt~ ha age. ~ e ~ ~at in~u os~s w e r e raise ~ s and an but ~fious s u ~ ~e~a~c_y for human ~ (l~um-o, i 972; SC~oenbaua~ & 1972; ek, 1975; ~Vei & S c h e ~ 1975). i n early s~adies o f t _ ~ we t o in t h e f r e q u e n ~ o f rot-asby ha~e t~e B. d ~ p~r but we found that lar~ even of heated rapidly ~Hed rr~t of our f r o m amJte t o x i ~ t y . N e O n (1967) ~ a t tI-do ~ z t e n ~ a t e d t h e toxicity o f peritoneally * $ (~92-II$71~7
for publieatio~ 19 ~alth +~
1977. ri~. 45 P o p ~ Rcatd,
$02.00}0 ~ T h e
~.aI
Victoria 3012, ~ of B i o l o ~
267
C~ B L A S K E T T E T AL, administered vaccines, ~ we inv~tigated ~ e effe~ o f thiome~oaI in our , and ~s d that it fvotentiated the oco~rre~nce o f int~ssusce e 1). Injection of both ~ . p~tusais and ~ i o m e ~ was al for the development of intussusceptions. T h e mLqimum in~ssusceptogenic of t h i o m e r ~ w ~ approximately 20 tzg Fmr mouse, w ~ a reasonably good d s e in Lh¢ 2 0 - I ~ vtg th per mouse but intus~susccptions never o c c u ~ d in 1@3% of ~ice, whatever the dosage combina~on. T h e lack of intussusceptions La mice ~ e most thiomersal ( 2 ~ 5 0 0 vtg) c~,mot presently be explained. By manipulation of the t h i o m e ~ l ~ d B. # y e n to of mice, we ~ u s e d a p r o ~ r t i o n of animals to have inra~usceptions. Hence, the provides an experimental modal for anyone "÷A~shing T^~LE 1. Cons~
.~t, per~us~ ( × 10 ~') ~ r mouse
mers~
mo~e 19 20-- 4-3 5 ~ 90 1~0-190
sception data (October I975-Apr:~ I977): ~g tb.iomers~ ~ r , ~ B. dc~s~
~-05 0/482 0/894 2/1269 0/137
= = = =
0.0% 0-0% 0-16% 0-0%
0/80
=
o.0%
2 [ ~ 6 2 ----0-07%
0~06~3"9
1~
0/2036 ~ 0"0% 0/40 = 0"0% 17/2578 = &66%
111085= 0.09% 34/3309 ~ 1.03% 74/I914 ~ 3"9%
1/3~3 3 93/5761
= 0.03% ----0-80% ~ I'6%
13/177 0/70
13/334
~ 3-9%
WI~
= 0-0%
0]20
~ 0-0%
0/40
~ 0"0%
~ 7-3% 0~0%
=
I7/4714 = 0"36% 122/65S5 ~ 1.86%
141/14 131 = I - ~ %
to study t~h~ relatively un but ~ r i o u s ~ , i ~ c o n d ~ o n . By g the thiomer~l d to a ~ vtg m m per mouse, we d Me p of mice d;dng from sception during vaccine T h e occurrence of inoa ions in mice ~ v e n ~ntaiv~ng B. e.g. D P T , the diphth "triple " vacch'~e, is not ~ly ~ated "~ith our mice or our V,re L n d u ~ d / n t u s suscep~ons ~ t h both l o ~ ~ d ed -~ and the p enon h ~ ~ n observed ~depende~-~xry ~ D mid t o r i ~ ~ . E. Ch~stensen and J. Camet~n~ p e ~ n M catior~, 1 Cul n, & Ens (1963) Me U.S.A. also observed. ~ B. dosed mice, bowel I which could c o n ~ i c a b l y precede ptions and 2.oi (1971) noticed i n t c ~ s ~ l in h a m s t e ~ repeatedly ~ j e c t e d (route unstated) ~ i t h thiome~q_l d D~..We ~ n s i d e r e d Me ~gssibili~ that the L ~ s : s u s ~ - t i o n s might have a ~ n by the ~ i o m e ~ B. pertus~;s mLT~uare caush'~g some local ~ l e s i ~ ~v,~ch d norm~ ~st.a_Isis. I n support Of this ig w e k_new that thi and B. p e r t u s ~ could enhance each other's n toxAdties for Wainea-pig ~ a (BI , Gul ar~ and Fulton, u shed r~sults) and we had to believe that needle a to the g~at, xsith pen o f injection re into the bowel ~al], v~ould oco.tr in a minor p o f mice injected peritoneally r, er & G I969). H e n ~ we felt ~J~at. ~ i t h appropriate ~ . pert e~.~l ~ x t u ~ = , lesions could develop in the bowel wall at s i t ~ of e ~ d pro-v~d¢ foal of pe e for the ini of Lug, &
