'AD[U-k j

REOTNo. >2-03-12 (F--inal iheuort)

~CONTRACT No. DA-92-7E-84'

ELCR~?CR05/307E STL"DY C' T1E IU'FECTIOU27 A':D jERI H-EFATIfIS

byGonpachiro Yasuzuni, M;.!).

1rofessor of' Anatomy'ara Nedical 'University

k ashihra, 'iara, Japan

October 196?

U. S. ARMY RESEARCH AND DEVELOPMENT GROUP +FAR EAST

APO San Francisco 96343 F)D- cNOYr 1 96

L Ut

ICFSI

DDC DISTRIBUTION ANT) AVAILABILITY NOTICE

Distribution of this documnt is unlimited. Qualified requesters mayobtain copies of this report from Defense Documentatioi. 'enter, CanerorStation, Alexandria, Va. 22314.

DISPOSITION INSTRUCTIONS

Destroy this report when it is no longer needej. Do not return itto the originator.

The findings in this report are not to be construed as an officialDepartment of the Army position uniss so designated by other authorized

documents.

Abstract

In 26 cases oi serum hepatitis from the observatiow: of80 cases of serum hepatitis and infectious rlepatltis, 1hedense virus-like particles appro.xdmately .0 A. . inC diameter,isolated or in clusters, were -bserved in the cytoplasxa o -fthe hepatic parenchymal cells and of the dev -.lping flibro-blasts. Moreover, the intranuclear inclusion bodieS fcr-Medby invagination of the nuclear envelope were found in Severalhepatic parenchymal cells in cases of serum hepatitis. Faniyof the inclusion bodies were revealed to contain denseparticles which were similar or smal~er as 'compared withth virus-like particles appeai~ng in the cytoplasm. Thedense particles within the inclusion seemed to appearassociated with the AT~ase reaction product.

CONTENT

1. Purpose.of the Investigation

2. Materials and Methods

-R. esults

L. Discussion-

5 ASu wary

6. Literatures Cited

. Explation of Figures

8, Figures 1-10

-2--

4~Iin

1. Purpose of the Investigauion

The purpose of the 7tudy is to identify viral particlesfrom biopsy material or corpse materials in cases of infect-ious ov serum hepatitis with the aid of electron m roscopyand x-ray scanning microanalysis or electron probe micro-analysis. Moreover, the structural changes of the liver cellsoccurring ir. such diseases are revealed in relation with theliver function.

During the past one year we have tri-d to observe severalenzyme activities of cell organelles in the parenchymal cellsof human liver biopsy materials from patients with serum orinfectious hepatitic. In the course of such study, severalunusual bodies of different sizes and forms have been foundin a series of the parenchymal cell nuclei which were prepar-ed to observe their ATPase activities. The descripti ofsuch structural pattern and a discussion of its functionalsignificance form the topics of this report.

2. Materials and Methods

Biopsy specimens of the human liver were available forelectron microscopy from patients those were admitted to NaraMedical University Hospital or to Osaka Prefectural Hospital,and diagnosed as serum or infectious hepatitis from the clini-cal findings and from the results of the liver function test.The liver biopsy materials were cut with a razor into smallerblocks and fixed either in 1.25 % glutaraldehyde or in 6.25lo glutaraldehyde followed by 1 % osmium teroxide (Ml), eachbeing adjusted to pH 7.2 with 0.1 H cacodylate buffer.

Small slices or frozen sections of the single fixationmaterial were treated according to a iodifided (4 ) Wachstein-Meisel procedure (36). Some materiai was incubated insubstrate-free medium as control, and subssequently immer -,edin lead nitrate. The specimens incubated wei'e then w aedfor 5 minutes in 7.5 % sucrose an ' postfixed for 1 'our in1 % osmium teroxide buffered to pH 7.2 with 0.1 M cacodylatebuffer. After fixation all the specimens were dehydrated ina series of increasing concentrations of alcohol and embeddedin epoxy Epon resin (25). Thin sections were cut on a Porter-Blum microtome or an LKB ultrotome i ,h glass knives. Thtwywere mounted on Formvar-coated copper specimen grids andsteined either singly with saturated aqueous uranyl acetate

(37) or doubly with saturated aqueous uranyl acetate followedby lead nitrate (28). Thse sections were examined in anelectron microscope of Hitachi Co., model HU-11C, or an elect-ron microscope of Japan Electron Optics Co., model JEM-7, oran Akashi electron microscope, model TRS-80.

