Chronic experimental allergic encephalomyelitis in strain 2 guinea pigs

Journal of Neuroimmunology, 4 (1983) 187-I99

ElsevierSciencePublishers

Ig7

Chronic Experimental Allergic Encephalomyelitis in Strain 2 Guinea Pigs Absence

of Resistance to Nervous System Changes Dependence of Clinical Disea~se

and Sex

Sanford H. Sto~.'e, Ute Traugott and Cedric S. Raiae Laboratory of Microbial Immunity. National Institute of Allergy and lnfectimts DLwos~. lqafiost~ ! ~ of Health, Bethesda, MD 20205; and Departments of Pathology (Neuroparlmlo~). Nem,olbU awl Nenroscience, and the Rose F. Kennedy Centerfor Research in Mental R,~tardationand H ~ Development, Albert Einstein College of Medicine, 1300 Morris Park Avent~. The B~,zlr. lqr 10461

( U.S.,4.) (Received22 September,1982)

(Revised,received29 No-~ember1982/3i January, 1983) (Aocepted I February,1983)

Summary

Experimental autoimmune (allergic) encephalomyelitis (F.AE) was induced in Strain 2 guinea pigs, a strain usually regarded as resistant to EAE. The developmem of disease in groups of juvenile male or female Strain 2 / N guinea pigs was Lrrqgalar with clinical EAE manifesting only in some groups of females. All animals examined morphologically 0 2 females and 9 males), between 3 and 18 months posfinoculation, showed extensive changes in the central nervous system, although 12 of these had displayed no neurologic signs. This silent central nervous system disease, reminiscent of similar phenomena in man, indicates that Strain 2/'5I is fully competent immunologically at the level of the target organ. Key words: Central nerpou,= system - Experimental allergic encephal~myelitis Guinea pigs - S e x dependence

This work was supportedin part by USPHSgrants NS-08952,NS-11920,and NS-0/095; and Igaat RG 1001-D-4fromthe National Multiple Sclerosis,Society. Corr~pondenceto: Dr. SanfordH. Stone, Laboratoryof MicrobialImmunity,lthdklialg5, Room2~t, NIAID, NIH, Bethesda,MD 20205, U.S.A. 0165-5728/83/$03.00 ~ 1983 ElsevierSciencePublishers

188

Introduction Early studies on strain differences in susceptibility to acute experimental allergic encephalomyelitis (EAE) showed that while adult Strain 13 guinea pigs were highly susceptible, adult Strain 2 animals were resistant, and in general, immature guinea pigs of both st.rains wc,'e relative2y r~,sistant (Stone 1962; Stone et al. 1969). Previous research had shown that Strain 13 newborns would regularly deve|op a chronic, delayed-onset form of EAE following sensitization with whole central nervous system (CNS) tissue, while Strain 2 juveniles were comparatively insusceptible to t~ds chronic disease (Stone and Leraer 1965). However, in a previous ultrastructura! study on chronic EAE in Strain 13 and Strain 2, some Strain 2 guinea pigs unexpectedly displayed chronic disease (Raine et al. 1974). This finding recalled earlier experiments in which some clinicaily healthy sensitized Strain 2 animals also displayed inflammation within lhe CNS of an intensity comparable to that found in Strain 13 guinea pigs with signs of EAE (Stone and Lerner 1965). In view of the unexpected susceptibility of Strain 2 to chronic EAF., the present stu.Jy was conducted. It was found that a significant percentage of Strain 2 fema!es are susceptible to clinical signs of chronic F.AE induced by whole CNS tissue while morphologic changes consistent ,~,ith a long-standing demyelinative pcocess were evident in all inoculated animals of both sexes, irrespective of clinical history.

Materials and Methods Immature Strain 2 / N guinea pigs ( < 250 g) (from the Animal Preduction Unit, NIH, Bethesda, MDI~ were immunized as previously described by a single intracutaneous sensitization with 0.5 ml cf an emulsion containing guinea pig spinal cord in saline and coraplete Freund's adjuvant (CFA) containing 2.5 mg of Mycobacterium tuberculosis (Stone and Lerner !965). A total of 92 juvenile Strain 2 guinea pigs was inoculated and studied for up to 18 months postinoculation (p.i.) (Tables I ;rod 2).

