Induction of Chronic Lethal Australia Antigen Hepatitis in Mice Lewis A. Johnson, MD, and Emil Wirostko, MD
ClinicallI. idiopathic uv eitis may be associated with chronic active hepatitis B. In searching for a possible cause of the uveitis in 6 patients having concurrent chronic iridocvclitis and chronic active hepatitis with serum Australia antigen (AA). the aqueous humor from each patient was analyzed for AA, passed through 220-mg filters and inoculated directlv into the livers of mice. The animals were observed for spontaneous mortality for 12 months, at which time the remaining animals were sacrificed. The liv ers of all animals were examined for hepatitis and AA Although the aqueous humor from only 1 patient was found to contain AA, all six aqueous specimens produced a lethal viral hepatitis-like disease with liver AA The results suggest that the six positive aqueous specimens contained a viral infectious agent, which may have been the core of the Dane particle, and that the mouse is suitable for the laboratory inv estigation of Type B hepatitis by the technique described. (Am J Pathol 82:85-100, 1973)
U VEITIS is defined as inflammatory disease in the deeper ocular structures such as the choroid, iris, and ciliary bodv. The disease may be either exogenous or endogenous.1 In the exogenous form, the responsible agent gains entry to these deeper structures from the external surfaces of the eve. In the latter form (endogenous). it is assumed that the exciting factor arises from wvithin the eve or elsewvhere in the body.2 In most patients hav-ing endogenous uveitis, no infectious agent is either clinically implicated or isolated in the laboratory, and the inflammation is considered to be idiopathic.3 Although idiopathic uveitis (IU) usually occurs in a patient without evidence of other systemic disease, it is frequently seen in association wvith other systemic diseases such as ankvlosing spondvlitis, sarcoidosis, and multiple sclerosis.4 Recently it has been reported that chronic active hepatitis (CAH)5 and serum Australia antigen (AA) may be associated wvith lU.6 Serum AA was described many years ago in Australian aborigines.7 Since then, extensive evidence links AA wvith the agent of viral hepatitis Tx-pe B.89 Electron microscopy of serum positive for AA demonstrates a 40-mm spherical body-, the so-called Dane particle,j0 which is believed to be the intact infectious agent. with a 27-nm electron-dense inner core F1rom the Department of Pathology. Columbia-Presb%terian NMedical Center. Columbia Unisersit\ (G)llese of Ph\-sicians and Surceons. Nes- York. Nesv X-ork Xccepted for publication Xugust 21. 1975. Xddress reprint requests to Dr Les-is X Johnson. Department of Pathology. Columbia University (ollege of Ph-sicians and Surgeons. 6:30 West 168th Street. Nev- York. N\' 10032 85
86
JOHNSON AND WIROSTKO
American Journal of Pathogy
composed of double-stranded DNA molecules." In addition, other particles have been described in these sera-a 20-nm sphere and an elongated tubule or filament 20 nm in diameter and variable in length. 2 Within the nuclei of infected liver cefls in acute hepatitis B, one can often demonstrate a fourth particle-a 27-nm spherical structure-the inner core of the Dane particle representing the nucleocapsid.'3 It is presently believed that AA is the outer lipoprotein that covers the inner electrondense 27-nm portion of the complete Dane particle."4 Recently it has been shown that these two portions of the assembled Dane particle are antigenically distinct and that each is capable of inducing distinct antibodies. Antibodies to AA react not only with the surface component of the Dane particle but also with the 20-nm spherical and tubular particles. Antisera to the nucleocapsid of the Dane particle fail to react with AA."5 Over the past 30 years there have been many attempts to transmit serum hepatitis to laboratory animals with equivocal results. Unreported studies conducted in our laboratories, in which young mice were inoculated either intravenously or intraperitoneally with AA-positive aqueous humor obtained from patients having concurrent active IU and CAH with serum AA also gave unconvincing histologic evidence of hepatitis. Recently, however, purified human serum AA has been transmitted to nonhuman primates, African green monkeys, and passed two times, the resultant level of animal serum AA being greater than that expected on the basis of dilution.'6 More recently, it has been reported that the inoculation of human hepatitis B material into chimpanzees has resulted in hepatitis and liver cell AA.14 The purpose of this report is to document that although AA can be detected in only an occasional aqueous humor specimen from patients with IU having concurrent CAH with serum AA, the direct liver inoculation of these aqueous humor specimens into young mice consistently produces a lethal chronic active viral hepatitis-like disease with liver AA. Materials and Methods Desripto of te Patien Podaion Six patients having acute exacerbations of chronic IU (iridocycitis) and concurrent CAH served as the source of the experimental material. The patients varied in age from 11 to 62 years; 2 were males and 4 females. All 6 patients had had needle biopsies of the liver interpreted as CAH by established histologic criteria,'7 and all 6 had serum AA. Each patient was subjected to repeated anterior chamber paracentesis with a maximum of aqueous humor (0.1 to 0.73 cu cm per tap) being withdrawn.