3. Results

In the present materials obtained from 26 cases of serumhepatitis, which were selected from the observations of 80cases of serum and infectious hepatitis, the dense particles

approximately 200 A in diameter have been observed in thecytoplasm of the 'epati! parenchymal cell and the develop-ing fibroblast. These particleb are worth demonstratinghere, for purpose of comparison wi';h particulate elementsappearing.in the intranuclar inclusion bodies. Suchdense particles appear isolated or 'n clusters iii the cyto-plasmic vesicles or in the cytoplasmic matrix which is oftensurrounded by a triple-layered unit membrane (Figs. 2 and 5).The particles are clearly differentiated from the glycoLengranules which are characteristically organized into 400 to600 A rosettes (Final Report: J-203-2). These particles aresimilar in size and locus to the virus-like particles describedrecently by Babudieri and coworkers in cases of human infect-ious hepatitis (5).

In several cases of serum hepatitis, intranuclear inclusionbodies have been found in the hepatic parenchymal cells, whichare usually round or oval in shape, and seldom guiter-shaped.They range from 1.0 to 2.5 4 in size (Figs. 5, 4, 7 and 8),but the guiter-shaped is 6.5 4 in length, and 2.5 U and 3.2 Uin each diameter (Fig. 9). All the inclusion bodies arebounded by a distinct double-layered membrane which is dis-continuous in a similar way as in the nuclear envelope, andits outer layer appears associated occasionally with thecondensed chromatin masses (Figs. 3 and 4). The inclusionbodies are characterized by containing degenerated mitochon-dria (Fig. 4) and vesicular elements of the agranulr orranular endoplasmic reticulum in different shapes and sizesFigs. 3, 4 and 8), sometimes showing a parallel arrangement

of cisternal elemen+q (Fig. 7). As the inclusion bodyincreases in size, becomes more complicated in strnct.re,containing lipid droplet-like dense bodies, less densemicrobody, and vesicular or vacuolar structures of varyingsize and form (Fig. 8). At the stage when the inclusionbody becomes larger to show a guiter-shape, the includedelements appear to be different from the morphologically well-defined cell organelles in the cytoplasm (Figs. 9 and 10).The most striking abnormal elements of the contents are denseparticle-containing vesicles. The particles are variable insize between 140 A and 200 A in diameter (Figs. 4, 8 and 10).

The roughly round-shaped nucleoius 1.4 to 1.7 11 in diameter,appearing in the nucleus, which contains an inclusion body,consists mainly of tangled, dense nucleolonemata. It is noti-ceable that such nucleolus seems to have no particular rela-tionship with the inclusion body (Figs. 5 and 7).

The karyoplasm containing an inclusion body is character-ized by the relatively clear appearance, consisting of dis-persed chromatin elements which are aggregated only in smallamount, but not aggregated to form condensed chromatin mass-es (Figs. 5, 7 and 9).

