Time and dosoge dependence of immunoenhancement by murine type II interferon preparations

CELL,-LAR

111 \I I.N

Time

OLOGY

40, 285-293 ( 1978)

and Dosage Dependence of lmmunoenhancement Murine Type II Interferon Preparations1

by

Preparatio1ls of Type II immullr induced interferon cnllanced tile pIa(lue forming cell response of mice to sheep red blood cells both iu c~kw and in Tvitrm The enhancrment of the antibody response 1, as dependent on the tlos;cge of interferon used and the time of administration of interferon. The expession of the antivid nntl immunoenllancing- activities of Type II interferon prq)nrations shared several physical-chemical properties. including pH 2 lability and heat stability. The plaque forming cell l-c‘sponse to IipoI~olysaccharitle, a T-intlepentlrnt antigen, could not tx enhanced hy treatment wit11 Tyl)e 11 interferon. In addition. trcntment of spleen cell cultures of nude thymic deficient mice u ith Type TI interferon could not cause an twhanccmeut of the plaque forming cell response to lipopolysaccharide. These data suggest that the immunoenhancing effect of Type I I interferon on antihotly responses is protlucetl hy an effect on T Iymphocytes in contrast \vith the illlrnuliosui)l)r’ssi~~ cffcct \vhich appe;l~-s to he mediated through an effrct on I3 l?;n~l~hoc~trs.

Jilterferon, originall\. tlcscril~etl as 2111antiviral agetit. 1~s r?celltly IKYYI slu~~~n to have several non-aiitiviral activities incltding regulation of the imnunc resplse ( 1 ). ‘I% majority of the iiiimtiiioregtil~it~r~~ activities that have Ivxn tlescril)etl have been ili~li~uliosul)l)ressi~~, I)~it several nmrkers have slio\vn tllat tlic intluctioti of interfrron (2 ) or atltlition of inter~rron to spleen ccl1 cultures after atltlition iIf sheep red I~lootl cells (3 j ca~~setl enliaticenictit of the iminune respi~se. Inmm10enliancenieiit Ius also occured \vhen iiiterferou uxs injected into liiice after sellsitimtim of tlic Illice with slieel) ret1 I~lootl cells (SliIlC‘) 2 (4 ), 7‘ylx I1 immuiie induced interferon has rccrntly I)een slio\vii to have ml iniliitinosqq~rcs5ive activity. \\‘hen Type L[ interferon preparations wrre atltletl to splcw cell culturt~s 24 hr before atltlition of SI
s from the I,.S.P.EI.S. (.\I 03029) and hy the National .\et-onautics under Interchange SCXZ-OR7-15~801. this paler : RC(;. Racillc C:dnwttc rt (;uerin ; l/.\ ratio. ratio of antiviral titer ; I. I’S, lipo~~ol~s~tccl~a~-i~le; KS. not signific;ttnt ; SKRC,

285 OOOS-S739/78,/0102-0285$02.00/O Cwyright 0 1978 hy Academic Press, Inc. All rights of reproduction in any form reserved.

286

SONNENFELD,

MANDEL,

AND

MERIGAN

required was 2.50 times less than the dosage of Type I interferon required for a similar effect (5). In view of this very potent imnmnosuppressive activity, the present study was undertaken to determine if Type II interferon preparations also contained an iiiimunoeiihai7ciiIg activity. The results of this study indicate that Type II interferon preparations do have an inmunoenhancing activity. The expression of this activity is dependent on the time of interferon administration in relation to sensitization to SRBC, and to the dosage of interferon administered. The antibody response of BXLB/c mouse spleen cell cultures to lipopolysaccharide, a T-independent antigen, was suppressed by addition of Type II interferon 24 hr before antigen, but was not enhanced by interferon addition 48 hr after antigen. Similarily, the antibody response of spleen cultures of thymic deficient nude mice to lipopolysaccharide was suppressed by interferon addition, but could not be enhanced by addition of the interferon preparation. Therefore, the suppressive effect of Type II interferon preparations appears to be an effect on B lymphocytes, while the enhancing effect appears to be an effect on T lymphocytes.

