THE CANADIAN MEDICAL ASSOCIATION

JOURNAL :vsOAMAIDHNSIB

DECEMBER 23, 1961 * VOL. 85, NO. 26

EXPERIMENTAL IMMUNOLOGICLIVER INJURY AND THE CONCEPTOF AUTODESTRUCTION*

(PART I)

JAN W. STEINER, M.D.,JOHN S. CARRUTHERS, M.D.,

REUBEN BAUMALt andSLIMEN R. KALIFAT, M.D.,: Toronto

IN PREvious papers we examined the clinical,"', 62

immunologic and morphologic60 evidence for andagainst a concept of autoimmunization as a me-

chanism of tissue injury. We concluded that therewas ample clinical and immunologic evidence thatsuch a process may play a role in the initiationand/or perpetuation of some disease processes. Wecame also to the conclusion that pathologic evi-dence of autodestructiveness or autoclasia was atpresent lacking, despite the availability of fairlyconvincing experimental models.

Investigations of immunologic tissue injury em-

ploy various methods for the induction of lesions:non-specific immune systems (e.g. injections offoreign proteins), heteroimmune anti-tissue anti-bodies (e.g. injections of rats with rabbit-anti-ratkidney antisera), isoimmune systems (e.g. injec-tions of rabbits with rabbit tissue homogenates)and finally autoimmunizing procedures (e.g. in-jections of guinea pigs with a homogenate of one

of their own adrenals). The lesions which resultfrom these procedures are thought to be due eitherto circulating antibodies (immediate, Arthus or

anaphylactic type hypersensitivity reactions) or to"cell-bound", non-circulating antibodies or to cellsof the reticuloendothelial system (tuberculin, de-layed or cellular-type hypersensitivity reactions).Evidence is now available3, 4, 23, 54, 64, 65 that thesetwo types of fundamental immunologic responses(i.e. classical antibody production and the tuber-culin response) and their corresponding hyper-sensitivity reactions overlap. Thus a more unitarian

*From the Department of Pathology, University of Toronto,Toronto. Original work reported in this paper has beensupported by a grant-in-aid of the Medical Research Councilof Canada (MA-785).tThird-Year Medical Student, University of Toronto, Recipientof a Summer Research Scholarship of The Medical ResearchCouncil of Canada.tMedical Research Fellow of The Canadian Arthritis andRheumatismn Society.

concept of immunologic tissue injury is graduallyemerging. It is being realized that the factorswhich determine the character of immunologiclesions may be quantitatively rather than quali-tatively different.Autoimmunity lies at the crossroads between the

classical antibody and the tuberculin responses.

The former is emulated by the development ofcirculating autoantibodies, and the latter by our

ability to transfer the response to normal recip-ients by means of 'lymphoid" cells.48 The characterof suspected autoclastic lesions is reminiscent ofthe tuberculin-type hypersensitivity reaction.69

In the light of the above considerations, investi-gations of experimental autodestructive tissuelesions can no longer be analyzed as isolated phe-nomena; the evidence must be integrated into thescheme 'of immunologic tissue injury in general.Organs are capable of only a limited response to

injury. Therefore, when assessing the significanceof a given alteration, changes produced by otherthan immunologic means must be examined andthe specificity of the alleged allergic tissue injuryconfirmed, denied or left in abeyance.Autoimmune mechanisms in liver disease have

acquired the quality of a popular fad in clinicaland pathologic investigations. The purpose of thesepapers is to review critically the available mor-

phologic evidence of experimental immunologicliver injury and to assess its specificity. The rele-vance of the findings to the concept of autode-structive liver disease will be examined.

THE PURPOSE OF INVESTIGATIONS OF ALLERGICLIVER INJURYThe liver occupies a unique, strategically impor-

tant position in the organism from an immuno-pathologic point of view.