Prior to the onservations on ATPase activities of thenucleus containing inclusion body, the activities have beenobserved in the nucleus which contains a large nucleolusconsisting of tangled nucleolonemata, but has neither anyinclusion bodies nor condensed chromatin masses. Such nucleus

-4-

_________T ____

r ______________________

appears to show clearly the deposits of lead phosphate in thenuclear pores (Fig. 5). In a tangential section through thenuclear envelope, the fine dense deposits of reaction productare observed on the nuclear envelope, and the most importantfeature is globular deposits measuring 460 to 560 A in dia-meter. It is certain that these reaction sites correspondto the nuclear pores, on the basis of previous reports (41,4 ). Although the deposits are too heavy to observe indetail the structure of the pores, many of the depositsseem to occur remarkably at the peripheral part of the pore,leaving a lighter inner space. But, one or two small depos-its are found occasionally at a central point of the pore.Moreover, the deposits occur at the part surrounding theperipheral dense deposits, showing a less dense radial appear-ance (Fig. 6). Such appearance suggests that the ATPasereaction product has a close relationship with the structuralelements constituting the pore, since Gall (14) has recentlydemonstrated by negative staining of isolated nuclear envelopesof several animal oocytes that the nuclear pores are oc.,ago.alrather than circular.

The ATPase activity is clearly detected also in the nuclearpores of the nucleus containing an inclusion body (Figs. 7,9 and 10) in a similar way as in the nucleus containing noany inclusion bodies (Fig. 5). The limiting membrane and itspore-like spaces surrounding an inclusion body appear to beactive sites of ATPase (Figs. 7, 8, 9 and 10), but depositso the reaction product are smaller in amount as comparedwith those in the nuclear envelope (Figs. 5,'7 and q). Theprecipitate occurs also in association with the membranes;lining vesicular or vacuolar structures (Fig.. 7, 9 and 10),and with the limiting membrane of lysosome-like bodie', (Fig.10). In an enalarged electron micrograoh (Fig. 10) oC a partof the guiter-shaped inclusion body Fig. 9), it may be seenthat unusual particles 140 to 200 A in diameter appear associ-ated with the deposits of ATPase reaction product: the parti-cles are embedded in the dense deposits; they arn attachedclosely to the depsits; and they are surrounded by the deposits.

4. -Discussion

Intranucle'r inclusion bodies have already been reportedin the human and several animal hepatic parenchymal cellsunder a wide variety of conditions (1, 1, , 7-),

C-8, 30, 35-35, ;8-40). On the other hand, a cc.siderablenumber of electron microscope studies have been done in aneffort ';o identify the unknown agents of human inf'e'tiousor serum hepatitio in liver cells or body fluids of patientswith viral hepatitis, and such studies have been suamnerizedby Steiner et al. (52) and B~budieri et al. (_ ), but theintranuclear inclusion bodies have never been reportei in the

liver cells in such cases.Recently, ATPase activities have been reported for the first

time by the modified procedure (45) of Wachstein-Meiel method(36) within and adjacent to the nuclear pores of epitnelial

-5-

Cells of the choroid plexuns from an adult mouse brain. Morerecently, such acuivities have been detected in the nuclearpor 2s of the Leydig cell in the young human testis, whileit has been failed to reveal in the nuclear pores of thecell containing intranuclear inclusion bodies (41). Conse-quently, it seems pertinent to attampt a study devoted toATEase activities within the pores of the nuclear env-lopeof nuclei which contain inclusion bodies in several shapesand sizes.

All of the intranuclear inclusion bodies demonstrat-dhere were completely surrounded by a clearly discernible1ouble-layered membrane. Although it was not demonstratedthat this double-layered membrane was continuous with thenuclear envelope at one point, and that an opening waspresent connecting the interior of the inclusion with thecytoplasm, the outer membrane surrounding the inclusionbody appeared to be associated with the chromatin elements,and ATPPse activities were revealed at the points whereseemed to correspond to the nuclear pores. Therefore, itseems that all the inclusion bodies examined representinvaginations of the nuclear envelope.

