Proc. Natl. Acad. Sci. USAVol. 91, pp. 614-618, January 1994Medical Sciences

Chronic active hepatitis in transgenic mice expressinginterferon-y in the liver

(viral hepatitis/major histocompatibility complex class I)

TETSUSHI TOYONAGA*t, OKIO HINOt, SATOSHI SUGAI*, SHOJI WAKASUGI*, KUNIYA ABE*,MOTOAKI SHICHIRIt, AND KEN-ICHI YAMAMURA*§*Department of Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 4-24-1, Kuhonji,Kumamoto 862, Japan; tDepartment of Metabolic Medicine, Kumamoto University School of Medicine, 1-1-1, Honjo, Kumamoto 860, Japan; andtDepartment of Experimental Pathology, Cancer Institute, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170, JapanCommunicated by Baruch S. Blumberg, September 22, 1993

ABSTRACT Interferon-y may play an important role inthe immune response and in inflammatory diseases, includingchronic active hepatitis. To understand the role of interferon-yin the regulation of inflammation and to establish a mousemodel of chronic active hepatitis, we produced transgenic micein which the mouse interferon-y gene was regulated by aliver-specific promoter, the serum amyloid P component genepromoter. Four transgenic mouse lines were generated, andtwo of these lines expressed mRNA of interferon-y in the liver.Levels of serum transaminases increased gradually as a func-tion of age and were significantly higher than those of inter-feron--negative littermates after 4 weeks after birth. Onetransgenic mouse line showed a histology of chronic activehepatitis similar to that found in human patients, althoughcirrhotic changes such as fibrosis were scarce. Thus, theliver-specific production of interferon-y is sufficient to inducechronic inflammatory disease and this mouse is a transgenicmodel of chronic active hepatitis.

Chronic hepatic diseases, such as chronic active hepatitis andliver cirrhosis, are public health problems of worldwideimportance and are major causes of mortality in certain areasof the world. Clinical and epidemiological studies (1-4) haveclearly shown the importance of hepatitis B and C viruses(HBV, HCV) in chronic active hepatitis as well as hepato-cellular carcinoma (HCC). Furthermore, studies ofpathologyrevealed that HCC arises in a cirrhotic liver and that chronichepatitis is prerequisite for the development of HCC. Themechanisms responsible for HBV- or HCV-induced hepato-cellular injury are not well understood. However, it is gen-erally accepted that HBV is not directly cytopathic and thatliver cell necrosis is dependent upon the host's immuneresponse, directed at viral determinants on the hepatocytemembrane (5, 6). This immune response is mainly mediatedby cytotoxic T lymphocytes (7).

Several transgenic models of hepatic disease have beendescribed. Chisari et al. (8) demonstrated that transgenicmice expressing the HBV large envelope polypeptide undercontrol of the albumin enhancer/promoter develop liverlesions due to accumulation of long filaments composed ofHBV surface antigen (HBsAg) in smooth endoplasmic retic-ulum (ER). Dycaico et al. (9) reported that transgenic miceexpressing the mutant a1-antitrypsin gene display intracellu-lar globules within the hepatocyte rough endoplasmic retic-ulum (RER) that contain mutant protein, leading to neonataland adult hepatitis. In addition, Sandgren et al. (10) showedthat transgenic mice expressing the albumin-plasminogenactivator gene develop progressive degenerative change inthe liver due to accumulation ofRER-bounded multivesicular

bodies. However, the principal lesion in these models ap-pears to involve the hepatocyte secretory pathway and thusis different from that found in human patients.A model of acute hepatitis can be produced by adminis-

tration of chemicals. However, remaining hepatocytes areinduced to proliferate until the liver regains its originalweight. Shull et al. (11) produced transforming growth factor(TGF)-,81-deficient mice. These mice developed an inflam-matory liver disease similar to that found in HBV infection.However, about 20 days after birth these mice died due to awasting syndrome. Mori et al. (12) demonstrated that liverchanges histologically mimicking human hepatitis were pro-duced in the mouse liver after repeated immunization withsyngeneic crude liver proteins. However, it is not knownwhether hepatitis continues long after the final immunization.Thus, a mouse model for chronic active hepatitis has neverbeen produced, so far. It is a well-known fact that liver inmice and rats can regenerate and return to normal size evenafter removal oftwo-thirds of it. Furthermore, Sandgren et al.(10) demonstrated that a small number of liver cells caneffectively reconstitute functional liver mass. These suggestthe remarkable regenerative capacity ofindividual liver cells.This might be the reason why a mouse model for chronicactive hepatitis is difficult to establish.