1. It contains components of the reticuloendo-thelial (RE) system and therefore plays a role inthe mechanisms concerned with the metabolism ofantigens. It may, under pathologic circumstances,elaborate antibody.12

2. Antigens may reach the liver either throughthe portal or systemic circulation and persist therefor many months or years.9

3. By virtue of its dual blood supply it may beexposed to circulating antibodies arising in the RE

1369

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VA000CUAT)T0N bff IDUCAILIR

1370 STEINER AND OTHERS: IMMUNOLOGIC LIVER INJURY

tissues of the gastrointestinal tract as well as toothers produced by the RE system at large (e.g.spleen, lymph nodes).

4. The liver is concerned with detoxification.Noxious substances produced in the course of im-munologic reactions throughout the organism mayreach the liver for catabolism and disposal.

5. The liver produces most of the circulatingplasma proteins.46 Any failure on the part of theliver to discharge this function adequately mayrelease into the circulation proteins which are po-tentially "foreign", i.e. antigenic.

It is therefore reasonable to assume that theliver, concerned as it is with vital immunologicfunctions of the organism, should be protected insome special way from injury resulting from im-munologic happenings. Being a reticuloendothelialas well as an epithelial organ containing connec-tive tissue, its response to allergic injury shouldbe more complex than that of other organs be-longing to one or other of these groups. Theseconsiderations and speculations have stimulatedmany investigators to explore the response of theliver to various immunologic stimuli.The more immediate aims of such investigations

and the problems which required elucidation fallinto two groups: (a) To establish the morphologiccriteria of the response of the liver to immunologichappenings in general and to learn to recognizethe features of allergic inflammation in particular.(b) To explain the role of immunologic processesin the causation and/or perpetuation of some dis-eases of the liver. The first of these problems willbe discussed in detail later; the second will bereferred to briefly at this stage.

THE PossIBmE RoLE OF ALLERGIC PROCESSES INHUMAN LIVER DISEASE

The interest in this subject is largely of recentorigin. This may be gauged from a perusal ofPopper and Schaffner's book of pathology of theliver, published in 1957.49 Less than a page at thevery end of the text is devoted to hypersensitivityin a volume of 777 pages.We may trace the resurgence of recent interest

in the subject to several events:1. The realization of the mystery of the cau-

sation and/or perpetuation of some liver diseases,e.g. active chronic hepatitis,40 portal cirrhosis5'and piimary biliary cirrhosis.26

2. The emergence of lupoid hepatitis as a sepa-rate and distinct entity.33 36,09

3. The formulation of the concept of self-per-petuation in liver disease.41

4. The characterization of anti-liver autoanti-bodies.7' 15, 20-22, 32, 37, 38, 53, 55, 56, 66

5. Drug-induced liver injury which is thoughtto have an allergic etiology.30' 31, 47, 50

6. The realization that granulomatous lesions ofthe liver may be caused by allergic processes.24' 25

Canad. M. A. J.Dec. iz, 1961, vol. 85

The clinical problems have been adequatelyreviewed by Goldgraber and Kirsner,25 Vorlaen-der,67 Mackay and Wood4' and by Mackay.40 Someaspects of the matter were also explored by Ket-tler34 and by ourselves.62The pathologic aspects of suspected human

autoimmune liver disease have been reviewed byPopper.5'

EXPERIMENTAL IMMUNOLOGIC LIVER INJURYInjury by non-specific antigen-antibody systemsThe designation "non-specific" indicates that

neither the antigens nor their corresponding anti-bodies possess any known affinity for liver tissue.Experimental prototypes of this injury serve todelineate the criteria of the immediate-type(anaphylactic, serum sickness, Arthus-type) al-lergic tissue injury. Anaphylaxis per se will notbe included in the discussion, since it contributeslittle to the understanding of the morphologic fea-tures of the response of the liver to allergic stim-uli. (This subject has been adequately reviewedby others.)25 34

These experiments fall into four distinct groups:(1) The production of classical Arthus lesions

where the antigen and antibody find themselvestheoretically on opposite sides of a vascular bar-rier.

(2) The production of serum-sickness-type le-sions probably resulting from the interaction invivo of antigen and antibody in the hepatic cir-culation.