The cytoplasmic componeii's engulfed by the invaginationswere similar to the cell organelles but were sometimes notidentical with those in the cytoplasm. Tentatively, basedon the static electron micrograpbs, a line of developmentof inclusion bodies may be supposed as follows: the inclu-sion bodies seen in Figs. 5 and 4 represent early stages,Fig. 9 an advanced stage, and the other figures intermediatestaGes, since the bodies hecame more complex in structureconcurrently with the increa.iing of their sizes. Engulfedcell organelles within the inclusion in advanced stages mighthave beon degenerated as a result of such unfavorable enviroument as the decreased ATPase activity in the invaginatednuclear envelope. In the isolated rat livers perfused 'ithgiucagon, Ashford and Porter (2) found hepatocyte lysosomescontaining cytoplasmic components in various stages of break-down or hydrol$sis. Some of the contents appearing in theGuiter-shaped inclusion body seem to be similar to lysosomesdemonstrated by the authors mentioned above. But, it is wellknown that acid phosphatase is found exclusively in lysosomessince the work of De Duve (19). Therefore, whther or not thelysosome-like bodies are of acid phosphatase activities isa problem for further investigation.

Although the evidence presented did not provide an answerto the question of why the invagination of the nuclear enve-lope containing the cytclasmic organelles or their derivati-ves occurred, at least one possibility will be consideredbriefly. The formation of invagination of the nuclear envelopeis speculated to be dependent on an excellent mechanism bywhish the ratio between the surface of the nucleus and itsvolume is increased to perform nucleocytoplasmic exchanges ofmac:romolecules in more active condition, as already suggestedby Leduc and Wilson (21, 22). Such speculation may be supportedby the presence of a large nucleololus consisting of densenucleolonemata and by the clear appearance of karyoplasm,

-- -

containing only a few aggregated. 'omatin elements. Bearcroft(4), by histochemical techniques, showed t'at the nucleolarenlargement in yellow iever could be correlated with increasedpr-otein synthesis and probably represented the first reactionof injury. Yasuzumi et al. (42) suggested that the occurrenceof tangled nucleolonemata might be associated with the proteinand nucleic acid syntheses, on the basis of observations ofnucleoli in several animal tissue cells under a variety ofphysiological conditions. By means of autoradiographic tech-nique, it was suggested by Hay and Revel (16; that the dis-persed DNP gel, diluted chromatin, is the metabolically activeform of the DNP meshwork and that the condensed chromatin isrelatively inert, at least with respect to nucleic acid. andprotein synthses. Thus, it may be concluded that the nucleuswhere the invagination of nuclear envelope occurrel is in ametabolically active state, so far as the present material isconcerned.

It was assumed by Yasuzumi et al. (41) that one of thecauses leading to the formation of inclusion bodies in theaging human Leydig cell nuclei might be due to deficiency ofATPase activity within or adjacent to the nuclear pores.Hcwever, such case was clearly different from the present one,since abnormal protein or new and different protein wasproduced in the Leydig cell nucltuE as a result of unfavorableenvironment.

Although it was assumed that the invagination of the nuclearenvelope occurred to perform a more active metabolism, suchformation is not compatible with current concepts of normalnuclear structure or function. Such structure maV be theexpressimn of nu, lear modifications related with the viralparticleis. Many of the inclusion bodies were revealed tocontain dense partcles which are similar or smaller in siz-eas ,'omr-ared with the virus-lik , Particles appearing in thecyt opl 'ism. I i- intere-eting hat the particles in theinciuS ion were associated with the ATPase :'eac tion product,As reported byv Epstein xd llolt in herees virius paw'ticles.1C) and by De The in av;: n tumor vi tms particles (1)

In the intrac{ytoplasmic inclusion bodies cepn in neurons-ad microflial ,ells from trhe thal.ums of mi-e infek ted with

apanese encelhalit-is vims.';, den.;e p.trticles §1( to 75o Ain diaumeter were foun , and they were considered to be -re-zature virus particles, since matur-e ones were 70 to -0Ain diuneter ( 44). In a similar way, the particles found inthe intranuclear inclusion bodies maV be a precursor of thevirtis-like particles appearing in the hepatic parenchymalcol[ cytoplasm or in the developing fibroblast cytoplasm,though That must await further evidence.

The present study will be published in Experimental (ellResearch in near future.