1 6). 2~
INTUSSUSCEPTIONS
OF THE BOWEL IN MICE
T o ~ o l v e dais q u ~ t i o n , we do~ed ~ J ~ in tonealIy ~5th a specially prepared experimental :~ecme of unusually high Lntu~usccpt e f a v o r (IF) a c f i f i ~ ; we used a surgical iquc whereby the needle w ~ i d either over the Hver (thus avoidL-~g int~tinal ~ a u m ~ or over the ine~tines (thus exposing them to possible ~ u m a from the needle). Both methods y i d d e d a high proportion o f intussuscepdon~ (3/12 for over liver ~ d 2/5 for over the es). Hence, it w ~ t h e Lnjection mixt~a~ alone ~ a t m.itiated ~ e events Lu the ~ v i t y that led to Lntussusception, For a tLme we suspected that the I F was identical ~ t : h the lo#caJly active B. component recently t e ~ e d "perodssigen' by ~ I a n o z & Be~gman (1977). Heating of thiom rr-~rved B. susp indicated that, like p e r u , I F was labile at 75 °C for 30 ~ but re to 56 °C for a s i m i l ~ # o d . Furthe~ore, ~e pe~ hneiden~ o f intu ions and ~en in mice beth occurred about 5 days after dos~hqg ~ ~C~ B. pert iomersal m~xtures. Testing thio-treated s u f ~ m bacterid spedes Ia en-Iike such his e-ser,~itizkng ~ w e r (Munoz arid Bergmma, 1968), re that, of 107 anh'~xals which died and were autopsied, none showed e v i d e n ~ o f sception ( employed beL~.g S. p , L~th~ and B. ed b y routes other than (su b ~ t ~ e o u s , intravenous, i n ~ t h o r a ~ e ) ~ d u ~ d n o i n t ~ u s e e p ~ o n s in m i ~ , w h e r ~ ~ a s i t ~ t i o n to e can be reduced by various routes, of -which s is the m~st (rdunoz & Bergman, 1977). We also induced a few scepfions ~=ith 'C'-mode B. CLacey, t9~0) ~ d xdth ~ - g e d o s ~ o f ~ . neither o f w h i ~ p o s s ~ e d p e r t u ~ g e n . Th,.~s the nature o f I F remains unclear. Con , we know that it is an excellent c, superior to ~ l oLher p for r r ~ high protective ~ t e n c y in sLngle and ned whool~hng v a c d n ~ (Gardner & Pitt~m~, I965). I t ~ n also suPpros a reaction that develops m ~ 1 2 h in ~dce f o c a l i z e d B. cells toneally eton, 1967). H o w then it Lnt with ~ . p c~Hs to increase t o x i d ~ or produce hnru ions conje but the mcch~ism d in i ceptions ~ o b ~ o u s ~ ~ to the abdominal c a ~ . Although it is s that furuher r ~ a r c h is ne into the i n t e r a ~ o n thiomers~ ~ d bacterial v a c c i n e , no evidence h ~ arisen lhakmg int~assusception in ekildren with ~ . In Iar, the for t h e u ~ o f the peritoneal route t'or the induct/on o f scepdon in mice would seem. "~o rule out such a ~ s i b i l i ~ . ~ i n ~ y , idbo B. con vacchaes in the presence o f thio~ o u l d be a v ~ e that m o ~ d ~ t h s may occur due to in sc~ption of ~ e b~wd, mad that t h ~ d ~ t h s should ~-es be ~ s ed in the ~ of the B. ent.