flninrals. Randomly bred 6 to S-week-old female Swiss-14rebster (S/IV) mice were used for the passive transfer experiments and BALB/c mice were used for spleen cell culture experiments. The mice were supplied by Simenson, Inc., Gilroy, California. Nude (m/m) mice on a BALB/c background were obtained from ARS/Sprague Dawley, Madison, LVi. I~zterfrson /~rod~~ction. Mouse Type I interferon was produced in L-929 cells with the Herts strain of Newcastle Diseases Virus as previously described (6). After inactivation of residual inducing virus by pH 2 treatment at 4°C for 5 days, the tissue culture supernatant fluid was concentrated lo- to 15fold in a pressure cell employing a Uh4-10 filter (Amicon Corp., Cam~briclge, h1ass). Mock Type I interferon was prepared by using culture supernatants from uninfected cells. Mouse Type II interferon was produced by intravenous injection of SO mg old tuberculin (OT j into mice sensitized with M~~obncferiz~m bovis strain BCG (7). hIock Type II interferon was prepared by inoculation of 50 mg OT into unsensitized mice. In vitro Type II i~lterf~rolz ~‘~odllctioll. Type II interferon was produced in zlifvo by harvesting supernatants of spleen cell cultures of SwissfiVebster mice that had been sensitized with Mycobacferiz(uz boeais strain BCG and challenged lvith old tuberculin (5). Infcrfero+t assq. The antiviral activity of the interferon preparations was determined by means of a plaque reduction assay on mouse L-929 cells using the Indiana strain of bovine vesicular stomatitis virus as the challenge virus (8). The interferon titer corresponded to the reciprocal of the highest dilution of test sample that unit is equivalent to 0.9 reduced plaques by 50%. In this assay, one interferon reference standard units. were obtained from one sheep Sheep erytlzrocyfes. Sheep erythrocytes (SRBC) (#772 yellow) of Gibco Diagnostics, hladison, Wis. In vitro sensi&afio~z to SRRC. Spleen cells were cultured in vitro and sexisitized \vith sheep erythrocytes according to the method of Mishell and Dutton (9). Cultures were harvested 4 days after sensitization and plaque forming cells were expressed per lo6 trypan blue viable cells.

_I ‘he nunilwr 011 antil)otly producing cells \vas tletmnined Hclrlolj*tic- pltrqftr ~~.s.su~, l,y a modificatioil of the !clrilc l)lacl1le assay ( IO). blouse sl)lecns mw-e rclnOvCt1 nlltl strninetl into single ccl1 sus~~ensi0ii.s or sl)leell cell cultures were liar\-estetl ant1 1)~ iiiculmtioll for .1 Ill- at 37°C‘ \vith direct lhcptc forming cvlls \vcrc’ tlevclolwl guinea l)ig ccmll)leiiiellt ((;il,co, licrlieley, (‘alif.). Lip(~ih”l?‘sclc-~l~~~~.;~~~,. I,il~o~~~~l~s;~~ch;iritlc ( I 1I ‘S ) fl-crni Isc.hc,,-ic-hicr c-o/i ( I )ifco J,ahratories, Iktroit, \lich. ) \ms tlilutetl to ;i ccmcentrntiot~ of l(x) g/n11 in i)lioil)li~ite-l)uI‘rerctl s;llinr ant1 soiiicate(1 for 3 niiil. Fivr Iilicrograllls 0i 1,1’S were atltletl I)er sldecn cell culture fiu- scllsitization. Shwl, er!diroc~tes \vrre contetl \vitli J,I’S 1)~ Iiiixiiig 100 iii1 of I -I’S sclltition (0.1 mg/nil) \vitli 2.j lul of SI
~11 2 irrcrc-tiwtior~. Interferon preparations were lmql~t to l)H 2 lvitll 1 .V TIC1 for 1 hr ant1 then neutralized with 1 ;Y NaOH mtl assayed for resitlual activity. G-100 Scpllcrtlr~.r coll/,rrn ~~hroIrltrto~r~aplr!‘. .\ 90 X 2.3 cm column of Sepliatlex ( I’lm-ma& Fine Chemicals, T’iscatau.a!,, S.J.) was prepared at 1°C: \vith plmsIhate lm~feretl saline. (he telith nlilliliter of 5s’; plienol red \vas atltlvtl to ;L 2.5 ml iracti0n5 to tlw colun~n. Five iiiilliliter niouse serum sniiq)le l)eforr its alq)lication were collectrtl aiitl tile al~4~1-l~a1~c~at 280 tini \V;IS tletermind. l‘lie sunldes cvu-c tlialvzctl for 2 tl;iys agaiilst foul- cliang-es 0i Aliiiiliial lissential Llediulii ( Gihco, Tnc.: lkrkele~-, Calif. ) prior to study of their antiviral activity. .Z full profile of interferon activity ant1 protein content 0i all fractions tested \v;is lm5entetl elseu.here (5). Selected fractions \vere tested for tlirir iinln~unosu1~l~res~i~~ acti\.ity. .\‘/uti.stirnl c~~MI/~~.s~.s. 1’ values l\.ei-e tlcterniiiietl 1)~ ~iicxiis of a Stli(lent‘s t-trst.