(3) The production of serum-sickness-typelesions by the injection into the portal circulationof in vitro preformed complexes of antigen andantibody (at equivalence or in antigen excess).

(4) The production of serum-sickness-typelesions by the systemic venous injection of in vitropreformed complexes of antigen and antibodyconstituted in antigen excess.

1. Classical Arthus lesionsUsing horse serum as antigen, Choi'0 and Al-

lessio' hyperimmunized rabbits and then injecteda challenging dose of the specific antigen directlyinto the liver parenchyma. The rapidly developinglesion consisted of a hemorrhagic necrosis associ-ated with an allergic inflammatory reaction inwhich eosinophils'0 and plasma cells' were prom-inent.

2. In vivo interaction of antigen and antibodyin the hepatic circulation

As early as 1894, Flexner'8 noted hepaticnecroses in some rabbits after repeated intra-venous injections of horse serum, and Wells7' re-corded in 1903 that repeated administration ofpeptone in these animals led to the developmentof cirrhosis.

Canad ML A JDec. 25,19G;1, vol. s3 STEINER AND OTHERS: IMMUNOLOGIC LIVER INJURY 1371

Fig. 1.-Rabbit. Three months' administration of 10 mg. htiman gai-ma globulin everysecond day. Mesenchymnal cell proliferation in portal tract and infiltration of lining of bile duetby lymphoid cells. HPS, X 305. Fig. 2.-Same procedure as 'ig. 1. Exteni,4ve mesenchymnal celleggregates in periphery of lobule formiinlg a lynmphoid follicle. HIenatox lln and eosinstain, X 206.

Longcope3 undertooks an extensive investigationof the role of a complex (horse serum) and of aless complex antigen (egg albumin) in the de-velopment of hepatic lesions in guinea pigs, rab-bits, cats and dogs. The antigens were adminis-tered intravenously, intraperitoneally, subcutane-ously and by variotus combinations of these routes.Nearly all animals developed focal liver necrosescharacterized by an intense eosinophilia of theaffected cells and by karyolysis ("eosinophilicnecrosis"). Diffuse mesenchymal cell infiltrationwas present in the liver, but there was only ascanty cellular reaction in the margins of the ne-crotic lesions. The sinusoids between necrotic cellswere widely patent. Rabbits and some cats de-veloped a finely nodular "cirrhosis" characterized byan overgrowth of dense connective tissue. Thisdid not develop in dogs or guinea pigs. Similarresults were obtained when foreign sera were ad-ministered repeatedly intraperitoneally into guineapigs.'13

Hartley and Lushbaugh27 hyperimmunizedguinea pigs and rabbits with egg albumin andthen injected a challenging dose of the antigeninto radicles of the portal vein. A similar experi-ment using horse and porcine serum was carriedout by Watjen.70 The resulting eosinophilic ne-

c:oses were absent from centrolobular areas andwvere found in the periphery and midzones oflobules."7 Most of the lesIons were repaired with-ouit trace in 10 days btut a few animals developedfibrosis. In these experiments thrombosis of sinu-soids was noted in and around areas of necrosis.These and the accompanying polymorphonuclearreaction were interpreted as happenings secondaryto necrosis.Not all antigens produced liver injury. For ex-

ample, a single injection of whole bovine serumor of bovine serum albumin led to no discernibleliver injury in rabbits."9 On the other hand, asingle injection of bovine gamma globulin pro-duced focal parenchymal necroses and prolifer-ation of monocytes and lymphocytes beneath theendothelium of sinusoids in centrolobular areas.Such lesions could only be found before the 14thday after injection.2"9

Epstein1 characterized the cells which aggre-gate in the liver of hyperimmune rabbits whenNvhole porcine serum was used as antigen. Herecognized the following: (a) large basophilicrouLnd cells, (b) macro-histiocytic cells, (c)pseudo-eosinophilic leukocytes, (d) histiocyticgiant cells and (e) cells resembling megakaryo-cytes. Since precipitin tests in these animals in-