REFERENCES Cart, J.B., Luft, F. C., Hamb~-ger. R. J. & Kleit, S. A. (1976). I ~on m cKronlc renal of I11, 866. Cul~rtson, C. G , H o ~ , D. H. & En P . W . (I 963). r~on of tOX/C[W o f S preparatlo~ in ¢onventior~ ~ d g e r m - ~ e ~ and the ~ o n of the pa~Cnologi~ bas~ for ~-is on. Idd, ., 21 Jl~3, I , IS3-d66.
269
A. C. B L A S K E T T
ET A~
G a r d n e r , R. A. & Piu:rr~n, M~ (1965). R ~ v e 0mb~/~es o f ~ r r a s s i s vaccLne p r e s e r v e d ~'ith me~.ioLate, ~ o n i u m c~g.loHde, or t h e ns. A_~lied ~dicroMolo_~, 13, 56~569. Laeey, B. ~:/. (I960). ~ f i g ~ i c mod~on o f Bo~r~tetla po'tussf.~, f f o u ~ l of e, e ~ , 57-93. M i n e r , N . A . Koehler, J. & Greenawa~; L. (I969). Intraperitoneal i n j e ~ o n o f r n i ~ . Applied biology 17, 2 5 ~ 2 5 1 . . ..................
MLhnOZ, J. J. & B e r g m a n , R. K. (1977). BordeteIla pert~.o~: Immunol~cal and o ~ Biological ~cti'~iti~, 1st ed., C h . 2 a n d 3, p p . 47--107. N e w York, B ~ e l : M a g i M u n r o , K . M. H . (I972)~ C a ~ ~ d ~ t , h ,m m a t e d female ~r~.ice. ~ o r a t o r y 6, }67-74. l'~eLson, E. A. & G o t ~ . ~ , B.. Y. (1967). ~ d t ~ d ~ y f o r m f i ~ o f p e ~ s i s -cackles wh~ pr~erved wi~ merthidate...qpplied togy 15, 5 ~ 5 9 3 . P o l l ~ k , W. B. (1975). Prolapse o f ated colon t h o u g h ~ e anus in g o l d ~ han~ste~ (~ge~o~etus ~ r a ~ ) . t o ~ Ammat e ~ , 334--336. S~ba~n% M . , F~lopfe% U . & Egyed, ~I. ~-~. (I972). S p o n ~ e o ~ ~t-~s~cep~on of t h e s m ~ intestine h'~ g ~ , p i ~ . L ~ o r a t o ~ . ~ m a ~ 6, 327--330. ~VeisbroC% S. H . & So,her, S . (1975). F a ~ m ~ u s ~ e p ~ o ~ ~ t e d ~th L~Jt~ cocddiosis (Eim~a p in a rabbit, ~ o r ~ o r y An~.~l ~ c ~ c e ~, 7 ~ I . ~ltow~, A. (1971). R e s u I ~ o f h-~tensive i ~ u ~ _ ~ t i o n o f r o d e n ~ - ~ t h t h e d i p h ~ e d a . te~n~-pert~is ~e..4 lmmu et Theeapiae ~ I9, 7 4 9 765.
2~
of
~tan
1 ~ 8 6, 267-270
om
tolye, p
l
vaccine b m mtce
A.C. and I!_. C.
t N.B. t
o f the
t I . O.
