Archiv fiir die gesamte Virusforschung 30, 147-- 154 (1970)
Interaction between DEAE-Dextran and Nucleic Acids I I . E f f e c t on I n t e r f e r o n
Production
in v i v o
By R. F. MA~s and M. V. FERNANDES1 With 4 Figures Received August 26, 1969
Summary The double-stranded polymer of poly-inosinie acid and poly-cytidylic acid protects mice during five days against foot-and-mouth disease infection. The peak protection is obtained one day after the inoculation of the interferon inducer. When one nucleic acid strand is coated with DEAE-Dextran before complex formation into a double-strand, the peak of protection against viral infection is delayed by two days. When the two nucleic acid strands are coated with DEAEDextran before complex formation into a double-strand, the induction of protection appears at its maximum only 5 to 9 days after inoculation of the inducer. An enhancement of the inducer effect is observed when the nucleic acid is coated in the double-strand form by the DEAE-Dextran. The peak of protection is obtained in this case one day after inoculation of the polymer.
1. Introduction The use of interferon for therapeutic purposes has only limited applications because of the general species specificity of this protein (1). Lately, substances highly efficient in the induction or release of interferon in the animal host have been found of whom poly-inosinic acid forming a complex with a poly-cytidylic acid was the most satisfactory (2). However, interferon production by this system is usually limited to 2 to 3 days after the injection of the polymer. A pyran eopolymer used for interferon production has been shown to have long lasting protective effects, but the compound is slightly toxic (3). A system where the production of interferon with double-stranded polynucleotides is enhanced and/or prolonged is desirable. It has been demonstrated that coating of single-stranded RNA b y DEAEDextran protects partly the nucleic acid against the digestive action of RNase (4). Since the biologic activity of poliovirus RNA is enhanced by its coating with DEAE-Dextran it seemed of interest to test if this polycationic polymer would have the same protective and enhancing effect on the double-stranded nucleic acid used in the production of interferon. 1 Pan American Foot-and-Mouth Disease Center, Pan American Health Organization, P.O. Box 589-ZC-00, Rio de Janeiro, Brazil. 10"
148
R . F . MAES and M. V. FERI~ANDES: 2. Materials and Methods
Th~polynuclcotides poly-C and poly-I were purchased from P-L Bioehemicals, Inc. The D E A E - D e x t r a n (M.W. 2.10 e) was purchased from Pharmacia. The single stranded nucleic acid polymers were dissolved b y gentle heating and stored as stock solutions of 1 mg/ml at --20~ in Tris-HC1 0.02 M, p H 7.0 and 0.15 NaC1 unless otherwise stated. The D E A E - D e x t r a n stock solution was 20 mg/ml in water. Before use, the polymers were diluted in a solution 0.15 M in NaC1, 0.02 M in NaCI, 0.02 M in Tris-HC1, p H 7 and mixed at room temperature. No less a concentration than 200 9g/ml of the polynucleotides (single or double-stranded) was diluted slowly with an equal volume of suitably diluted D E A E - D e x t r a n , so t h a t complex formation or precipitation could be visualised easily. Adult female white Swiss mice of 5 weeks weighing 10-- 12 g were inoculated intraperitoneally with 0.2 ml of the polymer solutions. 6 to 8 mice were used per dose and time. Challenges were performed one to eleven days later b y intraperitoneal inoculation of 0.1 to 0.3 ml of a foot-and-mouth disease virus suspension containing 20 to 60 LD 5ofor adult mice. The virus used was t y p e " 0 " Vallde, subtype Ox (Campos, Brazil) adapted to adult mice (6) and the animals were examined daily during 6 to 12 days. The cumulative number of mice surviving every d a y from the third d a y on after virus inoculation was recorded (Z S [3 to x]). This number was usually divided b y the cumulative number of mice surviving in the uninfected