Interferon (IFN) seems to be an important regulator of thelocal immune response in the liver of patients with chronichepatitis B, because cells expressing IFN-a and -y arelocalized in the liver tissue of these patients (13). In addition,two transgenes, the IFN-ygene and the tumor necrosis factor(TNF)-,8 gene, have been shown to induce chronic inflam-matory lesions in Langerhans islets of the pancreas (14, 15).However, in this case, the expression of TNF-,8 in islets isshown to be insufflcient to cause insulin-dependent diabetesmellitus. On the basis of these facts, we attempted to estab-lish a mouse model of chronic active hepatitis by producingtransgenic mice carrying the mouse IFN-y gene linked to thepromoter of serum amyloid P component (SAP) gene. Wepreviously showed that this SAP promoter could direct theliver-specific and developmental expression of the heterolo-gous gene (16, 17). We report here the transgenic mousemodel of chronic active hepatitis.

MATERIALS AND METHODSConstruction of SAP-IFN-y DNA. The SAP gene promoter

is derived from the Lm hSAP-8 genomic DNA clone of the

Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus;HCC, hepatocellular carcinoma; HBsAg, HBV surface antigen;IFN, interferon; SAP, serum amyloid P component; GOT, glutamic-oxaloacetic transaminase; GPT, glutamic-pyruvic transaminase; RT-PCR, reverse transcription-polymerase chain reaction; MHC, majorhistocompatibility complex.§To whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 91 (1994) 615

human SAP gene (18). We used a 0.6-kb HindIII-Avr IIfragment. The Avr II site is located 14 bp upstream from thestart codon. This fragment was inserted into pUC19 digestedwith HindIII and Xba I to produce the plasmid pUC-SAP. ABamHI-HindIII fragment from this pUC-SAP was insertedinto pLG1-SAP, a plasmid carrying 1.2 kb of the rabbitf3globin gene. The cDNA encoding mouse IFN-,y was in-serted into the EcoRI site of pLG1-SAP, resulting in con-struction of the plasmid pLG1-SAP-IFN-y (Fig. 1). Prior toinjection into fertilized mouse eggs, pLG1-SAP-IFN-y wasdigested with HindIII and Xho I, and a resulting 2.4-kbfragment, the SAP-IFN-y gene, was isolated and used formicroinjection.DNA Injection and Screening of Transgenic Mice. BDF1

(C57BL/6 x DBA/2) mice were used to obtain fertilizedeggs, and several hundred molecules of the SAP-IFN-yfragment (2.4 kb) were microinjected into the pronucleus offertilized eggs, as described (19). When the mice were 4weeks of age, total nucleic acid was extracted from a piece ofthe tail of each mouse and used for Southern blot analysis(20). The BamHI-Xho I fragment was used as a probe (Fig.1).RT-PCR Analysis. Total RNA was extracted from various

tissues by using the guanidine thiocyanate procedure. Fivemicrograms oftotalRNA was reverse transcribed into cDNAby using SuperScript RT (BRL SuperScript PreamplificationSystem). To avoid detection of mouse endogenous IFN-ymRNA, two primers were synthesized for PCR; one corre-sponded to the IFN-y gene and the other corresponded to the3' untranslated region of rabbit f,Bglobin gene (Fig. 1). ThePCR was performed in 20 ,u of solution consisting of 1 x PCRbuffer, 200 ,uM dNTPs, 5 pmol of primers, and 1.25 units ofAmpliTaq DNA polymerase (Perkin-Elmer/Cetus). Mineraloil was overlaid on the reaction mixtures and the cDNAswere amplified on a heating block (ASTEC PC-700). Cyclingparameters were as follows: denaturation at 94°C for 1 min,annealing at 64°C for 2 min, and extension at 72°C for 2 min.RNA samples from the SAP-IFN-y 5 line or the SAP-IFN-,y44 line were submitted to 25 or 30 cycles of amplification,respectively. After amplification, the PCR products wereelectrophoresed on a 5% polyacrylamide gel and stained withethidium bromide. These gels were subjected to Southernblot analysis using the BamHI-Xho I fragment (Fig. 1) as aprobe.