I’u.~sky tr-oll.sfm o/ 7‘yp I/ jkft(‘l.fc’w12 ~~e~czl-ntio~rs. Several pi-epr;ltioiis containing different doses of 7‘yl)e I I interferon \vere ntliniiiisteretl intravenously to normal S\viss-\!‘ehster mice to cleterinine if extraneously added interferon could effect the irr zkw antilmtly reslmbe to SlII3c‘. l‘n-enty-fmr hours after atlministration 0i the interferon l)rep:irations. the niicc were ititrnvenously injrctetl lvitli STII
288

SONiVENFELD,

MAiVDEI.,

AND

TABLE

MEKIGAN

1

Effect of Passive Transfer of Interferon 24 hr Before SKBC Sensitization in Virlo Production of Antibody Forming Cells Interferon dosage (units)

Number

of mice

Mock interferon

Interferon

Type I 1.58 x 105 1.1 x 101

2 2

2 2

Type 11 1.4 x 7.0 x 1.75 x 3.5 x

5 2 5 8

4 3 8 10

104 102 10” 102

PFC,‘lOG cells ZIZSE Mock interferon

8-l 357 f 45 1411 +

1 31

f f f f

87 1877 f 7.5 833 f 69 708 f 188 463 &

228 126 147 123

P

-45 +017


$303 $136 + 79 + 1

< 0.0005
313 It 31

26

SRBC onI>.

‘;I Difference

Interferon

645 f 695 * 466 3.53 394 454

on

NS = not significant.

ter dosage of Type I interferon was required to produce measurable levels of circulating interferon (12). When 1.2 X lo4 units of Type II interferon were intravenously administered 48 hr after sensitization with SRBC, an 877 o increase in the number of plaque forrning cells was observed (Table 2). Administration of 6 X 10” units of Type II interferon after antigen had no significant effect on the nunber of antibody forming cells (Table 2). In vifro enhancing effect of Type II illfeyjerou /we/mvfions. 14’hen preparations containing Type II interferon were added to cultures of BALB/c mouse spleen cells 2 days after addition of SRBC, 3657 0 more antibody forming cells were observed (Table 3). These data are similar to data previously obtained with Type I interferon (3). \f:ith Type II interferon, addition of as little as 3.5 antiviral units was TABLE

2

Effect of Passive Transfer of Interferon 18 hr After SKBC Sensitization in Viao Production of Antibody Forming Cells

on

-.~ Number

Interferon dosage (units)

Mock interferon

of mice

Interferon

I’FC/lOfi

cells f

SE

Mock interferon

Interferon

‘;; Ijifference

P

Type I 1.2 x 105 1.2 x 10”

2 2

2 2

337 f 332 f

35 5

497 f 484 f

23 43

f-l7 +a6

<0.025
Type II 1.2 x 104 6 X 10”

3 2

3 3

212 * 229 f

17 13

397 f 208 f

18 81

+87

<0.0025 NS

NS = not significant.

-10

sttfficiettt to Iuwtlt~ce a significant increase in the number of anti-SIZ HC: antilml~ protlttcing cells (Talk 3 ) Ii Type II interferon uxs added to spleen cell cultures 72 hr after addition of SIIBC, no significant effect \vas observed on the antiSRB(: plaque forming cell response (5). In a previous study, we have shown that addition of Type II ittterferon 21 hr before or sitittiltaneottsly with atltlition of SIIRC to spleen cell cttlturcs causetl a mat-ketl sttlqxessimt of the anti-SIi HC‘ antilmtly response (5). I
of T~lpc

II

iritcr-fcron

jol-ruing ccl1 ~~~.~pu~~sc to li/wpOly.strc-

011 the plaque

clzn~itlr. .1tltlition of ‘l‘ylw TI intrrferott to spleen cell cultures of liAl~R/c mice 24 hr I)efore sensitization \vith I,PS significantly suppressed the plaque forming- cell response to 1.1’S (Table 1). 12’hrn T!y IT interieron \vas atltletl to the cultures ati effect on the antibody response to I _I’S \vas tiot d38 hr after sensitization, served (Talde 4 1. I’assive transfer of 1.2 X 10’ units of Tyl)e II ittterfero~t to S\viss/\Z7el)stcl- mice 18 hr after iu 7bo sensitization to I .1’S did ttot sig:uificat1tl! increase the ttttnil~er of anti-I,l ‘S ~~laqtte forntittg cells.