TABLE 1 CLINICAL SUSCEPTIBILITY OF STRAIN 2 GUINEA PIGS SENSITIZED FOR EAE WITH SPINAL CORD IN CFA Group

Sex

No. o~: animals

Acute

Chronic

! !1 ill !~ V V! VII

F F F F F M M

21 10 19 5 8 20 9

0/21 2/10 4/19 2/5 I/8 0/20 I/9

12/21 0/10 1/19 2/5 2/8 0/20 0/9

TABL~

2

MO~°HGLOGIC FOR EArl" Aahntl

Se~

3 $

F F F

8

F

4

ANALYSIS OF CHRONK~

~

~ M ~ .~N S ~ . , A ~

C~ak~

4

3 3

-

++ -

-

+

÷÷

~*

++

*

÷~

*

÷+

~

~

+ ÷

" ~

~

7

+

++

++

9

F

7

+

++

•

10

F

I0

+ +

+

+ +

il 12 13

F

!2 18 ~75

-

-

M

-

14

M

6

.

IS

M M

6

16

6

.

17

M

6

.

18 19

M M M M

6 7 7 7

.

F

21

~

-~

-

-

~

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-

-

~ +

-

.

.

.

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.

+ +

.

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-

.

.

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.

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~ ÷~ ÷

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• . A n i m A k . . m ~ i l i z e d b e t w e e n 3 a n d 6 w e e k s ot" q ~ b + - ~ki

paraparesis;

+ + - moderate pmp~ests

md

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Detailed morphologic examination was carried out on 12 fmmk: ~ Stn~in 2/N g.inea p i p (Tabte 2). The 9 i n k s re,died ~ listed group

in Table

I as Stoup

V in Table

VII.

!. Animals

Four were

of the :mapled

females between

came

Loan

3 weeks

~ ~fd

when they were anesthetized with ether, pedesed with I t l m m . k ~ k .

g ma~ m

i ~

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for electron microscope (EM) study by techaiques described ~ ~ 1974). Brain, spinal cord, and lumbar spinal nerve roots wcJe examined ~ animal. Light microscopy was performed on H and E sectkms and on l.~tm stained with toluidine blue. Sections for EM were placed on ,mcoatcd ~ with u r a n i u m a n d l e a d s a l t s , c a r b o n - ~ o a t t ~ , and ~amted in a ~ I0|.

Resdts Clinicalfindings Table i shows that in addition to an extremely variable cliakal ottt~me

• each

190

.'.~o ., ~

Figures I to ,~ and 12 to 16 are light micrographs o/ I .pm Ioluidine blue.stained epoxy sections, Fig. 1. Low power view of an area of midbrain from a female S;rain 2 guinea pig (no. 80/962) with clinical signs of chronic EAE at 5 months p.i. This animal had demonstrated 3 clinical relapses. The 4th ventricle lies above. N~tc the multiple punctuate dcm'jelinated lesions (arrows). The large lesion on the left is active and inKammatory, the rest are mostly gZ.otic and sile~,.~. ×40. Fig. 2. Detail from active lesier, in Fig. I. Note tile extensive perivascular cuff of hematogenous cells and the, many r, ~ked axons cut It ~':~. ~rsely. ×250.

Strain 2 guinea pigs, there is clear evidence of a se~: difference in ~g-cV~b~h~, ~ observed previously (Stone et al. 1969). Males were resistant to cl~m~,d ~ ~q chronic EAE except for one juvenile male which developed transient ~ e ~ 3 weeks p.i. which remitted completely over a 7-day period ~Tabl¢ I, g r ~ V[|; Table 2, animal no, 13). In the 5 lVoups of juvenile females g/yen a singge i u o c ~ g ~ of spinal cord in CFA, about 25% developed chronic EAE. Tiffs ~ ~ m ~ r ~ f ~ cli~fically by hind limb paraparesis, some wasting, and urinary and fe¢.~ ~ u ~ : ~ neace. Only rarely did animals die as a result of complications caused b? c ~ EAE. Up to 3 clinical relapses were observed in some animals over an ~ = ~ , ~ u ~ period of study. Acute signs (2-3 weeks p.i.) were infrequent and ra/ld ar,-d ~ exception (see above), were limited to females. The higher degree of d~¢~:~ i u ~ - tion from group to group of females was not always reproducible. For ¢ x ~ ¢ . ~ ~ one group (Table 1, group II), of 10 animals, two developed mild, tranuem ~ g ~ acute EAE 2-3 weeks p.i., but none showed chronic EAE. Yet in group L ~2 ~ 2~ females developed chronic EAE. Analysis of the clinical and histolog;.c evidence of CNS mvolveracnt m a group of 21 animals (Table 2) showed a lack of correlaticn between c[/nu:~ u ~ and CNS lesions in that irrespec6ve of clinical picture, all animals d / : ~ , ~ morphologic evidence of disease.