controls (E So [3 to x]). The quotient indicates degree of protection ranging from 1.0 (full protection) to 0. Since almost all the control mice inoculated with FMD virus died within two days, the control mice surviving more than 2 days after viral inoculation were considered a background and always substraeted from the da~a. I n this work I is poly-inosinic acid, C is poly-eytidylic acid and a colon indicates complex formation between nucleic acids. D E A E - D is D E A E - D e x t r a n . (I) and (C) indicate the single-stranded nucleic acids eomplexed with an equal amount (w/w) of D E A E - D e x t r a n . (I:C) indicates the double-stranded nucleic acid complexed with D E A E - D e x t r a n . PBS is phosphate buffered saline and Tris is a solution 0.02 molar in Tris-HC1 p H 7.0 and 0.15 molar in NaC1. 3. Results 3.1. Dose Response P r e l i m i n a r y e x p e r i m e n t s showed t h a t I, C a n d these p o l y m e r s c o m p l e x e d w i t h a n e q u a l a m o u n t (w/w) of D E A E - D e x t r a n were unable, a t a dose of 50 ~g t o p r o t e c t mice significantly a g a i n s t a virus challenge of (60 LDs0 ) a n d (20 LD5o ) a t 1 d a y a n d 4 days, respectively. I t is shown in Fig. 1 t h a t p r o t e c t i o n a g a i n s t t h e same challenges is o b t a i n e d w h e n c o m p l e x f o r m a t i o n was allowed to occur b e t w e e n t h e free p o l y n u c l e o t i d e s as well as t h e D E A E - D e x t r a n coated polynucleotides. W h e n t h e challenge was m a d e one d a y a f t e r t h e a d m i n i s t r a t i o n of t h e p o l y mers, t h e b e s t p r o t e c t i v e effect was seen w i t h free I : C. A n a l m o s t similar response was o b t a i n e d w i t h 50 ~g a n d 25 ~g of this p o l y m e r , t h e lower dose being p e r h a p s t h e b e t t e r , a n d a n effect could still be o b s e r v e d a t 6.25 ~zg. A t one d a y t h e c o a t e d double-stranded polynucleotide (DEAE-D~-I):(C~DEAE-D) d i d n o t show m u c h of a p r o t e c t i v e effect, if any. A challenge m a d e four d a y s after t h e i n j e c t i o n of t h e p o l y m e r s showed t h a t t h e c o a t e d p o l y m e r s were able to induce i n t e r f e r o n p r o d u c t i o n . A good p r o t e c t i v e effect was o b t a i n e d w i t h a low dose of 12.5 9g. The p r o t e c t i v e effect o b t a i n e d a t 4 d a y s w i t h t h e D E A E - D e x t r a n c o a t e d p o l y m e r was a c t u a l l y b e t t e r t h a n t h a t o b t a i n e d w i t h free I :C, a n d was best a t t h e highest dose used (50 tzg).
Interaction between DEAE-Dextran and Nucleic Acids. II.
149
This experiment thus established the practical doses needed to analyse the effects of DEAE-Dextran on interferon production with a double-stranded nucleic acid. A more detailed analysis of the interferon production in the course of time induced by I:C coated in various ways by DEAE-Dextran was then initiated. 3.2. T i m e S e q u e n c e Former experiments indicated that both single-stranded and double-stranded nucleic acids can interact with DEAE-Dextran. In the case of a single-stranded nucleic acid, a 1/1 (w/w) relative concentration of the DEAE-Dextran would induce the formation of a colloid, leaving the bases of the nucleic acid free (4). In this case, complex formation between two polynucleotides will occur after attachment of the single polynucleotides to DEAE-Dextran. This case was analysed first.
/8
Zd
/
~
/8
I. ~m Fig. 1 : Dose response of i n t e r feron production by I : C and ( D E A E - D + I) : (C + D E A E - D ) . Challenge a t 1 a n d 4 d a y s a f t e r t h e a d m i n i s t r a t i o n of t h e polymers. Ordinate: Cumulat i v e n u m b e r of s u r v i v i n g mice, f r o m 3 to 6 d a y s a f t e r cha]lengc w i t h 60 a n d 20 LDs0 F I ~ D V . A b s c i s s a : Doses (i~g] m o u s e ) of t h e i n j e c t e d polymers. I:C, 1 day:O O. I : C , 4 d a y s : A . . . . . A. ( I ) : ( C ) , 1 dayl I I . (I):(C), 4 d a y s : 9
........