5. Summary

A series of intranuclear inclusion bodies appea-ing in thehepatic parenchymal cells in cases of serum hepatitis were

- f

studied under consideration of ATPase activities on theelectron microscopic level, in which virus.-like particles

200 A in diameter were round in the cytop'asm, and bimilaror smaller particles in size within some inclusion bodies.All the inclusion bodies were formed by invagination of thenuclear envelope, in which the pores of the nuclear envelopeand its invaginated one were revealed to be active sites ofATPase. Possible function and relationship to viral infect-ion of the intranuclear inclusion bodies were discussed.

6.

1. Andrew, W., Brown, H. H. and Johnson, J. B.: Senilechanges in the liver of mouse and iran, with special refer-ence. to the similarity of the nuclear alterations. Am. J.Anat. 72, 199-221. (194>).

2. Ashford, T. P. and Porter, K. R.: Cytoplasmic compo-nents in hepatic cell lysosomes. J. Cell Biol. 12, 198-202 (1962).

5. Babudieri, B., Fiaschi, E., Naccarato, R. and Scuro,L. A.: Presence of viius-like bodies in liver cells ofpatienLs with infectious hepatitis. J. clin. Path. 19,

577-58d (1966).e. Leacroft, W. G. C.: (ytologica] and cytochemical

studies on the liver cells of yellow-fever-infected rhesusmonkeys. J. Path. Bact. 80, !9--Ui (1960).

5. Berg, W.: Uebtr Fett- und Pigmenteinschlsse in denLeberzellkern des Menschen. Ihre Entstehung und ihreAusschei.dung. Z. miKr.-ana . Forsch. 8, 644-F.65 (19>5).

6. Caza] , P. et irouze, J. : Le. noyauz va'uolaire.Press. Med. 59, 571-57 11951).

7. Cowdry, E. V. : The problem of intranuclea, inclusionsin viru< diseases. Ar .h Path. 18, 5:7-542 ( 1 n);4).

9. Cowdry, E. V., luca:;, A. M. and Fox, H .: Di:t.ributionli*Ucllesr inclusions inL wild :nimuils. Am. J. PFth. 11,,7--52 i<)

. P:id, H. : Die I,(-. Akademie-Verlcg. Berlin, 1964.10. De ve, C. : Lysos:omes, a new group of cytoplasmic

;<arti les in Subc.ellular Particles. 1. 1, 8. Hayash , T.ed. ), iionild Press Co. , New York, 195'.

11. De-he, G., Heine, U., Sommer, J. R., Arvy, L.,Beard,P. and Beard , J. W. : Jltr'structural characters of the thymus. myclobIistosi s and of the adenoinetriphosphatase activity

-nthyr cells and associated virus. , Na,. Cancer Inst.~O, 1.-4% (1965)

1.. Ep'tc inl l A. and Holt, S. J. : Electron microscopeobservations on the surface adenosine triphosphatase-likeenzymos of IeLa cells infecte:! with herpes virus. J. Cell

i . Findlay, G. M. : Intranuclear bodies in the liver-cellsof mice. Brit. J. Ex,. Path. 1<, 1 2 e?-c ( ..•oa nula poes J. -, 1

14. Gall, J. G. : Octagonal nuclear pores. J. Celi Biol.: 591-400 (1967).

15. Gueft, F. and Kikkawa, Y.: The periodic structure ofnuclear and cytoplasmic crystals of dog liver aells. FifthInt. Congr. Electron Miicroscopy. Vol. 2, p. T-5 (Philadelphia).Academic Press, New York, 1962.

16. Hay, E. D. and Revel, J. P.: The fine structure of theDNP component of the nucleus. An-electron microscopic studyutilizing autoradiography to localize DNA synthesis. J. CellBiol. 16, 29-51 (1965).

17. Himes, N. and Pollister, A. W.: An electron microscopicstudy of intianuclear glycogen~ inclusion in tadpole livers.Anat. Rec. 132, 453-454 (1958).