Morphologic findings Strain 2 females displaying clinical signs of chronic EAE displayed large k ' ~ throughout the entire neuraxis. These lesions were deafly demyelinativ¢ and h ~ ~ e same morphologic picture as that described previously for Strain 13 anima[~ ~R~ne et al. 19'74). Lesions were also seen at early t i m q ,)~nts p.i. in f e m a ~ ~ h displayed no clinical signs of EAE. However, the most severe changes were s ~ n m relapsing animals (vide infra). Two females sampled at 10 months p.L and Ig months p.i. which had not displayed signs of EAE, had evidence of l o n g - s t a m ~ $ disease. In Strain 2 females with relapsing disease activity (Table 2, animals 7-101~ t h e ~ was a rostral spread of disease activity with time in that areas adjacent to the L~terd ventricles and beneath the floor of the 4th ventricle displayed exteas/v¢ ~ = ~ inflammation and ongoing demyelination in addition to a few e s t a b l ~ lcs/~g These were scattered throughout the white and grey matter and had a p u f f - b ~ appearance (Fig. 1). The largest lesions were active and heavily infiltr-~|ed b.~ hematogenous cells and contained large btmdles of demyelinated axons and

4 Fig,3. Detail of several perivascularcuffs and bundlesof naked axonbcut obliquelyfrom ~ q~g~,~t~$ cranial nerve, x 250. Fig. 4. A small, gliotic,incompletelydemyelinatedlesionis seen from a regton near the [k,~¢¢q[~¢ ventricle, x 120. Fig. 5. A group of neuronsfrom a nucleusnear the floor of the 4th ventriclelies amidst an aa~ o~ ~ demyelination. × 150.

192 •

.

~ ~

,., ~ . .

~

.

-

Fig. 6 Lower lumbar cord. A small round lesion Js shown within an anterior column, The arrow points (o the vessel shown in Fig. 7. × 120. Fig. 7. Detail from the area around the vessel (ar:ow) shown in Fig, 6. Note the widespread remyelination and the few persistent macrophages and myelin debris. × 250. Fig. 8. Upper sacral cord. A broad band of advanced remyelination (arrows) follows the margin of the spinal cord. × 2 0 0 . Fig. 9. Female Strain 2 guinea pig (no. 80/960). 7 months p.i., with clinical signs and one relapse. This electron micrograph is from the center o1 a demyelinated plaque from the spinal cord which displays many naked axons lying within a gliotic matrix. The body of a fibrous astrocyte, nucleus at (n), is shown. × 5250.

Fig. 12. Strain 2 guinea pig (no. 81/014), male, 7 monlhs p.i., no clinical signs. A large d c m ~ y ~ " plaque is seen at the subpial margin (above) of the lumbar spinal cord. Note the intense ~ w~ r~-~ meninges and around many parenchymal vessels, the widespread demyelination and $ ] k ~ ~ the n ~ recent inflammatory activity around some of the vesselb (v) at the edge of the leston. Some r e m ~ ¢ ~ ~ is seen to the upper right and around the margins of the lesion, ongoing myelin breakdo~'r~ ts ~ k ~ × 550. • Fig. 10. Same animal and lesion as in Fig. 9. At the edge of the lesion widespread rem~,~ek~|~ L~,¢ ~ l'.iote the disproportionately thin myelin sheaths and large diameter axons, x 8400. Fig. ! I. A small area from a macrophage lying at the edge of the lesion shown in F15. 9~ N ~ membranous channel containing a droplet of myelin and the closely assoctated ¢txtled ~1 ~_~,~.~, >: 80,000.