1372 STEINER AND OTHERS: IMMUNOLOGIC LIVER INJURY

Fig. 3.-Rabbit. Anaphylactic shock. The densely eosino-philic material almost occluding the lumen of the centralvein has been shown to consist of precipitated complexesof antigen and antibody. Note aggluttinated red cells insinusoids. These are matted together by the same material.Azur eosin, X 270.

dicated good antibody production, Epstein con-

cluded that the large basophilic round cells, whichhe thought to be derived from histiocytes of theliver, were the morphologic expression of antibodyproduction. Ehrich'6 subsequently identified thesecells in Epstein's drawings as belonging to the"lymphoid-plasma cellular series".

Although necroses and cellular infiltrations were

the hallmark of this form of liver injury whenforeign proteins as well as bacteria8 or bacterialtoxins19 were injected, some reports indicated thatoccasionally the injury was restricted to degener-ative phenomena of parenchymal liver cells. Forexample, some found fatty changes and vacuolardegeneration in rabbits and guinea pigs repeatedlyimmunized with egg albumin.14 Others noted thathorses receiving repeated injections of diphtheria,botulinum or tetanus toxins respectively developedfatty metamorphosis.68Our own experiments59 have shown that injec-

tions of small doses of a foreign antigen (bovinegamma globulin 10 mg. every other day for up

to three months) produced necrosis in rabbits onlyrarely but led to massive infiltrations of mesen-

chymal cells in lobules and portal tracts (Fig. 2).Segmental arteritic lesions were noted in branches

Canad. M. A. J.Dec. 23, 1961, vol. 85

of the hepatic artery, and in all animals mesenchy-mal cells were seen in the epithelial lining of bileducts (Fig. 1).The interpretation of the pathogenesis of the

lesions varied:(i) It was suggested by some that the necroses

were ischemic in nature, owing to the formationof intravascular hyalin thrombi8' 19 or to other dis-turbances of circulation.2

(ii) Ischemia was not considered to be thecause of the necroses by others, since the sinu-soids were usually empty or, if thrombosed, theprocess was considered to be secondary to cellinjury.27' 35

(iii) Others thought that the lesions resultedfrom the interaction of liver-cell-bound antibodywith circulating antigen.27

(iv) The interaction of antigen and antibodyin the circulation was implicated.29 3

Support for the allergic etiology of the hepaticlesions was claimed, since it was found that wash-ing through the isolated liver of hyperimmunizedanimals with fluid containing the specific antigen,produced a considerable yield of heparin and his-tamine in the suspending medium.57 Further proofwas forthcoming since it was found that the in-jected antigen (S35-labelled sulfanilic azo-ovalbu-min or I131-labelled ovalbumin) would persist inthe liver for many months and in large quanti-ties,9' 28 thus being available for interaction withantibody.

It is worth recording that, with the exceptionof the experiments of Hawn and Janeway,29 par-enchymal liver lesions were not mentioned in anyof the myriad of well-documented investigationsof serum sickness, despite the use of many andvaried antigens. We are obliged to conclude thateither they were not particularly searched for orthat the liver parenchyma occupies some specialposition in the organism when systemic im-munologic happenings affect almost all other tis-sues including the spleen and lymph nodes.52 Thelatter interpretation seems more likely in view ofthe thoroughness with which these investigationshave been pursued.

3. The effect of in vitro prepared antigen-anti-body complexes injected via the portal vein

Three observations prompted further investiga-tions of the immediate-type allergic liver injury:

(i) In rabbits anaphylactic shock is at timesthe result of intravascular precipitation of antigenand antibody.43 4 Such precipitates are found inthe hepatic sinusoids in addition to other sites(Fig. 3).