‘I‘AIII,E: Effect

of Addition (‘ell

of TJ pe I I Interferon I
4

on the Anti-I.15 l’hclue c Mouse Spleen (‘ells

Forn\ing

- 24

24 2.4 0.24

2 2 2

240 f 227 + 22.5 *

i 12 14

06 f Ii9 f 232 f

24 0 17

-60 -21 + 3

+4x

24 2.4 0.24

2 2 2

252 * 240 f 227 f

30 38 2

250 f 243 * 222 +

IT 17 4

+ -

NS = not siplificant.

1 1 2

< 0.0025 < 0.01 NS ss NS NS

290

SONh-ENFELD,

MAiWEL,

.4&-D

RIERIGAN

Time of interferon addition in relation to antigen (hr)

Interferon units added

-24

24 2.4 0.24

2 2 2

241 zt 30 211 f 23

103 f 163 f

24 2.4 0.24

2 2 2

208 i 233 f 228 f

202 f 11 220 f 14 231 + 20

h-0. of expts.

Mean PFC,/106 cells f standard error

+48

P

Interferon

Mock

199 i

(,L Change

13

21f

10 11 1.5

1

-89

30

-57 -23

1.5

-3 -6 +1

<0.0005
-.

KS = not significant.

plaque forming cells than cultures treated \vi:ith mock interferon (Table 5). I,ate addition of Type II interferon 48 hr after LPS sensitization did not cause a significant enhancement of the plaque forming cell response to LPS (Table 5). Passive to nu/nu mice A!? hr after sensitransfer of 1.2 x lOa units of Type II interferon tization i~z r~bo to LPS did not increase the number of anti-IJS plaque forming cells. Z~Z.zr’tro characteri3~tiorz Of ‘I’_vpc II illtrrfmwI acfizitirs. Preparations containing Type II interferon activity were suhjectecl to physical-chemical characterization in order to further define the relationship between the antiviral and immunoenhancit~g activities. The expression of both the antiviral and immu~loenhancing activities of Type II interferon was resistant to heat treatment at 56°C for 1 hr (Table 6). hlaintenance of Type II interferon preparations at pH 2 for 1 hr inactivated the expression formed untreated

of both between

Type

the

the

antiviral

and

immunoetlha~~ci~lg

immunoenhancitg

II interferon

and

preparations,

Characterization

of Type II Interferon

Treatment

Control pH 2 (1 hr, 23°C‘) Heat (1 hr, 56°C) Sephadex fraction 11 Sephadex fraction 29 Sephadex fraction 39 In nitro produced Type II interferon

.activities.

\Vllen

a ratio

is

(I/A ratio) of the that ratio is 2.7. Type II interferon that antiviral

Immunoenhancing titer” 6500

< 100 3900

< 10 65

activities

Activities Antiviral titer 2405 40

1600


1000

3000

~-

= Immunoenhancing titer = greatest dilution of interferon number of plaque forming cells/106 spleen cells. Ihta is representative of duplicate experiments.

that causes a 505; change in the

I.\1 \I I’N0l~SII.\S(‘E\1I~N1‘

Ii\’

IS’r1-I
Antiviral titer

Itlltllunoenh;~l~cill~ titer’l

‘l‘reatlllent

.zo

40,000 30.000 2,000

IX < IO ” Immunoenh;lncing titer = greatest dilution of interferon number of plaque forniing cells 10” spleen cells. Ik~ta is representative of duplicate esperitllents.