Vol. 82, No. 1 January 1976
AUSTRALIA ANTIGEN HEPATITIS IN MICE
87
All specimens from each patient were pooled giving a total of six pools, which were stored at 2 to 6 C while the specimens were being collected. Latory Stuies Performed on the Aqueous Humor Spec
Aliquots of each of the six pooled specimens were tested for the presence of AA by counterimmunoelectrophoresis (CIEP)"8 and by radioimmunosassay (RIA).1' The RIA assays for AA were perforned at the Pioneer Blood Bank, Inc., New York, N. Y., using the Ausria 11-125 equipment, supplies, and method supplied by Abbott Laboratories, North Chicago, Ill. just prior to animal inoculation, each of the six pooled specimens was diluted 1:30 with sterile isotonic saline, passed through a 0.220-M Milipore filter, and tested for final bacterial sterility using sheep's blood agar, aerobically and anaerobically at 37 C for 48 hours. No growth occurred from any of the six filtrates at the end of 48 hours. Description of the Animas
One hundred eighty male, CD-I strain mice, 12 to 14 weeks old and 15 to 20 g in weight at the time of inoculation, were obtained from the animal care facility of the Institute for Cancer Research, ColumbiaPresbyterian Medical Center. Descripio of tie Anima Inoco
The inoculum, 0.3 cu cm, was injected directly into the liver of each mouse using a tuberculin syringe and a needle through a blind transperitoneal anterior right subcostal approach. One patient's filtrate was inoculated into each of 10 mice, with 1 mouse receiving the filtrate from 1 patient only for a total of 60 mice. Normal control aqueous humor was removed from 6 grossly normal eyes from an Eye Bank (average 5.0 cu cm/eye) and consisted of an aqueous-vitreous mixture. After assaying for AA by both CIEP and RIA, the aspirate from one normal eye was inoculated into 10 mice for a total of 60 mice. Sixty mice received sterile isotonic saline. All animals were inoculated on the same day. Desiption of the Aninal Studies Peformed
The 10 mice receiving the filtrate from 1 patient's aqueous specimen were placed together in one box for a total of 6 boxes. The 10 mice receiving the normal aqueous from one Eye Bank eye were likewise
88
JOHNSON AND WIROSTKO
American Journal of Pathology
placed in one box for a total of 6 boxes. The saline-inoculated mice were placed in 6 boxes of 10 mice each. All the animals were fed a standard pelletized diet and were housed in the same room throughout the experiment. They were observed once daily, 5 days a week, for their general appearance, spontaneous activity, and mortality. At the end of 12 months the experiment was terminated, and all living mice were sacrificed using Fluothane anesthesia. All animals, those dying during the experiment and those sacrified at the end, were subjected to a complete autopsy. Animals dying during the week were autopsied within 24 hours of death. The bodies of those animals dving on weekends were placed in a 2 to 6 C refrigerator within 24 hours of death. On the following Monday they were autopsied. The liver of each animal was totally removed and examined for evidence of gross disease. One random fresh sample, approximately 500 mg, from each liver was stored in a sterile glass test tube at 2 to 6 C for up to 48 hours, emulsified in 2.0 cu cm of sterile distilled water and analyzed for AA by CIEP. To eliminate bias, the CIEP analyses for AA on both the human and animal materials were conducted by hospital blood bank personnel, who were unaware of the experiment. The instrument, method, and materials supplied by Ortho Diagnostics, Raritan, N. J., were used for the CIEP determinations. Four random sections of each liver w%ere fixed in 10% formalin, processed for routine paraffin histologic sections, stained with hematoxylin and eosin, and examined for evidence of hepatitis. All liver sections showing evidence of hepatitis were stained X ith the Giemsa stain and examined for evidence of Toxoplasma gondii. Portions of all other organs were fixed in 10% formalin, sectioned, stained with hematoxvlin and eosin, and examined for disease. Results Hunan Studies Aqueous Humor Australia Antigen Assays
None of the aqueous specimens from the 6 patients with uveitishepatitis demonstrated AA by CIEP. Using RIA, however, the aqueous specimen from 1 of these 6 patients was positive for AA. No AA could be detected in any of the aqueous aspirates from the six eyes from an Eye Bank, neither by CIEP nor bv RIA. Anmal Studies