Assay of Serum IFN-y and Transaminase. Blood was ob-tained from the tail artery and was kept standing for 30 minat room temperature. Sera were separated by centrifugation(3000 rpm in a Tomy MR-150 for 5 min). Each serum (100 ,ul)was assayed for murine IFN-y in duplicate using an enzymeimmunoassay kit (Genzyme InterTest-y). Each serum wasassayed for plasma glutamic-oxaloacetic transaminase

Hind IlI BamHl Xholt t ~~~ATG TGA An

~- ~PCR primer

0.6kb 0.6kb 0.6kb 0.6kb

Human SAP gene promoterLII Rabbit p - globin exon and intron

Mouse IFN - y cDNA

FIG. 1. Structure of the SAP-IFN-y transgene. An, poly(A).Arrows indicate the sites of restriction endonucleases. Primers thatwere used in reverse transcription-polymerase chain reaction (RT-PCR) are also represented.

(GOT) and glutamic-pyruvic transaminase (GPT) by using theReflotron system (Boehringer Mannheim).

Histological Analysis. Tissues were fixed in buffered 10%formalin and embedded in paraffin, and 4-,m sections wereroutinely stained with hematoxylin and eosin.

Immunohistochemical Analysis. Tissues were embedded inTissue-Tek (Miles) and sectioned in a cryostat at -20°C at athickness of 6-8 ,um. Frozen sections of liver were air-driedfor 30 min and fixed in acetone/methanol at 4°C for 10 min.After a wash in phosphate-buffered saline, fixed sectionswere blocked with endogenous avidin and biotin by using aVector blocking kit. After incubation with 10% normal rabbitserum in phosphate-buffered saline for 20 min, sections wereincubated with an appropriately diluted anti-mouse-I-Ab an-tibody (rat IgG) at 4°C overnight. Sections were then incu-bated with an appropriately diluted biotinylated anti-rat-IgGantibody (rabbit IgG) for 30 min, with avidin-biotin-peroxidase complex, and subjected to the peroxidase reac-tions. Sections were counterstained with hematoxylin andmounted.

RESULTSEstablishment of the Transgenic Mouse Line Expressing

IFN-y. Four out of 56 mice, SAP-IFN-y 5, 27, 30, and 44,were shown to carry the transgene. SAP-IFN-'y 5 was maleand the others were female. The numbers of copies inte-grated were estimated to be 4, 4, 2, and 1, respectively. At8 weeks of age, these mice were mated with C57BL/6 mice.All lines of transgenic mice transmitted the transgene totheir offspring. These offspring were used in the followinganalysis. As most of SAP-IFN-y 5 mice that showed thehighest plasma transaminase level (see Table 1) died ataround 6 months of age under conventional conditions, allmice were transferred to specific-pathogen-free conditionsby using in vitro fertilization. However, about 80% oftransgenic mice died by 1 year of age even under specific-pathogen-free conditions.

Tissue and Developmental Specificity of Transgene Expres-sion. To examine tissue specificity of transgene expression,total RNAs were extracted from various tissues (liver,spleen, stomach, small intestine, pancreas, lung, kidney, andperipheral blood lymphocytes) of transgenic mouse lines

ssAN\ 0rA00t ? wS0~~~~bR4 %

C opQ

No 5

No 44

FIG. 2. Tissue-specific transgene expression detected by RT-PCR (SAP-IFN-y 5 and 44). RT-PCR was performed using PCRprimers located on mouse IFN-y and rabbit f-globin genes (see Fig.1), and these PCR products were electrophoresed on a 5% poly-acrylamide gel. Hae III-digested fragments of #X174 DNA wereused as DNA size markers. The bands are indicated by arrows. PBL,peripheral blood lymphocytes.