mad p~rtussis to ~ e eeptior~ ~ a ~f ~ o f t~es, ma by the in I ~ u t e . ~ e acti~qry i~ labile at 75 *O ~nd ~ absent frc~rr~comb of tbd with b a e t e ~ from other g ~ e ~ , I~a ~ e ent of waccines i~4~ B. p from ~S~ ~ fro~a ~ho~e due tO B, toxiedty. ~
D
control t e s s o n v a c c i n ~ ~ g cells p r e ~ r ' ¢ ~ ~ we have e~'ac~untered e e t e d d e a t h s in g r o u p s o f otShe g m i c e . S u c h deatahs p r o v e d ~arly t r o u b in m o u s e t-g~ tats wbAe.h o n e w ~ e n ~ m g to o h ~ ~rne ~d of q a d v e corn he ~ ~ r a t h e r ~ a n j u s t a 'p~ss" o r "fail' ~ u l t . W e i r ~ f i t u t e d r o u t i n e au of d i e d w ~ t s t o n t ~ t . S u d a autopsies that of the ~~e~ed d~s w e r e d u e to in 6oa, generally in the ecaA area, with obstr~j~on and w i ~ Ltltt~ ha age. ~ e ~ ~at in~u os~s w e r e raise ~ s and an but ~fious s u ~ ~e~a~c_y for human ~ (l~um-o, i 972; SC~oenbaua~ & 1972; ek, 1975; ~Vei & S c h e ~ 1975). i n early s~adies o f t _ ~ we t o in t h e f r e q u e n ~ o f rot-asby ha~e t~e B. d ~ p~r but we found that lar~ even of heated rapidly ~Hed rr~t of our f r o m amJte t o x i ~ t y . N e O n (1967) ~ a t tI-do ~ z t e n ~ a t e d t h e toxicity o f peritoneally * $ (~92-II$71~7
for publieatio~ 19 ~alth +~
1977. ri~. 45 P o p ~ Rcatd,
$02.00}0 ~ T h e
~.aI
Victoria 3012, ~ of B i o l o ~
267
C~ B L A S K E T T E T AL, administered vaccines, ~ we inv~tigated ~ e effe~ o f thiome~oaI in our , and ~s d that it fvotentiated the oco~rre~nce o f int~ssusce e 1). Injection of both ~ . p~tusais and ~ i o m e ~ was al for the development of intussusceptions. T h e mLqimum in~ssusceptogenic of t h i o m e r ~ w ~ approximately 20 tzg Fmr mouse, w ~ a reasonably good d s e in Lh¢ 2 0 - I ~ vtg th per mouse but intus~susccptions never o c c u ~ d in 1@3% of ~ice, whatever the dosage combina~on. T h e lack of intussusceptions La mice ~ e most thiomersal ( 2 ~ 5 0 0 vtg) c~,mot presently be explained. By manipulation of the t h i o m e ~ l ~ d B. # y e n to of mice, we ~ u s e d a p r o ~ r t i o n of animals to have inra~usceptions. Hence, the provides an experimental modal for anyone "÷A~shing T^~LE 1. Cons~
.~t, per~us~ ( × 10 ~') ~ r mouse
mers~
mo~e 19 20-- 4-3 5 ~ 90 1~0-190
sception data (October I975-Apr:~ I977): ~g tb.iomers~ ~ r , ~ B. dc~s~
~-05 0/482 0/894 2/1269 0/137
= = = =
0.0% 0-0% 0-16% 0-0%
0/80
=
o.0%
2 [ ~ 6 2 ----0-07%
0~06~3"9
1~
0/2036 ~ 0"0% 0/40 = 0"0% 17/2578 = &66%
111085= 0.09% 34/3309 ~ 1.03% 74/I914 ~ 3"9%
1/3~3 3 93/5761
= 0.03% ----0-80% ~ I'6%
13/177 0/70
13/334
~ 3-9%
WI~
= 0-0%
0]20
~ 0-0%
0/40
~ 0"0%
~ 7-3% 0~0%
=
I7/4714 = 0"36% 122/65S5 ~ 1.86%
141/14 131 = I - ~ %
to study t~h~ relatively un but ~ r i o u s ~ , i ~ c o n d ~ o n . By g the thiomer~l d to a ~ vtg m m per mouse, we d Me p of mice d;dng from sception during vaccine T h e occurrence of inoa ions in mice ~ v e n ~ntaiv~ng B. e.g. D P T , the diphth "triple " vacch'~e, is not ~ly ~ated "~ith our mice or our V,re L n d u ~ d / n t u s suscep~ons ~ t h both l o ~ ~ d ed -~ and the p enon h ~ ~ n observed ~depende~-~xry ~ D mid t o r i ~ ~ . E. Ch~stensen and J. Camet~n~ p e ~ n M catior~, 1 Cul n, & Ens (1963) Me U.S.A. also observed. ~ B. dosed mice, bowel I which could c o n ~ i c a b l y precede ptions and 2.oi (1971) noticed i n t c ~ s ~ l in h a m s t e ~ repeatedly ~ j e c t e d (route unstated) ~ i t h thiome~q_l d D~..We ~ n s i d e