18. KleinfeV- R. G., Greider, N. H. and Frajola, W. J.:Electron microbcopy of intranuclear inclusions found inntunan and rat liver parenchymal cells, J.? Biophysic. andBiochem. Cytol. 2, No. 4, quppl. 435-438 (1956).

19. Kleinfeld, R. G. and Koulish, S.; Cytological aspectsof mouse and rat liver containing nuclear inclusions. Anat.Rec. 128, 433-445 (1957).

20. Kleinfeld, R. G. and Lessler, N. A.: Progressive intra-cellular changes induced in rat liver by thtoacetamide admi-nistration. Am. J. Phyo~ol. 179, 651 (1955).

21. Leduc, E. H. and W-lsoh, J. W.: A histochemical st-2dy ofintranuclear inclusions in mouse liver and hepatom. J. Histo-chem. Cytochem. 7, 8-16 (1959).

22. Leduc, E. H. and Wilson, j. W.: An electron microscopestudy of intranuclear inclusions in mouse liver and hepatomaJ. Biophysic. and Biochem. Cytol. 6, 427-450 (1959).

23. Luft, J. H.: Improvements in epoxy resin embeddinclmethods. J Biophysic. and Biochemn. Cytol. 9, 409-414 1l961).

24. NWel, R. et Drouin, N.: Modificati~ns nucl6airesrAversibles apparues dans la cellule h6patique apr~s l'actionde ccrtains antithyroidiens de s7rnth6se. Compt. rend. Assoc.Anat. 59 1-5 (1952).

25. Olino, S. and Kinosita, R.: Morphology of intranuclearinclusions in liver cells infected with contagious caninehepatitis. Exptl. Cell Ries. 7, 578-580 (19545.

26. Olitsky, P. K. and Casals, J.: Certain affections ofthe liver that arise spontaneously in so-called normal stockalbino mice. Proc. Soc. Exp. B~iol. Med. 60, 148-51 (1945).

27. Rather, L. J. : Experimental alteration of nuclear andcytoplasmic components of' the liver cell with thioacetamide.I. Early onset and rev&ersolbility of volume changes of thenucleolus, nucleus and cytoplasm. Bull. johns HopkinsHosp. 88, 38-.58 (1951).

P8. Reynolds-, E. S.: The use of lead citrate at high pHas an e1l.ctron-opaque atain in electron microscopy. J.Cell Biol. 17, 208-212 (1963).

2.9. Richt t,'' W. R., Stein, R. J., Rdzok,E. J., Noise, S.M. and bischoff, N. B,: Ultrastructural studies of intra-nuclear crystalline inclusions in the liver of the dog.Am. J. Path. 47, 587-599 (1965).

G, Rubarth, S.: An acute virus disease with liver lesionsin dogs (Hepatitis contagiosa canis). Acta Path. Nicrobiol.Scaind. Suppl. 69, 1-222 (l1IV).

-9-

31. Sabatini. D. D).. Bensch, K. and Barrnett, R. J.: Cyto-chemi.-try anid electron miicroscopy. The preserva3tions ofcellular ultrastructure and enzymatic actUivity by aldehydef4.xation. J. Cell Biol. 17, "19-58 (1963).

32. Steiner, J. W., Jhz~quei, A.-M., Philips, M. J., Niyai,K. and Arakawa, K.: Some aspects of the ultrastructuralpathology of liver, in Progress in Liver Diseases. Vol. 2,

p. 05-72,Popper, H. and Schaffner, F (eds.), Grune &-Stratton, New York anid London, 1965.

55. Tajima, M. and Motohashi, T.: Electron micrographs ofintranuclear inclusions in hepatic cells of dogs withi-nfectious canine hepatitis. Am. J. Viet. Res. 19, 666-674(1958).

34. Thompson, S. W., Cook, J. E. and Hoeyr, H.: Histochemicalstudies of acilophilic crystalline intranuclear inclusionsin the liver and kidney of dogs. Am. J. Path. 35, 607-623(1959).