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a M. 1w 2w 3w 4w 5w 6w

b

8

0

o 0

o 0

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0 o

1o2 3 121 2 3 4 5 6 J12 2637 52

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FIG. 3. (a) Developmental specificity of transgene expression(1-6 weeks) of the SAP-IFN-y 5 line measured by RT-PCR. Glyc-eraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA was alsoamplified as an internal control. Lane M., Hae III-digested 4X174DNA. (b) Developmental expression of mouse IFN-y in the SAP-IFN-y 5 line. Serum IFN-y was assayed by using an enzymeimmunoassay kit (Genzyme InterTest-y). The negative littermateshad less than the detectable level (125 pg/ml) of IFN-v.

(SAP-IFN-y 5 and 44) when these mice were 6 weeks of age.Using these total RNAs, we performed RT-PCR. As shownin Fig. 2, the PCR products (265 bp) were detected only in theliver of the SAP-IFN-y 5 line and in the liver and pancreas ofthe SAP-IFN-y 44 line. To confirm that these bands areamplified IFN-y cDNAs, they were hybridized with the

600-

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so*0

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Table 1. Plasma GOT and GPT at 6 weeksActivity, units/liter

Line GOT GPT

SAP-IFN-y 5 (n = 24) 123 ± 10 69 ± 10SAP-IFN-y27 (n = 5) 71 ± 4 35 ± 2SAP-IFN-y30 (n = 6) 72 ± 5 31 t 1SAP-IFN-'y44 (n = 13) 97 ± 6 46 ± 3Negative littermates (n = 24) 68 ± 3 25 ± 2Results are mean ± SEM. The differences between SAP-IFN-y 5

and 44 and negative littermates were statistically significant by theStudent t analysis (P < 0.001). n, Number of mice.

BamHI-Xho I fragment (see Fig. 1) and autoradiographed.The bands were detected in the same tissues.To examine developmental specificity oftransgene expres-

sion, we performed RT-PCR using total RNAs from the liversof 1-, 2-, 3-, 4-, 5-, and 6-week-old SAP-IFN-y 5 mice. PCRproducts were detected in the livers at any age, as expected(Fig. 3a). No band was detected when PCR was carried outusing total RNAs in the absence of reverse transcriptase,suggesting that DNA was not contaminating the samples.

Concentration of Mouse IFN-y in the Serum. To examinewhether murine IFN-y was present in the serum of eachtransgenic line, serum IFN-ywas assayed when mice were at7 weeks of age. The mean concentration of IFN-y in SAP-IFN-y 5 mice was 3533 pg/ml, whereas the mean concentra-tions of other lines and negative littermates were below thedetectable level (less than 125 pg/ml). To examine develop-mental expression of mouse IFN-y in the SAP-IFN-y 5 line,we measured serum IFN-y concentration when mice were 1,2, 3, 4, 5, 6, 12, 26, 37, and 52 weeks of age. Serum IFN-ywasdetected already at 1 week of age, but stayed at a low leveluntil 3 weeks of age. After then, the level of serum IFN-yincreased gradually and reached the maximum at around 6months of age (Fig. 3b). The serum IFN-y in negativelittermates was below the detectable level.

Concentration of Plasma Tranaminase. To examine livercell injury, GOT and GPT were assayed. When mice were 6weeks of age, blood samples were obtained from the tail

GPT0

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FIG. 4. Concentration of plasma GOT (Left) and GPT (Right) in the SAP-IFN-y 5 line at various ages. o, Transgenic mice; e, negativelittermates. The differences in the levels of transaminases between transgenic mice and negative littermates were statistically significant byStudent t analysis after 4 weeks of age (P < 0.05). The transaminases were measured by the Reflotron system (Boehringer Mannheim).

IFN-y

G3PDH

E 4 _

m 104-a

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= 10 3EE0CD

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Proc. Natl. Acad. Sci. USA 91 (1994)

1.

Proc. Natl. Acad. Sci. USA 91 (1994) 617

artery of each line of transgenic mice. As shown in Table 1,plasma levels of both GOT and GPT were significantlyelevated in the SAP-IFN-y 5 line and were slightly elevatedin the SAP-IFN-'y44 line, suggesting the presence of liver cellinjury in these transgenic mice. The differences were statis-tically significant by Student t analysis (P < 0.001).We then examined changes of plasma transaminase levels

as a function of age in the SAP-IFN-y 5 line. When the micewere 1, 2, 3, 4, 5, 6, 12, 26, 37, and 52 weeks of age, plasmaGOT and GPT were measured. There was no difference in thelevels of GOT and GPT between transgenic mice and nega-tive littermates until 3 weeks ofage. However, both GOT andGPT levels increased gradually and were considerably higherthan those in negative littermates after 4 weeks of age (Fig.4). The differences in the levels of transaminases betweentransgenic mice and negative littermates were statisticallysignificant by Student t analysis after 4 weeks of age (P <0.05).