2’) 1

I’Klrl’.Zl~.\‘I‘IoNS

that

caused

a 50 ; change

in the

1~;~slmducetl i/r :Yt/*o (4 ) also contained ail iiilinunoenhancing activity bvlien atldetl to spleen cell cultures 4X lir after semitization \vitli sheep erythrocytes (Tal)le 6). \I’hell fractions of Type IT interferon that hat1 been olhned by passage through Se]hatlex G-100 columns \vei-e added to sl)]een cell cultures 48 lir after selisitization \vith sheep red Ihd cells, i~iiiiiunoenhancilig activity bvas onI!. 0l)servetl iI1 those fractions that also containetl an anti\~iral activity (Tal)]e 6). I/r

T’itl.0

c’htJl-nc-fc’J,i,-cJtioJJ

Of

7‘)‘pr’

/

irltC’JfCJ’OJJ

~J~~firfitir.S.

\\7]le~l

71‘y])c

1

iliter-

feron prel~arations \vere added to s])lccn cell cultures 48 Iir after sensitiz;ltioli \\-it]1 sheep erythrocytrs, 3000 units of interferon \\-ere requiretl to 0l)tain ;I st:~tistic;Jl! significant enhanccnlent of the plaque forming cell reslwnse. ‘file T/X ratio of the Type I interferon ln-eparations \vas 0.0008. lhth the antiviral ant1 the it111111111oedmncing activities of T!y)e 1 illtcrfcron L\~CI’C])H 2 stalde a11t1 .50”( ];ll,ile (‘]‘a])]e 7,.

l’re]~arations containing Ty])e If interfrron ap]war to have a conq)lex eff‘ect OII (1 ). Their regulation the immme response, similar to that of Ty]le I interferon of the antilmdv response of mice to sheep crvtlirocJ?es alqmtrs to I)e lwtli tiiiic ant1 dosage tlelwndent. \I’hen Type IT interferon ])re]m-ations \vere added to s])leen ccl1 ctdtures aftel smisitization nit11 sheep erytlirocytes. enliancment of the priniary antilmtly res])mlse to SKI(]y res]mise to the antigen (4, 13-16). Ty])e 1 I interferon suppressed the anti])ot]\- response to T,I’S, as can Type I interferon (11, lmt did not l)roduce rllha~~c~lllel,t 0i the response. Sutle niicr have a tliymic tleficienc\ ant1 therefore do not respond to antigcns that require interactions nit]1 ‘I‘ lymphocytes ( 14, 17 ) HoIvever, they are aide to de\-e]op a normal atltilmdy reslmnse to T,T’S ( 14 ) T]le ]~]acl~l~~ forming ccl1 reslmnse of nude iuice to 1,1’S I\-as su])pressetl ])y early at]c]ition of ‘I‘ype I I interferon to s])lcrll cell cultures ])tIt \vas not rnhancetl 1)~.]:ltr a[]t]ition
292

SONNEKFELD,

RIANDEL,

AND

MERIGAN

Type 1 1 intcrfwoii. ‘l‘licsc data thcrcfure q)port the hypothesis that the sulqjressive effect of Type 11 interfr’roil is ail eilect on 13cells. Addition of interferon after sensitization may not al‘rect previously sensitized 13 cells, Ijut may cause an enhancement of helper T cell activity or an inhibition of suppressor T cell activity (3). Since cyclic AMP can block the immunosuppressive activity of interferon, it is possible that Type II interferon may interact with suppressor and helper T lymphocytes by means Of a cyclic ,4MP mediated signal (18). The dosage dependence of the immunoenhancing effect of Type II interferon preparations may be related to the time dependence. Passive transfer of moderate doses of Type II interferon preparations both before and after sensitization of mice to SRBC caused a marked enhancement of the antibody response to SRBC. Similar moderate doses of Type I interferon have also been shown to cause immunoenhancement ill z%w (3). It2 e-h!0 passive transfer of large amounts of interferon may allow the interferon to reach local antibody producin g sites such as the spleen and lymph nodes before antigen, thus causing immunosuppressiot~. However, passive transfer of moderate amounts of interferon may delay the arrival of the interferon to the local immunologic site until after the arrival of antigen, causing the iminuiioenhance~lleilt which we observed. Small amounts ,of interferon may be metabolized and eliminated befol-e reaching local sites, and therefore have no effect. Unfortunately, higher titeretl preparations of Type II interferon are not available at this time to permit the determination of the effect of large amounts of Type II interferon of the ilz &V inimune response. The antiviral and inlmu~~oenhancing activities of Type II interferon both share the l)hysical-chemical properties that have been described for Type II interferon (19). The expression of both activities was pH 2 labile and resistant to heat inactivation at 56°C for 1 hr. In addition, the imnl~~lloenhat~cing activity was only observed in those Sephadex G-100 column fractions that also contained the antiviral activity. Therefore, it is likely that the same substance or that closely related substances are responsible for both of these activities. However, since the Type II interferon preparations used were not completely purified, it is likely that substances that are produced concomitantly with the antiviral activity of Type II interferon, such as migration inhibitory factor (7), were also present. The immunoenhancing activity of Type II interferon may also be associated with those activities. Further purification of Type II interferon will permit the determination of the identity of all of the activities found in Type II interferon preparations. The potent immuIloenhancing activity of Type II interferon is another expression of its non-antiviral functions. As has been described for the other inimunoregulatory activity of Type II interferon, the im~~lunosuppressive activity (5)) the imgreater extent by Type II intermunoenhancing activity is expressed to a mucl~ feron than it is by Type I interferon. In addition, Type II interferon preparations also contain other potent regulatory activities, such as an anti-tumor activity (20) and an anti-bacterial activity (21) . Therefore, Type II interferon may be produced to act primarily as a cell regulatory substance. The antiviral activity may thus be only a side effect of its potent regulatory action. ACKNOWLEDGMENTS The authors appreciate the expert technical Hofmeister, and Dr. Zenaida Zerrudo.