35. Thompson. S. W., Wiegand, R. G., Thomassen, R?. W.,Harrison, M. and Thlrbyfill, C. T-. : Am. J. Path. 55, 1105-1115 (1959).

6.Wachstein, M. and Meisel, E.: Histochemistry of hepaticphosphatases at a physiologic pH. Am. J. lin. Path. 27,153-257 (1957).

37. Watson, M. L.: Staining of tissuie sections for electronmicroscopy with heavy metals. J. Biophysic. and Biochem.Cytol. 4, 475-478 (19518).

58. Weatherford, H. L. and Trimble, H. C.: A further mor-phological and biochemical study on the intranu,:Ilear cry-stals in the hepatic cells of the dog--tne pure breed andhybrid flalmatGion. Anat. Rec. 77, 487-507 (1940).

39. Wegelin, C.: tiber des Vorkommen von Fett in Leber-zelikernen. Verh. Dtsc-h. Ge. Path. 23, 519-522 (1928).

40. Wilson, J. W.: Nuclear inclusions in the mouse liver.Anat. Rec, 118, 368 (1954).

41. Yasuzumi, G., Nakai, Y., Tsubo, I., Yasuda, M. andSugioka, T.: The fine structure of nuclei as revealed byelectron microscopy. TV. The intranuclear inclusion forma-tion in Leydig cells of aging human testis. Eyptl. CellRes. 45, 261-276 (1967).

42. Yasuzumi, G., Sawada,T., Su ,ihara, R., Kiriyama, N.and Sugioka, M.: Electron microscope researches on theultrastructure of nucleoli in animal tissues. Z. Zellforsch.48, 10-23 (1958).

43. Yasuzuni, G. and Tsubo, I.: The fine structure of nucleias revealed by electron microscopy. III. Adnosine triphosphatase-activity in the pores of nuclear envelope of mouse choroidplexus epithelial c, lls. Exptl. Cell Res. 43, 281-292 (1966).

44. Yasuzumi, G. and Tsubo, T.: Analysis of the developmentof Japanese B encephalitis (JBE) virus. III. Electron micro-scope studies on inclusion bodies appearing in neurons andmicroglial cells infected with JBE virus. J. Ultrastruct.Res. 12, 317-327 (1965).

-10-

7. Explanation of Figures

Fig. I.- Dense particles approximately 200 A in diameterisolated or in clusters are visible in the vesicles or in thecytoplasmic matrix surrounded by a triple-layered unit mem-brane (arrows). X 126,000.

Fig. 2. Dense particles 200 A in diameter appear in thedeveloping fibroblast. I:IMyelin-figure. X 90,000.

Fig. 3. Electron micrograph of a hepatic parenchymal cellnucleus, showing an intranuclear inclusion and a large nucle-olus. The pores (arrows) are visible in the nuclear envelopeas well as in the membrane surrounding the inclusion. Thechromatin elements appear attached closely to the outerlimiting membrane of the inclusion. X 34,000.

Fig. 4. An intranuclear inclusion contains several vesiclesof different sizes and shapes, and mitochondrion (M) whichseems to be in a stage of breakdown.. A particles 200 A indiameter is visible in a small vesicle (arrow). The pores (P)may be seen in the limiting membrane of the inclusion. Theperichromatin granules (PG) can be seen in the chromatinmass attached to the outer limiting membrane of the inclusion.

X 75,000.Fig. 5. Part of the nucleus of the hepatic parenchymal

cell incubated with ATP. The roughly round-shaped nucleolusconsists of tangled nucleolonemata. The ATPase reactiondeposits (arrows) occur in the pores of the nuclear envelope.

X 51,000.Fig. 6. Tangential section through the nuclear envelope

of the hepatic parenchymal cell incubated with ATP. ATPasereaction product occurs as fine granular deposits on thenuclear envelope and as more dense globular deposits 460 ,560 A in diameter in the pores of nuclear envelope. The lessdense deposits (arrows) appear in a radial appearance aroundthe dense precipitate. X'!95,000.