Histology. To examine whether pathological changes de-velop in the liver of each transgenic mouse line, tissuesamples were taken at 17 weeks of age. The livers from theSAP-IFN-y S and 44 lines showed histology similar to acutehepatitis, but not the livers of the SAP-IFN--y 27 and 30 lines.Pathological change in the SAP-IFN-y 44 line was lessextensive than in the SAP-IFN-y S line. No pathologicalchange was observed in other tissues.For sequential morphological observation in the liver ofthe

SAP-IFN-y 5 line, tissue samples were taken at 1, 2, 3, 4, 5,6, 12, 26, 37, and 52 weeks of age, from at least two animalsat each time point, including both sexes. Negative littermateswere analyzed in parallel as controls for the different stagesof liver diseases. Up to 2 weeks of age, no recognizablehistologic change was observed in the liver of SAP-IFN--y 5mice in comparison with negative littermates, althoughIFN-,y was expressed. During the third week, necro-inflammation began to appear randomly in the lobules. Thischange became prominent in subsequent weeks (Fig. 5a),reflected in the serum transaminase level. From 5 weeksonwards, lymphoid cell infiltration appeared at the portalarea (Fig. Sb). From 26 weeks of age, slight fibrosis appeared,and at 37 weeks of age, ductal proliferation was observed(Fig. Sc), reflected in increased serum transaminase level.These sequential observations were consistent in subsequentgenerations and were not influenced by the sex of the host.We examined more than 30 mice, and all mice older than 3weeks showed pathological changes in the liver. Thus, pen-etrance was 100% in transgenic mice. In contrast, negativelittermates did not show any significant histologic abnormal-ities up to at least 52 weeks of age.Immunohistochemical Analysis of I-Ab Molecules. IFN-y

can induce the expression of major histocompatibility com-plex (MHC) class II molecules in antigen-presenting cells andother cell types, including pancreatic ,B cells (14, 21). Thus,we examined the expression of MHC class II molecules.Frozen sections of liver were made from mice of the SAP-IFN-,y S line when they were 6, 12, and 26 weeks of age. Theexpression of I-Ab molecules was observed on the surface oftransgenic liver cells at any age examined but was notobserved in the livers of negative littermates (data notshown).

DISCUSSIONIn this report, we demonstrate that local production of IFN-yis sufficient to initiate and maintain a liver-specific inflam-matory disease in two independent transgenic mouse lines.After 4 or 5 weeks of age, transgenic mice showed continuouselevation of serum transaminases, indicating the presence ofpersistent liver cell damage. The liver cell damage is moresevere in the SAP-IFN-yS line than in the SAP-IFN-,y44 line.

FIG. 5. Sequential morphology in the liver of the SAP-IFN-y Sline. (Hematoxylin and eosin; original magnification x 100, finalmagnifi'cation x70.) (a) Necro-inflammation (arrows) is present(male, 4 weeks). (b) Lymphoid cell infiltration at the portal area(arrow) is present (female, 12 weeks). (c) Ductal proliferation (ar-rows) is present (male, 52 weeks).

This is consistent with the data that IFN-y expression ishigher in the SAP-IFN-yS line than in the SAP-IFN-y44 line.Histological analysis also revealed the progression ofchronicactive hepatitis-that is, initial necroinflammatory changefollowed by lymphoid cell infiltration at the portal areareminiscent of those seen in human viral hepatitis. However,the pathological changes in the mouse liver are modest, andespecially cirrhotic changes such as fibrosis are scarce com-pared with those in human liver. This could be due to theremarkable regenerative capacity of mouse liver cells. In anycase, this is a transgenic mouse model for chronic activehepatitis.

