EXPERIMENTAL ACUTE AND CHRONIC VIRAL HEPATITIS

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1 GASTROENTEROLOGY Copyright 1970 by The Williams & Wilkins Co. Vol. 58, 4 Printed in U.S.A. EXPERIMENTAL ACUTE AND CHRONIC VIRAL HEPATITIS Effect of antithyillocyte globulin GARY L. GITNICK, M.D., Roy G. SHORTER, M.D., AND LESLIE J. SCROENFIELD, M.D. Gastroenterology Unit and the Section of Organ and Tissue Transplantation Research, Mayo Clinic and Mayo Foundation, Rochester, Minnesota Antithymocyte globulin (ATG) significantly increased the mortality of mice with experimental acute viral hepatitis due to reovirus type 3 or murine hepatitis virus type 3 infection. In other experiments it converted a subclinical hepatitis to an overt illness with significant mortality. The clinical course of experimental chronic hepatitis induced by reovirus type 3 was not influenced by the administration of antithymocyte globulin. Bilirubinuria was detected throughout the acute and chronic phases both in treated and in untreated mice. The livers of mice receiving ATG during acute hepatitis showed more extensive hepatocellular necrosis and mononuclear cell infiltration than those receiving virus without ATG. In contrast, in chronic hepatitis the degree of hepatocellular necrosis and inflammatory reaction in the livers of mice was not altered by ATG. Fibrosis, but not cirrhosis, was found in the chronic phase. Viral isolation attempts were successful throughout the acute phase but not in the chronic phase. Complement-fixation, immunodiffusion, and immunoelectrophoretic techniques failed to demonstrate evidence of circulating antiliver antibody in the sera of mice having chronic hepatitis. In mice, reovirus type 3 (reo) produces acute and chronic hepatitis having some features similar to those seen in man. I,2 These include bilirubinuria, progressive hepatocellular necrosis, plasma cell and mononuclear cell infiltration, and, in the chronic phase, fibroblastic proliferation. Murine hepatitis virus type 3 (MHV3) also produces a similar acute hepatitis in Received June 17, Accepted November 5, Address requests for reprints to: Section of Publications, Mayo Clinic, Rochester, Minnesota This investigation was supported in part by Research Grants AM-6ooS, AM-5259, and AI-7S3S from the National Institutes of Health, United States Public Health Service. The authors are indebted to Miss Linda Livingston and Mrs. Linda Royer for their assistance in these studies. 516 mice. Autoimmune mechanisms have been postulated to be operative in chronic reo hepatitis in mice 2 and in chronic hepatitis in man. 3 In recent years, antilymphoid serum (ALS) has been shown to be a potent immunosuppressive agent in organ transplantation. 4, 5 Some investigators have suggested that immunosuppressive therapy with antithymocyte globulin (ATG) does not alter the immunological response to infections. 6 However, an increased incidence of polyoma virus-induced tumors has been reported in thymectomized mice and in mice treated with ALS.7 Similar findings have been demonstrated for simian virus type 40, tumors induced by adenovirus types 7 and 12,8 and Rauscher leukemia 9 and Maloney leukemia 7 viruses. Outbreaks of canine distemper virus infections lo and monkey B virus infections ll

2 April 1970 ANTITHYMOCYTE GLOBULIN IN HEPATITIS 517 have been thought, but not proven, to be related to ALB treatment. ALB has little effect on yellow fever12 or influenza 13 viral infections in mice, but newborn mice pretreated with ALB and infected with lymphocytic choriomeningitis virus develop persistent viremia. 14, 15 No information on the effect of ALB on hepatitis has been reported. Because of increasing clinical use of ALS in patients who are at high risk for hepatitis, the effect of ATG on experimental acute hepatitis in mice was investigated and, because of its effectiveness in the suppression of delayed hypersensitivity reactions, the influence of ATG on experimental chronic hepatitis in mice also was investigated. The results of immunosuppressive therapy with ATG in experimental acute reo and MHV3 hepatitis and in reo-induced chronic hepatitis in mice are described in this report. Materials and Methods Virus. Reovirus type 3 was obtained, after the sixth passage in rhesus monkey kidney cell culture, from Dr. William London (National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland). The virus was carried through two more passages in the same cell type. The virus pool used throughout these studies had an infectivity titer of % tissue culture infectious doses (TCID 50 ) and % mouse lethal doses (MLD60)' Neutralization tests using hyperimmune serum to reo were performed on this viral pool to show that it contained only this virus. Murine hepatitis virus type 3 was obtained as a lyophilized mouse liver preparation from the American Type Tissue Culture Collection. The lyophilized preparation was reconstituted in Hanks' balanced salt solution to yield a final virus pool with a titer of 10-'" MLD60. Isolation procedures. Isolation attempts were made on livers from infected and noninfected mice. Livers were minced and then homogenized. A 10% (v/ v) suspension was made in tryptose phosphate broth (Difco Laboratories, Inc., Detroit, Mich.). Virus isolations and titrations were done in roller tube cultures of monkey kidney cells using Eagle's basal medium (Grand Island Biologicals, Grand Island, N. Y., dry type) with 5% fetal bovine serum, penicillin (100 U per ml), and streptomycin (100 p.g per ml). These roller tube cultures were inoculated with 0.3 ml of each tissue suspension and were examined for cytopathic change every other day for 21 days. Three roller tubes were used for each specimen. Specimens failing to show cytopathic change were subcultured two times at 14-day intervals. Virus isolated from these specimens was shown to be reo by neutralization tests with hyperimmune serum. Rabbit anti-mouse thymocyte globulin (ATG). Two batches of two-pulse rabbit antimouse thymocyte serum were made by a method described previously.'o Each batch then was absorbed three times with normal mouse erythrocytes (1 volume of cells to 4 volumes of serum) to remove hemagglutinating and hemolytic activity and once with normal homogenized mouse kidney (1 volume of kidney homogenate to 4 volumes of serum) to remove anti-kidney antibody. The y-globulin fraction of each whole anti thymocyte serum then was separated by ammonium sulfate precipitation and subsequently was reconstituted in 0.85% saline to the original concentration in the serum. During the dialysis of the ammonium sulfate-precipitated globulin, tetracycline (50 p.g per 100 m!) was added to the dialysis fluid to prevent bacterial contamination. The globulin preparation was tested by agar gel diffusion against normal mouse serum and was found to be free of antibodies to mouse serum proteins. Immunoelectrophoresis confirmed the presence of y-globulin. After filtration through Millipore filters (0.45 p.), the ATG solutions were found to be free of bacteria as determined by inoculation on blood agar plates and thioglycollate broth. Viral isolation studies in HeLa cell, human diploid lung cell, and monkey kidney cell cultures demonstrated the absence of cytopathic agents. The protein nitrogen content of lot 1 was 1.7 mg per 100 ml and of lot 2, 2.1 mg per 100 m!. The lymphocytotoxicity for murine lymphocytes in vitro of lot 1 was 1: 16,000 and of lot 2, 1: 4,000. The immunosuppressive effect on skin allograft rejection was tested for each batch of ATG, with Swiss ICR HA mice as recipients and C57BL/6J black mice as donors. Eight treated and 8 untreated mice were used to test each batch ; untreated mice rejected their skin grafts within 7 days while grafts survived more than 20 days in all treated mice. For control purposes, two separate batches of normal rabbit serum globulin were prepared and tested in the manner described above and did not demonstrate lymphocytotoxicity in vitro or suppression of allograft rejection in vivo. Experimental procedures and design. Acute

3 518 GITNICK ET AL. Vol. 58, 4 TABLE 1. Experimental design: acute and subclinical hepatitis group Total Treatment groups dose of ATG mice per group reo hepatitis reo hepatitis MHV3 hepatitis ml of I Acute I Subclinical I Acute Reovirus type 3 and ATG treatment 0.05 ml ATG 24, 48, and 72 hr before virus inoculation ml ATG 24 and 48 hr before virus inoculation ml ATG 24 hr before virus inoculation ml ATG on days 4, 6, and 8 after virus inoculation ml ATG on days 4 and 6 after virus inoculation ml ATG on day 4 after virus inoculation Reovirus type 3 without ATG treatment Reovirus only Same as above but normal rabbit globulin instead of ATG Control treatment without virus No treatment, no virus Culture medium control, 0.05 ml modified Eagle's basal medium ip ml ATG, one injection ml ATG, two injections at 48-hr intervals ml ATG, three injections at 48-hr intervals.., a Represents 0.05 ml of normal rabbit globulin on days 4, 6, and 8 after virus inoculation. mice Hepatitis (Overt and Subclinical). Pregnant Swiss ICR HA mice were obtained from the Mayo Foundation breeding colony (Institute Hills Farm). At 4 days after their birth, 620 