(ii) Complexes of antigen and antibody formedin vitro in the presence of moderate antigen excess(as compared with the amount of antigen presentat equivalence in the precipitin reaction) possessa phlogistic potential. They are capable of initi-ating an Arthus-type lesion in the skin of rabbits,1"

Canad. M. A. J.Dec. 23, 1961, vol. 85 STEINER AND OTHERS: IMMUNOLOGIC LIVER INJURY 1373

Fig. 4.-Rabbit. Portal vein injection of preformed precipitated comiiplexes of anitigen andaitibody (boN-ine gaiunna globulin-rabbit ainti-bouvine gaiii,iia globulin)-24 hoturs. Focal miiid-zonial area of celltular infiltrationi. Azur eosin, X 220. Fig. 5.-Same procedture as Fig. 4-48 houirs.Extensix e imiidzonial coagulative niecrosis with a relatively sli,ht l eactioii in its periphery.Azuii eoshi, X 130.

serum sickness in mice45 and acute glomerulone-phritis in rats.5

(iii) The liver differs histologically from allother organs in the body. Electron microscopicobservations have shown that parenchymal livercells are in direct contact with the bloodstreamWithout an intervening continuous endothelium ora basement membrane.(13 Thus antigen-antibodyreactions within the sinusoidal circulation wouldexert their effects not only on Kupffer cells butalso directly on parenchymal liver cells.63

In all previous investigations in which hepaticnecroses were produced by the injection of foreignproteins, the relative proportions of antigen andantibody interacting in the hepatic circulationw.ere unknown. Wlhen precipitates, formed by theinteraction of antigen and antibody in vitro, wereinjected into the portal circulation of rabbits, 44necroses resulted wvhich were identical with thoseproduced in some of the previous experiments(Figs. 4 and 5). The precipitates were found byimmunohistochemical techniques to occlude radi-cles of the portal vein and sinusoids within lo-bules. Thus it was suggested that the variousprototypes of allergic liver necrosis, which wehave previously analyzed, were caused, at least inthe rabbit, by ischemia due to intravascular pre-

cipitation. This was in no way surprising, sinceit was known that particulate material injectedinto the portal vein could produce similar necroticlesions.42'Our own investigations63 have shown that

identical midzonal necrotic lesions (Figs. 6, 7 and8) could be produced by the portal vein injectionof p,reformed soluble antigen-antibody complexesconstituted in antigen excess, which are knowvnto possess a phlogistic potential.7 11 i5 When suchcomplexes were tagged with a fluorescent dye,they Nvere found as precipitates within sinusoidsat the site of the necrotic lesion. It was suggestedthat this precipitation was the result of antigenescape and equilibration of antigen and antibodyat this site. Electron microscopic examination ofthese lesions revealed that the earliest injury tookthe form of edema of the sinusoidal endotheliumand the surface microvilli of liver cells where theseare in contact with the bloodstream (in the spaceof Disse). Thus the conclusion was arrived at thatthe prirnary injury was confined to the blood-liverbarrier and that both thrombosis and parenchymalnecrosis were secondary phenomena. The lattercould result from interference with the diffusionof metabolites across an edematous blood-liverbarrier.

1374 STEINER AND OTHERS: IMMUNOLOGIC LIVER INJURY Canad. M. A. 3.Dec. 23, 1961, vol. 85

g...x ..bt

I*1 ijj>e Sj> R*bj FF9 s G a Fig. 6.-Rabbit. Portal vein injection of soluble atntigein-antibody complexes (human serum albumin-rabbit anti-human albumin) formed in vitro in 8X antigen excess-24 hours. Focal area of peripheral necrosis. Note sparingof liver plate. Hematoxylin and eosin, X 305.

l _ | I _ s Rr.R.N¢ .~J Fig. 7.-Same procedure as Fig. 6-24 hours. Edge ofarea of coagulative necrosis of parenchymal cells. Kupffer_11u.3e _';_7-............._cellsremain intact at this time. Note scanty predomi-nantly mononuclear reaction in margin. Azur eosin, X 290.

Fig. 8.-Same procedure as Fig. 6-14 days. A septumnof connective tissue joining two portal areas betweennecrotic lobules. Progression to fibrosis is found onlyrarely. Azur eosin, X 150.