assistance

of Ms. Barbara

Graves,

Ms. Barbara

l
JO~UIWII. 11. 11.. ;cl~tl I:;u-C,II. S.. /A’( ‘.S j. .l/ol. .\‘~~ii~,r~~~ 4, 50, 1076. Rraun. W.. and lxvy. H. B., 1’vo~. S‘r>r. Erl) l?io/. dlrd. 141. 769, 1972. Gisler, R. H., I~indal~l. I’.. and (iresser, I., J. Im~~ur~ol. 113. 438. 1974. Brodeur. B. R.. and Merigan, T. C.. .I. I~~rrrt~cuol. 114, 1323, 1975. Sonncnfeld. C;.. MantleI, A. I).. and Merigan. T. C.. Cc//. Iurrrl~tr~ol. 34. 193, 1977. Hanna. I,.. Slerigan, T. C.. and Ja\vt‘tz. E.. Pror. Sor-. Ex~. Biol. .Ilctl. 122. 417, 1966. Salvin. S. R.. Youtqqwr. J. S.. and Ixdcrer, iV, H., I~rfcrt. Imm~rrr. 7. 68. 1973. Brodeur, B. R.. and Mcrigan, T. C.. J. I/rrlrlurr~~l. 113. 1319. 1974. Slislwll. R. I.. and Ihtton. R. L$:.. .I. h-p. ,l/mf. 126, 423. 1967. Chester. T. J.. De Clcrccl. E.. and hlerigan. T. C., 1llfrc.t. Inrr~r~frl. 3, 516, 1971. Seter. E.. Westphal. 0.. I.uderitz. 0.. (;(,I-zynski. E. ;\., and Eirhmherger, E.. J. Iurrurr~r:~i. 76, 377. 19.56. 12. Chester, 1‘. J., I’aucker, Ii.. and Ilerigan. T. C.. A’b~:trt~,~c~ (I~r~rtlor~ ) 246. 92, 1973. 13. hloller. C;.. and Michael, (;.. (‘c/l. Iu~r1rfrt7~~l.2. 309. 1971. 14. hlatminy, J. K., Reed, S. I).. and Jutila. J. W., .I. Iu~m~rrwl. 108. 1370. 1072. 15. laden. I>. P., and Reed. N. I)., IIII~/~I/II~I/. (‘ommu~r. 2, 335, 1973. 16. ;\ndersson, B.. and Blomgrcn. H., C‘ril lur~r~urrol. 2, 41 1. 1971. 17. Wortis, H. I-I.. (‘/in. Exp. Inrrrtuuol. 8. 305, 1971. 18. Johnson, H. AI.. Rlalock. J, E.. atld Baron. S., ~-p/l. ~~~~~J~~~~J~/. 33, 170, 1977. 19. ~~oungnrr. J. S.. and Salvin, S. B.. J. IIII~~IWIO[. 111, 1914, 1973. 20. Salvin. s. B.. ~oungner. J. S.. Kishio. J., and Neta. H.. .I. h;at’l (‘UIICCI. Iu.Y~. 55, 1233. 1975. 21. Calvin. S. IL Sisllio. J.. md Slmnnard. J. T.. Ilrfcrt. IUJUIUJI. 9. 631, 1974.