It has been reported that IFNs (a, f, and 'y) play animportant role against viral infection (21). IFN-y activatescytotoxic T lymphocytes and natural killer cells and blocksviral protein synthesis by inducing (2'-5')oligoadenylate syn-thetase. Because of these biological effects, IFNs are bene-ficial in the treatment of chronic viral hepatitis. On the otherhand, Sarvetnick et al. (14) reported that the expression ofIFN-yin islets of Langerhans of transgenic mice results in aninflammatory destruction of the islets. They demonstratedthat engrafted histocompatible islets were destroyed and thatlymphocytes from the transgenic mice are cytotoxic to nor-mal islets in vitro. On the basis of these findings, theyconcluded that islet cell loss was caused by infiltratinglymphocytes, not through deleterious effects of IFN-y.IFN-y can induce the expression of MHC molecules inimmune cells as well as nonimmune cells. Actually, they

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showed that expression ofMHC class II molecules is inducedin acinar tissue, islets, and epithelial cells lining the ducts.The expression of MHC class II molecules may lead to thepresentation of self-antigens, resulting in the activation ofquiescent autoreactive cells and loss of pancreatic islettolerance (22). A similar mechanism may be involved ininitiation and development of chronic active hepatitis, be-cause high levels of I-Ab expression are observed in ourtransgenic mice after birth.

It is of interest that about 80% of our transgenic mice diedby 1 year of age. Pathology studies revealed that these micedied of bacteremia, not of liver cell damage. As periodicalcomprehensive serology and histopathology revealed no in-dication of pathogens, the bacteria may have been enteric.This is consistent with the data indicating that IFN-y plays asignificant role in the pathogenesis of Gram-negative sepsis(23).We previously showed that chromosomally integrated

HBV genome suffices to allow viral replication in the liver ofthe transgenic mice (24). But the transgenic mice did notdevelop hepatitis, because of tolerance to viral gene prod-ucts. However, these transgenic mice can produce antibodyto HBsAg after immunization with this antigen (unpublisheddata). Double transgenic mice carrying both the SAP-IFN-ygene and the HBV genome will be a useful mouse model ofviral hepatitis, because the release of HBsAg by liver celldamage may enhance the immune response to viral antigen,leading to liver cell injury. On the other hand, it is shown thatchromosomal changes linked to HBV DNA integration occurfrequently in host DNA of human HCC (25, 26). Further-more, it was shown that HBV DNA has recombinogenicfunctions in the hepatitis-related proliferative state (27) andthat DNA rearrangement is involved in hepatocarcinogenesis(28). All these results suggest that genomic instability causedby HBV DNA integration to host DNA may play an impor-tant role in hepatocarcinogenesis. This possibility can betested by examining the development of HCC in doubletransgenic mice carrying both the HBV genome and theSAP-IFN-y gene.

We thank Dr. K. Kajino and Mrs. R. Yamamoto for technicalassistance. This work was supported by Grants-in-Aid for ScientificResearch on Priority Areas (K.-i.Y.) and a Grant-in-Aid for CancerResearch (K.-i.Y. and O.H.) from the Japanese Ministry of Educa-tion, Science and Culture.

1. Szmuness, W. (1978) Prog. Med. Virol. 24, 40-69.2. Beasley, R. P., Hwang, L. Y., Lin, C. C. & Chien, C. S. (1981)

Lancet i, 1129-1133.3. World Health Organization (1983) WHO Tech. Rep. Ser. 691,

1-30.4. Saito, I., Miyamura, T., Ohbayashi, A., Harada, H.,

Katayama, T., Kikuchi, S., Watanabe, Y., Koi, S., Onji, M.,Ohta, Y., Choo, Q.-L., Houghton, M. & Kuo, G. (1990) Proc.Natl. Acad. Sci. USA 87, 6547-6549.

5. Mondelli, M., Vergani, G. M., Alberti, A., Vergani, D., Port-

mann, B., Eddleston, A. L. W. F. & Williams, R. (1982) J.Immunol. 129, 2773-2778.

6. Moriyama, T., Guilhot, S., Klopchin, K., Moss, B., Pinkert,C. A., Palmiter, R. D., Brinster, R. L., Kanagawa, 0. &Chisary, F. V. (1990) Science 248, 361-364.