9
(Z.~.,.,=...~,_,
~8
(z).,(c)~~. ,FO
Z&
. ._ IZ5
8Z5
31B5
/.5
gose of fnterferon _TndL/cer
a) Coating o/the polynueleotides with DEAE-Dextran before complex ]ormation. Besides complex formation between the free polynucleotides, two combinations are possible. The first, where one polynucleotide strand only is first coated with DEAE-Dextran, and the second, where the two polynucleotides are first bound to DEAE-Dextran. These two combinations were tested. A small precipitate occurred sometimes upon complex formation between bound polynucleotides, due probably to a slight deficiency of DEAE-D and was disregarded. These polynucleotides were injected at a dose of 50 ~g, and virus challenges were done at 1, 3, 5, 7 and 9 days. It is seen in Fig. 2 that the protective effect of free I:C is highest 1 day after the injection of this polymer and is almost nil 7 days later. A delaying effect takes place with (DEAE-D~-I):C, where the peak of resistance occurs 3 days after the injection of the polymer. With (DEAE-D-~I) : ( C ~ D E A E D) the delay is shifted towards even later times with the maximum protection occurring 5 days after the administration of the polymer. b) Coating of the polynueleotides after complex formation. B y the use of singlestranded DNA, it has been shown earlier that the molecular weight of the nucleic acid has an influence on the amounts of DEAE-Dextran needed to precipitate i~
150
R . F . MA~s and M. V. FERNANDE8:
or form a colloid (4). I n the case of I : C, it was f o u n d t h a t n o f u n d a m e n t a l differences i n this respect existed between this complex a n d n a t i v e DNA, b u t the proportions of D E A E - D e x t r a n needed to show characteristic effects were m u c h lower: a d d i t i o n of D E A E - D e x t r a n to I :C i n relative final proportions (w/w) of 0.1, 0.2, 0.4 a n d 1.0 resulted i n the f o r m a t i o n of a colloid for the two first proportions; a h e a v y precipitate occurred at 0.4 a n d the colloidal state was regained. a t 1.0. 48
OZ
Q7
/o,\ / /"
08
"\
\
/'
'\
/
06
4U
.~ ",,~...,
/
" "
'/
/
"
\
.,/
/"
//'
'\
..."~z).(C)
",/
./o
'\'\ / /(I) ."C "v"
8,3
I 4, ~. ~1~ 4J
ot"
"\\.\
/
.o.~176 02
,'/';"""'. " "\.
4/L I
3
5
Bays of dk~llenQe~ Fig. 2
Y
F ~7
m~].,~/ $/~ 4
l'/b
2'
",..... \
./..,."'~
",, '\ z.'"
I . /:d
""'It/~ /'~,,5
Ratio #ucleikAaid/zTeae-2 Fig. 3
I 1"1
Fig. 2 : Time sequence of interferon production by 50 ~g of (DEAE-D + I) : (C + DEAE-D); [] . . . . D. (DEAE-D +I):C, 9 . . . . . O and I:C, 9 -O. Challengesat 1, 3, 5, 7 and 9 days after the administration of the polymers. Ordinate : Survival quotient in the interval 3rd to 9th day after challenge Fig. 3 : Assay of different concentrations of DEAE-D on the interferon inducing activity of I :C. The polynucleotide double-strand was diinted with 1]10, 115, 1/2,5 and ]]1 amounts of its own weight of DEAE-Dextran, and challengesmade 1 day (o o), 3 days (9 . . . . . 0) and 5 days (a . . . . m) later F o r t y ~g of I :C were injected i n t r a p e r i t o n e a l l y in mice a n d compared with t h e same doses of the complex after its coating with D E A E - D e x t r a n , a t the abovem e n t i o n e d proportions. Challenge was done 1 to 9 days later. Since all responses a t 7 a n d 9 days were identical to the controls, these results were o m i t t e d from Fig. 3. I t is clear from the d a t a presented in Fig. 3 t h a t the proportions 0.4 a n d 1.0 do suppress the effect of I : C . Of the two r e m a i n i n g combinations, the 1:10 p r o p o r t i o n is a p p a r e n t l y the b e t t e r a n d is superior to free I : C i n giving b e t t e r p r o t e c t i o n to mice against challenge, u p to 5 days after its a d m i n i s t r a t i o n . H a v i n g established t h a t the best protection a g a i n s t virus challenge is o b t a i n e d when I :C is mixed with a t e n t h of its own weight of D E A E - D e x t r a n , a more
Interaction between DEAE-Dextran and Nucleic Acids. II.