194

Fig. 13. Strain 2 male guinea pi$ (no. 81/016), asymptomati,:, 7 months p.i. A recem cuff of inflammatory cells is situated around a vessel overlying the lumbar spinal cord. ×250. Fig. 14. Same animal as in Fig. 13. A long-standing inflammatory cuff is seen around a major vessel over the dorsal columns. Note bow the inflan~matory cells (most of which were plasma cells at hi[her magnification) lie in annular rings. EIsewhele, th,: meningeal space is fibrotic, x 120.

laden macrophages (Figs. 2 and 3). Older lesions occurred aL~gi ¢ ~ - ~ i ~ evidence of ongoing activity. They presented as areas of diffuse d e m ~ g ~ ~ were gliotic and contained some thinly myelinated (remyelinated] fib¢~ ~ ~ small oligodendrocytes (Fig. 4). Diffuse demyelination was also app~eu~ u~ ~ grey matter where there was preservation of nerve cell bodies (Fig_ 5~. ~ ~ caudally down the spinal cord, lesion age increased and. in g e n e r a l p t : ~ *~ pathologically silent and reparatory. The lesions were usually s u b p / ~ large, a ~ staining (Fig. 6). Higher magnification revealed the presence of m a n y r e m S ~ fibers, some gliosis, and persistent droplets of myelin, but no active m ~ m m ~ (Fig. 7). Large lesions were also present at lower levels of spinal cor-:l (Fi$~ ~i~ ~ addition to containing m a n y CNS-remyelina.ted and few demyel/nated ax~u~. ~ : ~ times contained CNS fibers myelinated by Schwann cells, a common p b e ~ chronic relapsing EAE (Raine et al. 1978; Snyder et al. 1975). EM examination of lesions from clinically-affected animals confinm:d a ~ ~ ence of areas of chronic demyelination and gliosis (Fig. 9)~ CNS remye[U~m~u ~[c~ 10); and at the margins of old lesions, ongoing m, elin breakdown. The [ ~ o ~ ' ~ ~ myelin breakdown involved encirclement of the myelinated fibers by a m a ¢ r ~ a n d the dissociation of myelin from the sheath to form droplets or v e s k : ~ i~:gAe~ (Raine et al. 1974) which were then phagocytosed via coated pits {Fig [ ! ~ Surprisingly, examination of the CNS of clinically-resistant Stra/a 2 ~ ~ vealed evidence of myelin damage in all cases. In most lesions, d e m y e l i a ~ re~ to inflammation was encountered and the spinal cord was more invol~ed ~ ~e brain. The spinal cord of 1 male sampled 2 weeks p.i. displayed a b u n d a n t m ~ tion and some demyelination (Table 2) indicating that the disease i ~ ' ~ s s ~ : ~ mences at a timepoint comparable to that of acute EAE (Ratine et aI. 1974~. F~¢ most c o m m o n appearance was large areas of chronic demyelinatma ~ perimeters of which ongoing myelin breakdown, inflammation, a n d some r e m ~ tion occurred (Fig. 12). Oligodendrocytes were few in n u m b e r in c h r o m ~ d~ myelinated areas b u t were present in normal amounts among r e m y e l h ~ .~r~ Not infrequently, the meninges displayed evidence of e~tensive fib~c~b~ |~

• Fig, D. Strain 2 male guinea pig (no. 81/012), 9' months p.i., no signs. A large demye~ued ~ ~ a lateral column. The meningesare fibrotic and severalo| the vessels(v) within the lcsk~ ~ ~e~.~ of recent inflammation, x 120. Fig. 16. Lumbar spinal cord, Strain 2 male guinea pig (no. 81/015). 7 n~>nthsp&. no ~ broad band of subpial remyelination (arrows) and the extensive fibrosis ~lhin tl~ ~ x 250.