Fig. 9.-Mouse. Three injections into tail veinis ofsoluble immune complexes (human gamma globulin-rabbit anti-human globulin) in a period of 24 hours.O ~~~~~~~~~~Sacrifice 24 hours after last injection. Marked aggregation- of mesenchymal cells (many of them plasma cells) in

A, portal areas. Hematoxylin and eosin, X 195.

,^_,Fs,t&t 4* w L > Fig. 10.-Same procedure as Fig. 9. Sacrifice 48 hoursa4W ~~~~~~after last injection. Accumulation of mesenchymal cells4;^ ;j around central vein. Note acidophilic (dark) degeneration

of some cells and presence of mesenchymal cell aggregatesin sinusoids. Hematoxylin and eosin, X 305.

Fig. 11.-Same procedure as Fig. 9. Sacrifice 24 hours, l after last injection. Eosinophilic coagulative necrosis intwo areas adjacent to central vein. Note scanty cellularreaction. Hematoxylin and eosin. X 270.

Fig. 12.-Same procedure as Fig. 9. Sacriflce 48 hoursafter last injection. Necrosis becoming almost liquefactivein character with intense cellular reaction. This type otlesion is seen rarely. Hematoxylin and eosin, X 305.

Canad. M. A. J.Dec. 23, 1961, vol. 85 STEINER AND OTHERS: IMMUNOLOGIC LIVER 'INJURY 1375

tr4 jK

* At '.

.. # 1

Figs. 9, 10, 11 and 12.-Legends on opposite page.

. .: *-4

1376 STEINER AND OTHERS: IMMUNOLOGIC LIVER INJURY Canad. M. A. J.Dec. 23, 1961, vol. 85

4. The effect of in vitro prepared antigen-antibody complexes injected systemically

Studies of the effects of systemically adminis-tered antigen-antibody complexes formed in anti-gen excess in vitro5' 45 did not refer to the pres-ence of lesions in the liver of mice or rats.Recent studies carried out in our laboratory9

show that mice injected with non-specific, solubleantigen-antibody complexes (human gamma glob-ulin - rabbit antihuman gamma globulin) formedin 8X antigen excess develop hepatic lesions (Figs.9, 10, 11, and 12). Three doses of such aggregateswere injected intravenously in the space of 24hours and the animals sacrificed 24 and 48 hoursrespectively after the last injection. Necrotic le-sions (Figs. 11 and 12) and cellular infiltrations(Figs. 9 and 10) were in no way different fromthose produced by portal vein injection of suchcomplexes.;3 However, instead of being localizedin the midzones or periphery of lobules, the necro-tic foci were predominantly centrolobular in dis-tribution (Fig. 11).

CONCLUSIONS

1. The hallmarks of experimental allergic liverinjury of the immediate or Arthus-type are:

(a) A mesenchymal reaction, particularly inlobules, with prominence of plasma cells.

(b) Edema of the components of the blood-liver barrier with distension of the space of Disse.

(c) Degenerative parenchymal changes includ-ing fatty metamorphosis and vacuolar degenera-tion culminating at times in

(d) Eosinophilic (focal, midzonal or peripherallobular) necrosis.

(e) Hepatic fibrosis may rarely follow this in-jury in some species.

These lesions either jointly or seveerally are nIotspecific.

2. The vasculitis occurring in the radicles of thehepatic artery differs in no way from the arteriallesions of serum sickness elsewhere.

3. By whatever route antigens reach the liver(via the portal or systemic circulation), their in-teraction in hepatic sinusoids with their homo-logous antibody may lead to the development ofsuch lesions. Not all antigens, hoNvever, are cap-able of evoking these changes.

4. The mechanism of the injury may be de-pendent in rabbits upon the formation of intra-vascular immune precipitates causing vascularocclusion and ischemia. In other species, as wellas in rabbits, the injury may also result from theformation of intravascular, phlogistic soluble anti-gen-antibody complexes in the presence of antigenexcess. Necrosis may then be the result either ofdirect toxicity of such immune aggregates or itmay be secondary to interference with the move-ment of metabolites across the damaged blood-liver barrier.