151
detailed analysis of the dose response and time sequence of this compound was initiated. For this purpose, mice were given (I:C)-{-1/10 DEAE-Dextran (w/w) intraperitoneally in doses of 25 and 50 ~tg, and challenged from 1 to 7 days later. The results of this experiment are presented in Fig. 4. Responses obtained by free I : C and bound ( D E A E - D ~ I ) :(C-~DEAE-D) in doses of 25 ~g are given as reference, the 50 ~g doses of these compounds are those of Fig. 2. I t seems that a dose of 50 ~g of (I : C ) ~ D E A E - D e x t r a n is superior to any other dose and combination tested, in giving immediate protection to challenge, and that the effect can be detected up to 7 days after its administration. On the other
Q8
OY
\ '\
O6
_T..d~5/x67 \
\
/
'\.
9rl...............
(IJ.KcJzm~
/\ (z..c)z.+/~ .i '\
:/
\
03
F i g . 4 : D o s e r e s p o n s e a n d t i m e s e q u e n c e of i n t e r f e r o n p r o d u c t i o n w i t h (T :C) + D E A E - D e x t r a n , compared to I:C and (DEAE-D +I):(C +DEAED). The polynueleotide double-strand was coated w i t h 1 / 1 0 t h i t s o w n w e i g h t of D E A E - D e x t r a n and injected intraperitoneally i n 50 ~ g d o s e s . C h a l l e n g e s w e r e m a d e 1, 3, 5 a n d 7 d a y s a f t e r p o l y m e r a d m i n i s t r a t i o n . ( I : C ) , 50 ~ g : O - - 9 ( I : C ) , 25 ~ g : 9 9 ( I ) : ( C ) , 25 ~ g : [] . . . . [] a n d I : C , 25 ~ g 9 . . . . . 9
Q~
r
I
3 D~y8
'~-- . . . . . 4 3 2
of dh~llenge
hand I: C lost all its protective power at 7 days at all doses. The response obtained with 50 and 25 ~g of free I: C and (DEAE-D~-I) : (C~-DEAE-D) are about similar, as described (Fig. 1). The reduced activity of the maximal doses may be due to saturation effects similar to those reported by WH~LOCK (7). In the case of (I:C)~-DEAE-Dextran, it seems that such a saturation effect does not take place and that the range of doses tested (e.g. 25, 40 and 50 ~g) is within the limits of the linear increase of the dose-response curve, with complete protection obtained at the highest concentration. 3.3. C o m p a r i s o n of t h e F o u r I n d u c e r s Throughout this study, the method used for the presentation of the results is heterodox. I t has the advantage to allow a clear graphic representation of the
152
R . F . MiES ~nd M. V. F:ERNANDES:
Table 1. Comparison o/the Effect of four Inducers of Interferon on the Pathogenicity of
F M D V in vivo
o......qo+oo. Io.1....... + ooooo..ooo...
t i m e of challenge after inducer inoculation Iday 3days 5days 7days 9days ,jo=oooeoe! ooooooool ooeooeoo] Toooeooeo[ Iooooooool 51
I
n
oo I
!
+
,{ooooeoooi 2/ooooooool +/ ooeooeu +/ oooool
IOOOOOOOOl o o o o o o o o l IOOOOOOOOl o o o o o o o o l Ioooooeooi ooooj
,I
~_
I
oooool
OOOl OOOl
I
ooo
I:C
oo...o...o....o.. io''., ..,, oi
9-
oool
ool
,ooooooool ooooooool ,- z oooooooq~ o o o o o o o o / "~ ~ o o o o o o o o l oooooool slOg OOOOOOI oooooo/
+,......., +Io.oo.oo.l +|OIIO tO Ol
o....., oo..on Ol Oil
elOOOO Ooool 91oooo go ooJ
oooooooo ~[oooooooo] o o o o o o o o l lOOoOOOO~!t ! o o o o o o o l oooool 0
oooi oaf
91 ~, ,I
ool eel
7]
/ /I
o.i1
OOOOl oooo~
I
,looooooool e o o o o o o o l "-- 21ooooooool OOOOOOOOl 3l o o o o o e l oooooe ,I ooooi oooe 9I ~I
ooooooo oooooOI ooooooe ooooe
]
ooooe ooool OOOOqI
ooo~ 0
oaf
ooooe ~ooe-
9I
OOOOOO01
oooooool
I I I I
ooool I
lOlOOlOt
oooooooo
OO0@OOL OOOOO01 OOOOOOI OOOOOOI ooooool 1
ooooool t
OOOOOOI OOOO0 OOO0 OOO0 oooo
oooo
o:= ::1 :;I (':c) OOJ
o00o00001 ooooooool ooeooool ooou ooool
,,........~ .......;< ...o.... ~
zlOOOOOO01 +I oooool +! OOOO01 5 OOOO01 OOO0{ 7 OOOOJ al ooooI
+
looooooool IOOOOOOOOl IOeOOOOOOi ioooooooel ( I ) : ( C ) I oooooool o o q~olI o o o o o o o l I oOOOl OOOl / oOOOl oeon t ,,eeel
o.