r e d Me ~gssibili~ that the L ~ s : s u s ~ - t i o n s might have a ~ n by the ~ i o m e ~ B. pertus~;s mLT~uare caush'~g some local ~ l e s i ~ ~v,~ch d norm~ ~st.a_Isis. I n support Of this ig w e k_new that thi and B. p e r t u s ~ could enhance each other's n toxAdties for Wainea-pig ~ a (BI , Gul ar~ and Fulton, u shed r~sults) and we had to believe that needle a to the g~at, xsith pen o f injection re into the bowel ~al], v~ould oco.tr in a minor p o f mice injected peritoneally r, er & G I969). H e n ~ we felt ~J~at. ~ i t h appropriate ~ . pert e~.~l ~ x t u ~ = , lesions could develop in the bowel wall at s i t ~ of e ~ d pro-v~d¢ foal of pe e for the ini of Lug, &
1 6). 2~
INTUSSUSCEPTIONS
OF THE BOWEL IN MICE
T o ~ o l v e dais q u ~ t i o n , we do~ed ~ J ~ in tonealIy ~5th a specially prepared experimental :~ecme of unusually high Lntu~usccpt e f a v o r (IF) a c f i f i ~ ; we used a surgical iquc whereby the needle w ~ i d either over the Hver (thus avoidL-~g int~tinal ~ a u m ~ or over the ine~tines (thus exposing them to possible ~ u m a from the needle). Both methods y i d d e d a high proportion o f intussuscepdon~ (3/12 for over liver ~ d 2/5 for over the es). Hence, it w ~ t h e Lnjection mixt~a~ alone ~ a t m.itiated ~ e events Lu the ~ v i t y that led to Lntussusception, For a tLme we suspected that the I F was identical ~ t : h the lo#caJly active B. component recently t e ~ e d "perodssigen' by ~ I a n o z & Be~gman (1977). Heating of thiom rr-~rved B. susp indicated that, like p e r u , I F was labile at 75 °C for 30 ~ but re to 56 °C for a s i m i l ~ # o d . Furthe~ore, ~e pe~ hneiden~ o f intu ions and ~en in mice beth occurred about 5 days after dos~hqg ~ ~C~ B. pert iomersal m~xtures. Testing thio-treated s u f ~ m bacterid spedes Ia en-Iike such his e-ser,~itizkng ~ w e r (Munoz arid Bergmma, 1968), re that, of 107 anh'~xals which died and were autopsied, none showed e v i d e n ~ o f sception ( employed beL~.g S. p , L~th~ and B. ed b y routes other than (su b ~ t ~ e o u s , intravenous, i n ~ t h o r a ~ e ) ~ d u ~ d n o i n t ~ u s e e p ~ o n s in m i ~ , w h e r ~ ~ a s i t ~ t i o n to e can be reduced by various routes, of -which s is the m~st (rdunoz & Bergman, 1977). We also induced a few scepfions ~=ith 'C'-mode B. CLacey, t9~0) ~ d xdth ~ - g e d o s ~ o f ~ . neither o f w h i ~ p o s s ~ e d p e r t u ~ g e n . Th,.~s the nature o f I F remains unclear. Con , we know that it is an excellent c, superior to ~ l oLher p for r r ~ high protective ~ t e n c y in sLngle and ned whool~hng v a c d n ~ (Gardner & Pitt~m~, I965). I t ~ n also suPpros a reaction that develops m ~ 1 2 h in ~dce f o c a l i z e d B. cells toneally eton, 1967). H o w then it Lnt with ~ . p c~Hs to increase t o x i d ~ or produce hnru ions conje but the mcch~ism d in i ceptions ~ o b ~ o u s ~ ~ to the abdominal c a ~ . Although it is s that furuher r ~ a r c h is ne into the i n t e r a ~ o n thiomers~ ~ d bacterial v a c c i n e , no evidence h ~ arisen lhakmg int~assusception in ekildren with ~ . In Iar, the for t h e u ~ o f the peritoneal route t'or the induct/on o f scepdon in mice would seem. "~o rule out such a ~ s i b i l i ~ . ~ i n ~ y , idbo B. con vacchaes in the presence o f thio~ o u l d be a v ~ e that m o ~ d ~ t h s may occur due to in sc~ption of ~ e b~wd, mad that t h ~ d ~ t h s should ~-es be ~ s ed in the ~ of the B. ent.