(To be continued)

REFERENCES

1. ALESSIO, F.: Pathologica, 21: 121, 1929.2. APITZ, K.: Arch. path. Anat., 289: 46, 1933.3. BENACERRAF, B. AND GELL, P. G. H.: Immunology, 2: 53,

1959.4. Idem: Ibid., 2: 219, 1959.5. BENACERRAF, B. et al.: J. Expe6-. .lIed., 111: 195, 1960.6. BENNETT, H. S., LUFT, J. H. AND HAMPTON, J. C.: Amiz.

J. Physiol., 196: 381, 1959.7. BJORNEBOE, M. AND KRAG, P.: Acta path. microbiol. sca nd-

inav., 24: 352, 1947.8. BOXMEYER, C. H.: J. Med. Res., 9: 146, 1903.9. CAMPBELL, D. H. AND GARVEY, J. S.: Interaat. Arch.

Allergy, 12: 70, 1958.10. CHoI, C. Y.: Tr. Jap. Path. Soc., 20: 587, 1930.11. COCHRIANE, C. G. AND WEIGLE, W. O.: J. Exper. Med., 108:

591, 1958.12. COHEN, S. et al.: Ibid. 111: 285, 1960.13. CORNIL, L. AND MOSINGER, AI.: C. R. Soc. Biol. (Par.),

121: 61, 1936.14. CROIZAT, P.: J. m4ed. Lyon., 10: 803, 1929.15. EATON, M. D., MORPHY, NV. D. AND HANFORD, V. L.:

J. Exper. Med.., 79: 539, 1944.16. EHRICH, W. E.: J.A.M.A., 135: 94, 1947.17. EPSTEIN, E.: Arch. path. Anat., 273: 89, 1929.18. FLEXNER, S.: Med. Nevs, 65: 116, 1894.19. Idem: J. Med. Res., 8: 316, 1902.20. GAJDIJSEK, D. C.: Nature, 179: 666, 1957.21. Idemn: Arch. Int. Med., 101: 9, 1958.22. GEAR, J.: Tr. Roy. Soc. Trop. Med. & Hyg., 39: 301, 1946.23. GELL, P. G. H. AND HINDE, I. T.: Inttervat. Arch. Aller-gy,

5: 23, 1954.24. GOLDGRABER, M. B., KIRSNER, J. B. AND RASKIN, H. F.:

Arch. Int. Med., 102: 10, 1958.25. GOLDGRABER, M. B. AND KIRSNER., J. B.: Am. J. Ml. Sc..

241: 109, 1961.26. HAMILTON, J. D.: Lab. Invest., 8: 7-01, 1959.27. HARTLEY, G., JR. AND LuSHBAUGIT, C. C.: Am. J. Path..

18: 323, 1942.28. HAUROWITZ, F. AND CRAMPTON, C. F.: J. Immunol., 68:

73, 1952.29. HAWN, C. V. AND JANEWAY, C. A.: J. Exper. Med., 85:

571, 1947.30. HERBUT, P. A. AND SCARICACIOTTOLI, T. MI.: Arch. Path..

40: 94, 1945.31. HOLLISTER, L. E.: Am. J. Mled., 23: 870, 1957.3 2. HIUNTER, F. M., SPARKS, R. D. AND SALZMAN, R. T.:

Gastroenterology, 39: 394. 1960.33. JOSKE, R. A. AND KING, W. E.: Laucet, 2: 477, 1955.34. KETTLER, L. H.: Ergebn. Ally. Path., 37/38: 1, 1954.35. LON'GCOPE, W. T.: Tr. A. Amt. Physicians, 28: 497, 1913.36. MACKAY, I. R., TAF-r, L. I. AND COWLING, D. C.: Lancet,

2: 1323, 1956.37. MIACKAY, I. R. AND GAJDUSEK, D. C.: Arch. Imbt. Med., 101:

30, 1958.38. MACKAY, I. R.: New England J. Med.. 258: 185, 1958.3 9. MACKAY, I. R., TAFT, L. I. AND COWLING, D. C.: Lancet,

1: 65, 1959.40. MACKAY, I. R.: Gastroenterology, 40: 617, 1961.41. MIACKAY, I. R. AND WOOD, I. J.: In: Progress in liver

disease, edited by H. Popper and F. Schaffner, ('runeand Stratton, Inc., New York. 1961.