~

Fig. 17. This electron micrograph comes from the edge of the lesion shown from th~ ~ y ~ (no. 81/014) in Fig. 12. Note the ongoing myelin breakdown around an axon {a},the ~ ~ ~ been invaded and separated by processes from a macrophage. The innermost laS,~'lrsc~ ~ vesicular disruption. Elsewhere, mactophage activity and myelin droplets can be see~ × t(}0~

~-

~'-~

Fig. 18. Detail from Fig. 16. The vesiculardisruption of myelin occurs between Ihe ia~-ad[~$~-¢~~ . - ~ which sometimescontain a coated pit (arrow). The axon is shown at (a). x400~0.

196 clinically-healthy males, these lesions were extensive and morphologically indistinguishable from those encountered in clinicaIiy-affected and healthy females. In the majority of healthy male Strain 2 guinea pigs, ongoing CNS disease could be documented either in the form of meningeal vessels surrounded by thick, recent inflammatory cuffs (F~ig. 13) or by long-standing cuffs which were well-formed and annulated (Fig. 14). Plasma cells were common constituents in these infiltrates. Sometimes, chron/c subpial lesions were heavily infiltrated by hematogenous cells (Fig. 15), suggestive of recurrent episodes of disease. As in females, narrow bands of subpial remyelination were common in the spinal cord (Fig. 16). EM examination of CNS lesions in clinically silent male Strain 2 guinea pigs confirmed the presence of established and ongoing demyelination. At the perimeters of most lesions (up to 7 months p.i.), ongoing myelin breakdown by macrophages was apparent (Fig. 17). This process, as in susceptible Strain 2 females, involved myelin dissolution, vesiculation, and phagocytosis via channels between macrophage cell processes anti coated pits (Fig. 18). The bulk of the lesion area was occupied by chronically demyelinated axons, while a rim of CNS remyelination was not uncommon at the margins. Some spinal nerve root involvement ~vas seen in ~11 groups in that remyelination and onion-bulb f,-,rmation occurred in both the anterior and posterior roots.

Discussion

The present study has demonstrated that a significant percentage of juvenile Strain 2 / N female guinea pigs are susceptible to clinical chronic EAE (.~:,metimes relapsing), that males are clinically resistant to chronic EAE, and that both sexes display a 100~ ability to develop CNS plaques, irrespective of clinical signs. These findings extend plevious observations on sporadic EAE in Strain 2 and also show that juvenile Strain 2 / N animals behave differently from Strain 13/N after sensitization for I~AE. It is interesting to note that the recently obse: ~cxl changes in disease pattern,, in some Strain 13/N animals sensitized for chronic relapsing EAE (Raine and Stone 19T'), in which an early onset severe EAE was reported (Stone et al. 1980, was not reflected in Strain 2/N. The clinical resistance of malts, as reported before (Stone 1962; Stone and Lerner 1965; Stone et ai. 1969), hints at possible sex steroid inhibition of the immune response; but this, as well as the invocation of inhibition b) suppressor cells, is doubtful in view of the cellular response and inflammator~ demyelination within the CNS of sensitized males. The prominent morphological changes imply that the resistance is reflected as a functional deficiency late in ~he process of CNS damage, and in fact, dilute theories of immunode.~ic~ent T or B cells in Strain 2 (Teitelbaum et al. 1977; Teitelbaum and Arnon 1981). This possibilit,, has been tested with lymph node cells from sensitized Strain 2 gu'~:~¢a pigs which were transferred to (2 × 13)FI hybrids (susceptible) to examine waetner EAE could be produced in the recipients. However, in our expene~ce (S.H. Stone, unpublished observations), this system causes lethal graftversus-he, st reaction in ~.he (2 × 13)F t hosts before F ~ E was clinically apparent.