7. Chisari, F. V., Ferrari, C. & Mondelli, M. U. (1989) Microb.Pathog. 6, 311-325.

8. Chisari, F. V., Filippi, P., Buras, J., McLachlan, A., Popper,H., Pinkert, C. A., Palmiter, R. D. & Brinster, R. L. (1987)Proc. Natl. Acad. Sci. USA 84, 6909-6913.

9. Dycaico, M. J., Grant, S. G. N., Felts, K., Nichols, W. S.,Geller, S. A., Hager, J. H., Pollard, A. J., Kohler, S. W.,Short, H. P., Jirik, F. R., Hanahan, D. & Sorge, J. A. (1988)Science 242, 1409-1412.

10. Sandgren, E. P., Palmiter, R. D., Heckel, J. L., Daugherty,C. C., Brinster, R. L. & Degen, J. L. (1991) Cell 66, 245-256.

11. Shull, M. M., Ormsby, I., Kier, A. B., Pawlowski, S., Diebold,R. J., Yin, M., Allen, R., Sidman, C., Proetzel, G., Calvin, D.,Annunziata, N. & Doetschman, T. (1992) Nature (London) 359,693-699.

12. Mori, Y., Mori, T., Yoshida, H., Ueda, S., lesato, K.,Wakashin, Y., Wakashin, M. & Okuda, K. (1984) Clin. Exp.Immunol. 57, 85-92.

13. Dienes, H. P., Hess, G., Woorsdorfer, M., Rossol, S., Gallati,H., Ramadori, G. & Buschenfelde, K.-H. M. (1991) Hepatol-ogy 13, 321-326.

14. Sarvetnick, N., Liggitt, D., Pitts, S. L., Hansen, S. E. &Stewart, T. A. (1988) Cell 52, 773-782.

15. Picarella, D. E., Kratz, A., Li, C.-B., Ruddle, F. H. & Flavell,R. A. (1992) Proc. Natl. Acad. Sci. USA 89, 10036-10040.

16. Iwanaga, T., Wakasugi, S., Inomoto, T., Uehira, M., Ohnishi,S., Nishiguchi, S., Araki, K., Uno, M., Miyazaki, J., Maeda,S., Shimada, K. & Yamamura, K. (1989) Dev. Genet. 10,365-371.

17. Zhao, X., Araki, K., Miyazaki, J.-I. & Yamamura, K.-I. (1992)J. Biochem. (Tokyo) 111, 736-738.

18. Ohnishi, S., Maeda, S., Shimada, K. & Arao, T. (1986) J.Biochem. (Tokyo) 100, 849-858.

19. Yamamura, K., Kikutani, H., Takahashi, N., Taga, T., Akira,S., Kawai, K., Fukuchi, K., Kumahara, Y., Honjo, T. &Kishimoto, T. (1984) J. Biochem. (Tokyo) 96, 357-363.

20. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517.21. Dinarello, C. A. & Mier, J. W. (1987) N. Engl. J. Med. 317,

940-945.22. Sarvetnick, N., Shizuru, J., Liggitt, D., Martin, L., McIntyre,

B., Gregory, A., Parslow, T. & Stewart, T. (1990) Nature(London) 346, 844-847.

23. Silva, A. T. & Cohen, J. (1992) J. Infect. Dis. 166, 331-335.24. Araki, K., Miyazaki, J.-I., Hino, O., Tomita, N., Chisaka, O.,

Matsubara, K. & Yamamura, K.-I. (1989) Proc. Natl. Acad.Sci. USA 86, 207-211.

25. Rogler, C. E., Sherman, M., Su, C. Y., Shafritz, D. A., Sum-mers, J., Shows, T. B., Henderson, A. & Kew, M. (1985)Science 230, 319-322.

26. Hino, 0. & Rogler, C. E. (1986) Proc. Natl. Acad. Sci. USA 83,8338-8342.

27. Hino, O., Tabata, S. & Hotta, Y. (1991) Proc. Natl. Acad. Sci.USA 88, 9248-9252.

28. Sandgren, E. P., Palmiter, R. D., Heckel, J. L., Brinster,R. L. & Degen, J. L. (1992) Proc. Natl. Acad. Sci. USA 89,11523-11527.

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