Fig. 7. Part of the nucleus of the hepatic parenchymal cellincubated with ATP, showing a large nucleolus and .n iclusionin the nucleus. The ATPase reaction deposits (arrows) occurin the pores of the nuclear envelope as well as on the surfaceof a vacuole occupying the most part of the inclusion. Theprecipitate (P) occurs also in a small amount on the membranesurrounding the inclusion. The peripheral part of the inclu-sion consists of the elongated granular or agranular endoplas-mic reticulum. X 45,000.

Fig. 8. An iiclusioi body in the hepatic parenchymal cellnucleus incubated with ATP. The ATPase reaction depositsoccur on the membrane surrounding the inclusion, and remarka-bly at a point marked by P. Most of the contents in the±nclusion are in a stage of breadown, though a degeneratingmwcrobody (MB) and the endo 'asmic reticulum are found.Dense particles 140 to 200 A in diameter (arrows) can beseen in small vesicles. A homogeneously dense body (L) maybe lipid body. X 56,000.

-11-

Fig. 9. Part of the nucleus containing a guiter-shapedinclusion body in a hepatic parenchymal cell incubated withATP. The nuclear pores show clearly the ATPase reactiondeposits, but the membrane surrounding the inclusion depictsa smdl] quantity of the deposits in its pore-like spaces(arrows). Numerous degenerating lysosome-like bodies orvesicular structures can be seen within the inclusion.

X 23,000.Fig. 10. An enlarged electron micrograph of a part of

Fig. 10. The ATPase reaction deposits occur in a smallamount in the pore-like spaces (large arrows) of the limit-ing membrane of the inclusion. Note that the deposits arefound at the periphery of the vesicular structures or thelysosome-lke bodies (small arrows). Dense particles 1'40-200 A in diameter (P) appear in association with the ATPasereaction product. X 90,000.

-12-

I<1

40

I

4

Deparment of atc coilsiie

Final Report No'. J , July 1966 -July 1967%. uT;4-6At(RQ7Vmktm a mdxi, 1.f &a um)

Yasuzumi, Gonpachiro

6. MNRPOOP bAYK?1 OA N.O A1 7&w.o ag

October 1967 20 44_____

C ONt 5%A CT 0 S4ft ZI WNO . 90. OWGINATOW111 MWPOMT %uU~ftl3I9

-92-.557 E-8436otiffROet I no. J-203-.12

2NO14501BWlD*Task 06. 4 T~"af Im ojwpi-1 (;6Wg' @6ev mebeft ".. MW Q*a*#

00 035FE10. W1TUOTO STAYEM6N4T

This document ha~s been approved for public release and sale; its distribution isunlimited.

If- SUFLCURITAM- mova is.APON01H Sn ITX Frnic 96343U.S. Army R&D Group (Far East)

In 26 cas~s of serum hepatitib from the cbservationa of 80 cases of serum.Ipaitib and infectious e~~t~ ,the dense virusa-iike particles approximately

,WC A in diameter, lsolatji or- in clusters, were observed in the cytoplasm off thehepatIc parenchymsi. calls andi of the developing fibroblasts. Moreover, the intra-nuclear inclusion bodiez, fo.,Ted by invagination of the nuclear envelope were roundi.n sv"ra~l hepatic parenThy'Mal e.ls in cases of serumn hepatitis. Many of theinclusion bocles w--re revealed to contain dense particles which were similar orsmiller a& compared with the~ virus-like paiticlss appearing in the cytoplasm. Thedense particles within the iniclusion seeimed to appear associated with tL ! ATPasereacticn product. (Author)

DO .466"N 473 sePaS~~1f Unclassified

Security ClavolfieaiIon

1 IV !

Hepaum iet i

L i v~e r

Pt omlc~

e%L, .~ny ]a- 1,Crs

M-Ca

71I