,.......+] IOOOOOO01
)oooooool
OOOOOOI 9 OOOOl oooooL OOOOOi oool
(I): C
ooot
Open circles: sick or paralytic mice. Closed circles: h e a l t h y mice. I : C is poly-inosinic acid complexed with poly-cytidylic acid. (I:C) is the same double-stranded nucleic acid coated with 1/10 (w/w) D E A E D e x t r i n , after complex formation. (I):C and (I):(C) are the same nucleic acid with one or two of the individual strands coated with an equal amount (w/w) of the polycation before complex formation.
d y n a m i c s of p r o t e c t i o n induction. This was done a t t h e cost of loss of information, n a m e l y t h e t i m e of a p p e a r a n c e of o v e r t signs of illness in t h e i n o c u l a t e d a n i m a l s a n d t h e s t a t i s t i c a l significance of t h e differences observed. I n T a b l e 1, t h e fate of i n d i v i d u a l animals t r e a t e d w i t h t h e four inducers a n a l y s ed has been represented. All a n i m a l s were t r e a t e d a t t i m e 0 d a y a n d challenged
Interaction between DEAE-Dextran and Nucleic Acids. II.
153
with infectious FMD virus 1, 3, 5, 7, or 9 days later. Closed circles represent healthy animals and open circles represent animals showing signs of sickness or paralysis. Eight animals were inoculated per sample/time and a dose of 50 ~g/ animal of I : C was used in all cases. Double-stranded nucleic acid was coated with 1 : 10 (w/w) D E A E - D e x t r a n -~ (I: C), and single-stranded nucleic acids were mixed with an equal amount (w/w) of the polycation = (I) : (C) and (I) : C. I t is seen in Table 1 t h a t the challenges, calculated to be 40 LDs0 per animal, were not uniform since the death rate of the controls at the ninth day was slower. Taking into account all living animals, the mean time of death of the control animals (40) was X -~ 3.9 with an sm ~-- 0.35. For groups of 8 animals, sm would be 0.79 days. Considering as significantly different all mean values larger than 3.9~-1.96 sin, e.g. 5.44 days, it will be found t h a t all boxes of Table 1 marked are positive. I n the graphs, values reported equal or inferior to about 0.2 will be statistically dubious. 4. Discussion These experiments describe 3 different actions of D E A E - D e x t r a n upon the polynucleotide polymers used for interferon production. First, the resistance to infection is enhanced immediately upon injection of the polymer: this happens when I : C is coated as a double-strand b y an amount of D E A E - D e x t r a n sufficient and necessary to form a colloid. I t acts probably b y favoring the absorption of the polynucleotide in the target cells, with perhaps an additional protective effect against nuclease degradation. I n this respect, binding of the D E A E - D e x t r a n to the pyrimidine polynucleotide m a y be more rewarding than the protection of the poly-inosinic acid. Secondly, excess D E A E - D e x t r a n results in the precipitation of the nucleic acid or binding of the nucleic acid in a maze of D E A E - D e x t r a n in such a way t h a t all production of interferon is suppressed. Since base and sugar composition of the nucleic acid has a well established influence on the degree of interferon production (2, 8) the implication t h a t the bases and sugar of the nucleic acid must be accessible at a certain stage of interferon production is unavoidable. The complete engulfment of the nucleic acid b y protein or polysaccharide, when these polymers litterally wind around the double-stranded nucleic acid, will be inhibitory. This second effect supports the assertion (2) t h a t the nucleic acid must be free of inhibitory proteins in order to induce interferon. The last mode of action of the cationic polymer is expressed as delay in the production of interferon. I n this case, one or both strands of the polynucleotide are coated with the cationic polymer be/ore complex formation. Since the polysaecharide chain used as a coat is made of glucose units, an explanation of the delay observed m a y be t h a t this polymer must first be digested or removed, before the nucleic acid initiates its interferon production. I t is clear t h a t a greater delay will occur when both polynucleotides have to be zipped free than when only one is. A marginal protective and absorbing effect of the D E A E - D e x t r a n m a y be envisioned in the later process. The mouse is not a good interferon producer (8). On the other hand, if the intraperitoneal route is better than the subcutaneous one (7 and personal observation), other routes such as intravenous (3, 8) and intramuscular m a y prove still better. The challenge virus used in these experiments is extremely virulent for the animal host. Mean survival times of 8 days have been reported for mice inoeu-
154
MA~Set al.: Interaction between DEAE-Dextran and Nucleic Acids. II.