REFERENCES Cart, J.B., Luft, F. C., Hamb~-ger. R. J. & Kleit, S. A. (1976). I ~on m cKronlc renal of I11, 866. Cul~rtson, C. G , H o ~ , D. H. & En P . W . (I 963). r~on of tOX/C[W o f S preparatlo~ in ¢onventior~ ~ d g e r m - ~ e ~ and the ~ o n of the pa~Cnologi~ bas~ for ~-is on. Idd, ., 21 Jl~3, I , IS3-d66.
269
A. C. B L A S K E T T
ET A~
G a r d n e r , R. A. & Piu:rr~n, M~ (1965). R ~ v e 0mb~/~es o f ~ r r a s s i s vaccLne p r e s e r v e d ~'ith me~.ioLate, ~ o n i u m c~g.loHde, or t h e ns. A_~lied ~dicroMolo_~, 13, 56~569. Laeey, B. ~:/. (I960). ~ f i g ~ i c mod~on o f Bo~r~tetla po'tussf.~, f f o u ~ l of e, e ~ , 57-93. M i n e r , N . A . Koehler, J. & Greenawa~; L. (I969). Intraperitoneal i n j e ~ o n o f r n i ~ . Applied biology 17, 2 5 ~ 2 5 1 . . ..................
MLhnOZ, J. J. & B e r g m a n , R. K. (1977). BordeteIla pert~.o~: Immunol~cal and o ~ Biological ~cti'~iti~, 1st ed., C h . 2 a n d 3, p p . 47--107. N e w York, B ~ e l : M a g i M u n r o , K . M. H . (I972)~ C a ~ ~ d ~ t , h ,m m a t e d female ~r~.ice. ~ o r a t o r y 6, }67-74. l'~eLson, E. A. & G o t ~ . ~ , B.. Y. (1967). ~ d t ~ d ~ y f o r m f i ~ o f p e ~ s i s -cackles wh~ pr~erved wi~ merthidate...qpplied togy 15, 5 ~ 5 9 3 . P o l l ~ k , W. B. (1975). Prolapse o f ated colon t h o u g h ~ e anus in g o l d ~ han~ste~ (~ge~o~etus ~ r a ~ ) . t o ~ Ammat e ~ , 334--336. S~ba~n% M . , F~lopfe% U . & Egyed, ~I. ~-~. (I972). S p o n ~ e o ~ ~t-~s~cep~on of t h e s m ~ intestine h'~ g ~ , p i ~ . L ~ o r a t o ~ . ~ m a ~ 6, 327--330. ~VeisbroC% S. H . & So,her, S . (1975). F a ~ m ~ u s ~ e p ~ o ~ ~ t e d ~th L~Jt~ cocddiosis (Eim~a p in a rabbit, ~ o r ~ o r y An~.~l ~ c ~ c e ~, 7 ~ I . ~ltow~, A. (1971). R e s u I ~ o f h-~tensive i ~ u ~ _ ~ t i o n o f r o d e n ~ - ~ t h t h e d i p h ~ e d a . te~n~-pert~is ~e..4 lmmu et Theeapiae ~ I9, 7 4 9 765.
2~