42. -MALLORY, F. B.: J. Med. Res., 6: 264, 1901.43. MCKINNON, G. E. et al.: Bull. Johns Hopkins Hosp., 101:

258, 1957.44. MCKIT.NON, G. E.: Int: Henry Ford Hospital International

Symposium: Mechanisms of hypersensitivity, edited byJ. H. Shaffer, G. A. LoGrippo and MN. W. Chase, Little.Brown and Company, Boston, 1959.

45. MCCLUSKEY, R. T. et al.: J. Exper. Med., 111: 181. 1960.46. MILLER, L. L. AND BALE, W. F.: Ibid., 99: 125, 1954.47. MYERS, J. D. et al.: Tr. A. Am. Physicians, 70: 243, 1957.48. PATERSON, P. Y.: J. Exper. Med., 111: 119, 1960.4 9. POPPER, H. AND SCHAFFNER, F.: Liver: structure and

function, Blakiston Division, 'McGraw-Hill Book Co.Inc., New York, 1957.

50. Idem: Ann. Int. Med., 51: 1230, 1959.51. POPPER, H.: Chicago Med., 63: 9. May 6, 1961.52. RICH, A. R.: Bull. Johns Hopkints Hosp., 91: 109, 1952.53. RISSEL, E., STEFFEN, C. AND WVEWjALXA, F.: Wien. kWin.

Wchnschr., 69: 885, 1957.54. SALVIN, S. B.: J. Exper. Med., 107: 109, 1958.55. SCHEIFFARTH, F. AND BERG, G.: Klin. Wchnschr., 31: 441,

1953.56. SCHEIFFARTHI, F., FRENGER, WV. AN-D BRICHZY, W.: Ibid.,

33: 445, 1955.57. SCROGGIE, A. E. AND JAQUES, L. B.: J. Imnmunol., 62:

103, 1949.58. SOOSTMEYER, T.: Arch. path. Anat.. 306: 554, 1940.59. STEINER, J. W. et al.: Unpublished observations.60. STEINER, J. W. AND VOLP1t, R.: Cantad. M. A. J., 84: 1165,

1961.61. Idem: Ibid., 84: 1227, 1961.62. Idem: Ibid., 84: 1297. 1961.63. STEINER, J. W.: Anm. J. Path., 38: 411. 1961.64. TREMAINE, M. M. AND JETER, W. S.: J. Iminunol., 74:

96, 1955.65. UHR, J. W., SALVIN, S. B. AN-D PAPPENHEIMER;, A. WM.:

J. Exper. Med., 105: 11, 1957.66. VORLAENDER, K. O.: Klin. W11chnschr., 31: 748, 1953.67. Idem: In: Immunopathologie in Klinik und Forschung,

edited by P. Miescher and K. 0. Vorlaender, GeorgThieme Verlag, Stuttgart, 10957.

68. WADSWORTH, A., HYMAN, L. WV. AND NICHOLS, R. R.:Anm. J. Path., 11: 419, 1935.

69. WTAKSMAN, B. F.: In: Ciba Foundation Symposiuml o)ncellulawr aspects of immlunity, edited by G. EC. AN.Wolstenholme and M. O'Connor, J{* & A. Churchill, Ltd.,London, 1960.

70. XVATJEN, J. W.: Verh. dtsch1. pathl. Ges., 30: 148, 1937.71. WEL,LS. H. G.: Tr. Chicago Path. Soc., 5: 240, 1901-3.