The results of the animal studies are found in Tables 1 to 3. Table 1 depicts the incidence of gross liver disease, liver AA, hepatitis, and mortality
Vol. 82, No. 1 January 1976
AUSTRALIA ANTIGEN HEPATITIS IN MICE
89
by type of inoculum. Table 2 relates the mortality, hepatitis, and liver AA to each of the six hepatitis-uveitis aqueous specimens. In Table 3, the mortality, liver AA., and hepatitis are related to the month followving inoculation of the uveitis-hepatitis aqueous specimens. Mortality
All of the animals survived the trauma of the inoculation, and all remained healthy during the first month followving inoculation. However, during the second month, accelerated mortality was noted in the mice inoculated w-ith the uveitis aqueous. This phenomenon continued until the end of the experiment. By the end of 12 months, 36 mice receiving the uveitis aqueous were dead. In comparison, 32 mice receiving normal aqueous humor and :34 mice receiving saline were dead at the end of 12 months. The 12-month mortality rate (53.3 to 36. 7%7 ) in the control mice Table 1-Results in the Animals by Type of Inoculum Inocula Normal aqueous
Uveitis aqueous
Gross liver disease Australia antigen
Hepatitis
4 60 19 6 22
656
Mortality
-
6 % 6.%
0 0
31.60
60
°% 0%
0
1.6%
0 0 0
1
36.6%
93.3%
1 =
1.6%
- =
53.3%
-
32
SE difference Saline
34 -
Probability
°% 0%
(DEEC
E
F+40'
P,.i,#
44...
p
. . ,.
V .1 ,. C.I.a >:. I3
7&*
o. jl
'-
.
Figure 7-Mouse hepatitis. Australia antigen was detected in this liver. The mouse died
during the third month following direct liver inoculation of uveitis aqueous. Portions of several hepatic lobules show intense polymorphonuclear leukocyte infiltration and hyalinized necrotic liver cell cords. (H&E, x 100)
ClinicallI. idiopathic uv eitis may be associated with chronic active hepatitis B. In searching for a possible cause of the uveitis in 6 patients having concurrent chronic iridocvclitis and chronic active hepatitis with serum Australia antigen (AA). the aqueous humor from each patient was analyzed for AA, passed through 220-mg filters and inoculated directlv into the livers of mice. The animals were observed for spontaneous mortality for 12 months, at which time the remaining animals were sacrificed. The liv ers of all animals were examined for hepatitis and AA Although the aqueous humor from only 1 patient was found to contain AA, all six aqueous specimens produced a lethal viral hepatitis-like disease with liver AA The results suggest that the six positive aqueous specimens contained a viral infectious agent, which may have been the core of the Dane particle, and that the mouse is suitable for the laboratory inv estigation of Type B hepatitis by the technique described. (Am J Pathol 82:85-100, 1973)
U VEITIS is defined as inflammatory disease in the deeper ocular structures such as the choroid, iris, and ciliary bodv. The disease may be either exogenous or endogenous.1 In the exogenous form, the responsible agent gains entry to these deeper structures from the external surfaces of the eve. In the latter form (endogenous). it is assumed that the exciting factor arises from wvithin the eve or elsewvhere in the body.2 In most patients hav-ing endogenous uveitis, no infectious agent is either clinically implicated or isolated in the laboratory, and the inflammation is considered to be idiopathic.3 Although idiopathic uveitis (IU) usually occurs in a patient without evidence of other systemic disease, it is frequently seen in association wvith other systemic diseases such as ankvlosing spondvlitis, sarcoidosis, and multiple sclerosis.4 Recently it has been reported that chronic active hepatitis (CAH)5 and serum Australia antigen (AA) may be associated wvith lU.6 Serum AA was described many years ago in Australian aborigines.7 Since then, extensive evidence links AA wvith the agent of viral hepatitis Tx-pe B.89 Electron microscopy of serum positive for AA demonstrates a 40-mm spherical body-, the so-called Dane particle,j0 which is believed to be the intact infectious agent. with a 27-nm electron-dense inner core F1rom the Department of Pathology. Columbia-Presb%terian NMedical Center. Columbia Unisersit\ (G)llese of Ph\-sicians and Surceons. Nes- York. Nesv X-ork Xccepted for publication Xugust 21. 1975. Xddress reprint requests to Dr Les-is X Johnson. Department of Pathology. Columbia University (ollege of Ph-sicians and Surgeons. 6:30 West 168th Street. Nev- York. N\' 10032 85