That Strains 2 and 13 differ significaptly at the immuncloMc k v d k B ~ established. These differences occur at the level of the Ir gene ~ arc ~ different la antigens (Rosentha~ and Shevach 1973; Burger et ~ I ~ | ) . ~ also manifest different responses to synthetic antigens (Ben-E/raSm et all I%7J such differences appear to be la-related (Clark and Shevach 1982). ha E A E ~ the situation is probably not analogous in that complex (multiple) ~ ~ b¢~ T and B cell systems ~re involved. A recent comparative study in S t m i ~ 2 amd ~3 ¢ i circulating and CNS-infiltrating T cell subpopulations and a n t i g e a - ~ so myelin antigens has demonstrated a selective migration of activ~od T ¢ ~ ~ ~ ¢ CNS and a decrease in TO cells in the circulation only in the E A E ~ $¢r~ 13 (Traugott et al., in preparation). In Strain 2, although T cells inFdrar~ ~ the distribution of activated T cells and To cells in the CNS was random ~ to levels in the circLdation where ~['~cells did not decrease. ~ t y to ~ oligodendrocyte antigens o~curred in both strains. Therefore, at the ~ of ~ T cell in EAE, Strain 2 guinea pigs were responders. These d~ffe~m:~ ~ B responses to synthetic antigens where Strain 2 are n o n - ~ (Cl~rk Shevach 1982) and the above described positive T cell responses to CNS ~ Strain 2, indicate a dichotomy yet to be clarified. Some reports exist where Strain 2 guinea pigs have been found to be to EAE (e.g. Hashim 1981). These might raise the question of gez~_k: ~ m certain commercially-available sto~ks. However, the Strain 2 / N gu~aea I z ~ ~ ~a this study, are the stringently monitored offspring of parents wSk:h w~¢ d ~ from the original colony at N.].H. and which therefore constitute 0~e ~ f~ Strain 2. Furthermore, the genetic: purity of the Straia 2 used was ~ ~ absence of a response in a raixed lymphocyte reaction with cells from other ~ 2 animals and the presence of Strain 2-specific Ia antigens (Shevach, per~m~ munication). These considerations emphasize the desirab'~llty of i n ~ m ~ ~ terminology analogous to that us~t for mice and rats. The apparent dichotomy between clil~cal and morphologic d ~ e a ~ poses intriguing questions, in particular, how animals can function m m m ~ ia the ~ c~ large areas of CNS involvement. An enisma similar to this also exists h: MS CNS involvement is invariably greater than that expected from the ~ dam ~ k in some cases, MS has bezn diagnosed only at autopsy (Ghatak ¢t ad. 1974; V ~ 1977). To what extent the benign or silent progression of ch,~ages in ~ 2 gwm~a pigs was related to the absence of a fulminant inflammatory event with edema, differences in blood-brain barrier permeability, steroid ~ m~l C ~ $ compensatory mechanisms, must remain areas of specalafio~ It is ~ ~o however, that the CNS can withstand significant demyelinatioo without the ment of overt, cfnical signs. The proclivity for this to occur ~ ~ m~ 2 males may provide an interesting model for physiologic inves6gaticm~ of ~ to MS in wl~ch both female predilection and subclin~cal lesions are Paterson (1976, 1982) believes that permeability to fibrin p~ys a r o k i~ induction of clinical sig~s; immunofluorescence e x p e m n ~ in t i ~ ~ warranted in Strain 2 animals. The increased severity and rostral spread of lesions is consistent with

198 findings on relapsing EAE in S.~¢ain 13 (Raine et al. 1974) and experiments on acute EAE in the rat (Levine et al. 1967) where repeated passive transfers caused an upward spread of lesion activity from the lower gpinal cord to the cerebellum. In its early stages, chronic relapsing EAE is most commonly a disease of the spinal cord and it is usually only after repeated episodes that plaque-sized lesions develop in the brain (Raine et al. 1980b). These observations might imply that during the early stages of the disease, lesions may show a predilection for the spinal cord due to a less efficient b l o o d - b r a i n barrier in this region and that with time, this possible microanatomical variation nught spread rostrally bringing with it concomitant inflammatory activity and active demyefination. In addition to the well-described cellular invasion of the myelin sheath by macrophages (Lampert 19671 Raine 1976) and myelin vesiculation (Raine et ai. 1974; Dal Canto et al. 1975), myelin breakdown involved a process of endocytosis described previously in MS (Prineas altd ConneU 1978) acute EAE in the mouse (Raine et al. 1980a), and JHM-vir~Js encephalomyelitis (Fleury et al. 1980). This previously de-emphasized mechanism may have more widespread significance in autoimmune demyelination.

Acknowledgements The authors thank Everett Swanson, Miriam Pakingan, and Howard Finch for technical expertise, and Virginia Shaw, Mary Palumbo and Agnes Geoghan for excellent secretarial assistance.

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