lated by PMV and 5 days after Columbia SK inoculation (2). The survival time of FMDV infected mice is barely 3 to 4 days. Sick mice were noted to recover in some instances, and this phenomenon should be taken into account when evaluating the usefulness of interferon as a therapeutic agent, especially for noncytocida] viruses and tumor viruses. Since the injection of DEAE-Dextran has been shown to have toxic effects (10), search for another suitable polycationie protective polymer m a y be of value. A double-stranded R N A polynucleotide could thus be coated with a polycationic polymer in order to have an immediate maximal build up of interferon, whereas this protective polymer attached to one or both polynucleotide strands before complex formation could be administered jointly for longer-lasting and delayed effects. After this work has been oompleted, a report has appeared showing the enhancing effect of DEAE-Dextran on interferon production by synthetic polynucleotides in vitro (11).
Acknowledgements We thank Mr. Jorgevalso Sales Santos for his expert technical assistance.
References 1. MnnioA~, T. C., C. A. WING~% and C. B. DIxoN : Purification and characterization of vertebrate interferon. J. molec. Biol. 18, 679--691 (1965). 2. FIELD, A. K., A. A. TYTELL, G. P. LAMPSON, and M. R. HILLEMAN: Inducers of
interferon and host resistance II. Multistranded synthetic polynucleotide complexes. Proc. nat. Acad. Sci. (Wash.) 58, 1004--1010 (1967). 3. ME~IGA~J, T. C., and W. REGELSO~: Interferon induction in man by a synthetic polyanion of defined composition. New Engl. J. Mcd. 277, 1283--1287 (1967). 4. MArts, R., W. SEDWICK,and A. VAHERI: Interaction between DEAE-Dextran and nucleic acids. Biochim. biophys. Acta (Amst.) 189, 269--276 (1967). 5. VAHERI, A., and J. PAGA~O: Infectious poliovirus RNA: A sensitive method of assay. Virology 27, 434--436 (1965). 6. CU~HA, R. G.: Demonstration of immune response to foot-and-mouth disease vaccine in protection tests in young mice. Proc. Soc. exp. Biol. (N.Y.) 108, 700-703 (1960). 7. W~EELOCK, E. F. : Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease Vh'us. J. Bact. 92, 1415-- 1421 (1966). 8. LAMPSON, G. P., A. A. TYTELL, A. K. FIELD, M. M. NEMES, and M. R. HILLEMAN: Inducers of interferon and host resistance. I. Double-stranded RNA from extracts of penicillium funiculosum. Proc. nat. Acad. Sci. (Wash.) 58, 782--789 (1967). 9. YOU~GE]~, J. S., and J. V. HALLU~: Interferon production in mice by doublestranded synthetic polynucleotides: induction or release ? Virology 84, 177--179 (1968). 10. CRAIGHEAD,J. E. : Effect of polycations on growth and dissemination of the encephalomyocarditis virus in mice. J. Virol. 1, 988--995 (1967). 11. VIL6EK, J., A. E. FRIEDMAN-KIEN,and T. KRAwcIw: Induction of interferon synthesis by synthetic double-stranded polynucleotides. J. Virol. 2, 648--650 (1968). Authors' address: Dr. R. F. MAES, Pan American Foot-and-Mouth Disease Center, Pan American Health Organization, P.O. Box 589-ZC-00, Rio de Janeiro, Brazil.