86
JOHNSON AND WIROSTKO
American Journal of Pathogy
composed of double-stranded DNA molecules." In addition, other particles have been described in these sera-a 20-nm sphere and an elongated tubule or filament 20 nm in diameter and variable in length. 2 Within the nuclei of infected liver cefls in acute hepatitis B, one can often demonstrate a fourth particle-a 27-nm spherical structure-the inner core of the Dane particle representing the nucleocapsid.'3 It is presently believed that AA is the outer lipoprotein that covers the inner electrondense 27-nm portion of the complete Dane particle."4 Recently it has been shown that these two portions of the assembled Dane particle are antigenically distinct and that each is capable of inducing distinct antibodies. Antibodies to AA react not only with the surface component of the Dane particle but also with the 20-nm spherical and tubular particles. Antisera to the nucleocapsid of the Dane particle fail to react with AA."5 Over the past 30 years there have been many attempts to transmit serum hepatitis to laboratory animals with equivocal results. Unreported studies conducted in our laboratories, in which young mice were inoculated either intravenously or intraperitoneally with AA-positive aqueous humor obtained from patients having concurrent active IU and CAH with serum AA also gave unconvincing histologic evidence of hepatitis. Recently, however, purified human serum AA has been transmitted to nonhuman primates, African green monkeys, and passed two times, the resultant level of animal serum AA being greater than that expected on the basis of dilution.'6 More recently, it has been reported that the inoculation of human hepatitis B material into chimpanzees has resulted in hepatitis and liver cell AA.14 The purpose of this report is to document that although AA can be detected in only an occasional aqueous humor specimen from patients with IU having concurrent CAH with serum AA, the direct liver inoculation of these aqueous humor specimens into young mice consistently produces a lethal chronic active viral hepatitis-like disease with liver AA. Materials and Methods Desripto of te Patien Podaion Six patients having acute exacerbations of chronic IU (iridocycitis) and concurrent CAH served as the source of the experimental material. The patients varied in age from 11 to 62 years; 2 were males and 4 females. All 6 patients had had needle biopsies of the liver interpreted as CAH by established histologic criteria,'7 and all 6 had serum AA. Each patient was subjected to repeated anterior chamber paracentesis with a maximum of aqueous humor (0.1 to 0.73 cu cm per tap) being withdrawn.
Vol. 82, No. 1 January 1976
AUSTRALIA ANTIGEN HEPATITIS IN MICE
87
All specimens from each patient were pooled giving a total of six pools, which were stored at 2 to 6 C while the specimens were being collected. Latory Stuies Performed on the Aqueous Humor Spec
Aliquots of each of the six pooled specimens were tested for the presence of AA by counterimmunoelectrophoresis (CIEP)"8 and by radioimmunosassay (RIA).1' The RIA assays for AA were perforned at the Pioneer Blood Bank, Inc., New York, N. Y., using the Ausria 11-125 equipment, supplies, and method supplied by Abbott Laboratories, North Chicago, Ill. just prior to animal inoculation, each of the six pooled specimens was diluted 1:30 with sterile isotonic saline, passed through a 0.220-M Milipore filter, and tested for final bacterial sterility using sheep's blood agar, aerobically and anaerobically at 37 C for 48 hours. No growth occurred from any of the six filtrates at the end of 48 hours. Description of the Animas
One hundred eighty male, CD-I strain mice, 12 to 14 weeks old and 15 to 20 g in weight at the time of inoculation, were obtained from the animal care facility of the Institute for Cancer Research, ColumbiaPresbyterian Medical Center. Descripio of tie Anima Inoco
The inoculum, 0.3 cu cm, was injected directly into the liver of each mouse using a tuberculin syringe and a needle through a blind transperitoneal anterior right subcostal approach. One patient's filtrate was inoculated into each of 10 mice, with 1 mouse receiving the filtrate from 1 patient only for a total of 60 mice. Normal control aqueous humor was removed from 6 grossly normal eyes from an Eye Bank (average 5.0 cu cm/eye) and consisted of an aqueous-vitreous mixture. After assaying for AA by both CIEP and RIA, the aspirate from one normal eye was inoculated into 10 mice for a total of 60 mice. Sixty mice received sterile isotonic saline. All animals were inoculated on the same day. Desiption of the Aninal Studies Peformed
The 10 mice receiving the filtrate from 1 patient's aqueous specimen were placed together in one box for a total of 6 boxes. The 10 mice receiving the normal aqueous from one Eye Bank eye were likewise
88
JOHNSON AND WIROSTKO
American Journal of Pathology
placed in one box for a total of 6 boxes. The saline-inoculated mice were placed in 6 boxes of 10 mice each. All the animals were fed a standard pelletized diet and were housed in the same room throughout the experiment. They were observed once daily, 5 days a week, for their general appearance, spontaneous activity, and mortality. At the end of 12 months the experiment was terminated, and all living mice were sacrificed using Fluothane anesthesia. All animals, those dying during the experiment and those sacrified at the end, were subjected to a complete autopsy. Animals dying during the week were autopsied within 24 hours of death. The bodies of those animals dving on weekends were placed in a 2 to 6 C refrigerator within 24 hours of death. On the following Monday they were autopsied. The liver of each animal was totally removed and examined for evidence of gross disease. One random fresh sample, approximately 500 mg, from each liver was stored in a sterile glass test tube at 2 to 6 C for up to 48 hours, emulsified in 2.0 cu cm of sterile distilled water and analyzed for AA by CIEP. To eliminate bias, the CIEP analyses for AA on both the human and animal materials were conducted by hospital blood bank personnel, who were unaware of the experiment. The instrument, method, and materials supplied by Ortho Diagnostics, Raritan, N. J., were used for the CIEP determinations. Four random sections of each liver w%ere fixed in 10% formalin, processed for routine paraffin histologic sections, stained with hematoxylin and eosin, and examined for evidence of hepatitis. All liver sections showing evidence of hepatitis were stained X ith the Giemsa stain and examined for evidence of Toxoplasma gondii. Portions of all other organs were fixed in 10% formalin, sectioned, stained with hematoxvlin and eosin, and examined for disease. Results Hunan Studies Aqueous Humor Australia Antigen Assays
None of the aqueous specimens from the 6 patients with uveitishepatitis demonstrated AA by CIEP. Using RIA, however, the aqueous specimen from 1 of these 6 patients was positive for AA. No AA could be detected in any of the aqueous aspirates from the six eyes from an Eye Bank, neither by CIEP nor bv RIA. Anmal Studies
The results of the animal studies are found in Tables 1 to 3. Table 1 depicts the incidence of gross liver disease, liver AA, hepatitis, and mortality
Vol. 82, No. 1 January 1976
AUSTRALIA ANTIGEN HEPATITIS IN MICE
89
by type of inoculum. Table 2 relates the mortality, hepatitis, and liver AA to each of the six hepatitis-uveitis aqueous specimens. In Table 3, the mortality, liver AA., and hepatitis are related to the month followving inoculation of the uveitis-hepatitis aqueous specimens. Mortality
All of the animals survived the trauma of the inoculation, and all remained healthy during the first month followving inoculation. However, during the second month, accelerated mortality was noted in the mice inoculated w-ith the uveitis aqueous. This phenomenon continued until the end of the experiment. By the end of 12 months, 36 mice receiving the uveitis aqueous were dead. In comparison, 32 mice receiving normal aqueous humor and :34 mice receiving saline were dead at the end of 12 months. The 12-month mortality rate (53.3 to 36. 7%7 ) in the control mice Table 1-Results in the Animals by Type of Inoculum Inocula Normal aqueous
Uveitis aqueous
Gross liver disease Australia antigen
Hepatitis
4 60 19 6 22
656
Mortality
-
6 % 6.%
0 0
31.60
60
°% 0%
0
1.6%
0 0 0
1
36.6%
93.3%
1 =
1.6%
- =
53.3%
-
32
SE difference Saline
34 -
Probability
°% 0%
(DEEC
E
F+40'
P,.i,#
44...
p
. . ,.
V .1 ,. C.I.a >:. I3
7&*
o. jl
'-
.
Figure 7-Mouse hepatitis. Australia antigen was detected in this liver. The mouse died
during the third month following direct liver inoculation of uveitis aqueous. Portions of several hepatic lobules show intense polymorphonuclear leukocyte infiltration and hyalinized necrotic liver cell cords. (H&E, x 100)