mice were redistributed randomly and groups of 10 were assigned to the experimental groups of three simultaneous Latin squares. The groupings within this experimental design are shown in table 1. Those groups receiving virus were inoculated intra peritoneally with 0.05 ml of a 10-4 dilution of the reo pool 4 days after birth. This virus dose (0.8 MLD50 ) previously had been shown to produce 30 to 40% mortality consistently during the first 14 days after inoculation. Those animals receiving ATG or normal rabbit globulin were inoculated subcutaneously with 0.05 ml of the undiluted pool. Previous in vivo titrations also had demonstrated that the administration of MLD50 of reo resulted in hepatic infection, as determined by viral isolation, unaccompanied by the other manifestations of reo infection, such as growth retardation, oily hair, yellow stools, or death. This virus dose was used in the studies of subclinical or inapparent hepatitis (table 1). The same experimental design was used in subsequent studies with MHV3. Mice receiving virus were inoculated intraperitoneally 4 days after birth with 0.5 MLD50 of the MHV3 pool and were distributed into groups receiving ATG, normal rabbit globulin, or no treatment (table 1). All surviving mice were killed 70 days after the initiation of the study. Chronic Hepatitis. A total of day-old mice were inoculated intra peritoneally with 0.8 MLD50 of the reo pool. Twenty mice were inoculated with uninfected tissue culture maintenance medium. Seventy days after virus inoculation the surviving mice with persistent bilirubinuria, ruffled hair, and growth retardation were distributed randomly among the groups shown in table 2. On days 74, 76, and 78 after virus inoculation, the mice received 0.1 ml of ATG, normal rabbit globulin, or no treatment. Forty control mice of the same age but not inoculated with virus also were distributed randomly to groups receiving ATG or no treatment. On day 110, all surviving mice were killed. In addition, groups of 5 previously infected, untreated, chronically ill mice were killed at 20-day intervals from day 70 to day 150. Observations and postmortem studies. All mice were examined daily for evidence of illness. Qualitative determinations of urinary

4 April 1970 ANTITHYMOCYTE GWBULIN IN HEPATITIS 519 bilirubin were performed at 7 -day intervals by the Ictotest method. l7 Autopsies and viral isolations from livers were performed on mice dying or killed during acute or chronic hepatitis. Histopathology. The livers from mice dying or killed during the acute and chronic studies were fixed in 10% buffered formalin. Five 4-p. sections were cut from each liver and stained with hematoxylin and eosin. The slides then were coded and, without prior knowledge of the treatment received, were examined by one of us (R. G. S.) j the extent of hepatocellular necrosis, mononuclear cell infiltration, Kupffer cell p r o l i f ~ r and a t i fibrosis o n, was graded 0 to 3 (0 represented normal). Complement Fixation and Immunodiffusion Studies. The sera of mice killed in the chronic hepatitis studies were tested for the presence of anticytoplasmic antibody by a mouse liver antigen prepared as described by Doniach et al.' Microtiter complement fixation tests as described b y Sever' were performed in triplicate. Each test was controlled by positive and negative rabbit serum aliquots. Gel diffusion and immunoelectrophoretic studies were performed u sing the same antigen, test sera, and controls. Statistical methods. The X' test was used to assess each of the indices."" Results Natural course. During the first 14 days after virus inoculation, all mice exhibited growth retardation, yellow stools, and oily hair j 70% had bilirubinuria. Of the day-old Swiss ICR HA mice inoculated with reo, 39% died during the first 28 days after virus inoculation, with peak mortality occurring between day 10 and day 14. Forty per cent of the original study group recovered and, by day 42, could not be distinguished from uninoculated control mice. The remaining 21 % (160 mice) developed chronic hepatitis. Viral isolation attempts were successful in 71 % of the livers of mice dying between day 10 and day 17 during the acute phase, but were uniformly unsuccessful from suspensions of livers of mice killed at 20-day intervals from day 70 to day 150 during the chronic phase. The sera of these serially killed mice were tested for the presence of complement-fixing anticytoplasmic antibody and all lacked such a circulating autoantibody. Attempts to T A BLE 2. Experimental design: chronic hepatitis group Conditions Total ATG dose mice _._ demonstrate precipitating anti-liver antibody by immunoelectrophoresis of these sera against mouse liver anticytoplasmic antigen also failed to demonstrate the presence of autoantibody. In all serological studies, appropriate positive rabbit control sera demonstrated the presence of anti-liver antibody. Experimental acute hepatitis. Administration of ATG on days 4,6, and 8 after reo inoculation resulted in a significant increase in mortality (table 3) together with the clinical findings of more severe cachexia, oily hair, and growth retardation. There was an 82% mortality among mice receiving virus followed by ATG, in contrast to a mortality of 34% among those receiving virus plus normal rabbit globulin or virus only (P < 0.01). Mortality was less than 5% among mice in the control groups. Similar results were obtained when the second batch of ATG was tested. Administration of A TG 24, 48, and 72 hr prior to viral inoculation resulted in a high mortality, equivalent to that seen with three injections after virus inoculation. Administration of A TG 4 and 6 days after viral inoculation or 24 and 48 hr prior to viral inoculation resulted in 100% mortality, in contrast to a mortality of 30 to 40% among mice receiving virus plus normal rabbit globulin (P < 0.01). However, a single A TG inj ection 4 days after or 24 hr prior to viral inoculation resulted in 30 to Chronic disease and ATG on days 74, 76, and 78 after virus inocu- lation...,.,......,.. '" Chronic disease and normal rabbit globulin treatment (instead of ATG) on days 74, 76, and 78 after virus inoculation Chronic disease and no treatment No chronic disease, ATG on days 74, 76, and 78 after initiation of study No chronic disease, no treatment ml

5 520 GITNICK ET AL. Vol. 58, 4 TABLE 3. Effect of ATG on mortality of mice with reovirus type 3 infection (deaths in first 28 days) Treatment Schedule ATGlot 1 ATG lot 2 Dead Dead dead/total dead/total % % Virus and ATG... 4, 6, and 8 days after infection 82 41/ / 10 Virus and N R. G.... ~ 4, 6, and 8 days after infection 34 17/ /10 Virus and ATG , 48, and 72 hr before infection / /10 Virus and NRG , 48, and 72 hr before infection 40 4/ / 10 Virus and ATG... 4 and 6 days after infection / / 10 Virus and NRG and 6 days after infection 40 4/ /10 Virus and ATG and 48 hr before infection 90 9/ /10 Virus and NRG and 48 hr before infection 30 3/ / 10 Virus and ATG days after infection Virus and NRG days after infection Virus and ATG hr before infection Virus only ATG only No treatment / 10 3/10 22/49 37/ 110 2/ 60 0/ / 10 4/10 4/10 4/ 10 2 /40 0/20 a NRG, normal rabbit globulin p<o.01 Grade :3 Virus + ATG Virus + Virus Normal globulin FIG. 1. Effect of ATG on hepatocellular necrosis in mice with acute reo hepatitis. Data are from 20 mice dying between days 10 and 17 in each group. 40% mortality, which was not different significantly from that in mice receiving only virus or virus plus normal rabbit globulin. Bilirubinuria was detected only in mice infected with reo and was not detected in uninfected mice receiving A TG or normal rabbit globulin. Bilirubinuria was found in 70% of infected mice and this incidence was not altered by the administration of ATG (lot 1 or 2) or normal rabbit globulin. The results of the hepatic histopathological evaluation are given in figure 1. The number of livers showing grade 3 necrosis was significantly greater among the mice receiving virus plus A TG than among mice in the other groups (P < 0.01). A similar pattern was observed when comparisons were made of the degrees of Kupffer cell proliferation and of mononuclear cell infiltration (P < 0.01). A TG treatment alone did not influence hepatic histology. Reo was isolated from 94% of the livers of mice which received virus followed by sequential injections of ATG and died during a 7-day period (days 10 to 17) in the acute phase. Similar results were obtained when comparisons were made of virus recovery during the entire acute phase. The effect of ATG on the mortality of mice with MHV3 hepatitis is illustrated in table 4. Mortality was 97% among mice receiving ATG injections on days 4, 6, and 8 after virus inoculation but, in contrast, 25% or less among mice in the other groups (P < 0.01). Experimental subclinical hepatitis. In mice with subclinical or inapparent viral

6 April 1970 ANTITHYMOCYTE GWBULIN IN HEPATITIS 521 TABLE 4. Effect of A TG on mortality of mice with murine hepatitis virus type 3 infection (deaths in first 28 days) Treatment Schedule Dead ATG lot 1 ATG lot 2 dead/total Dead dead/total --- % % Virus and ATG... 4, 6, and 8 days after infection / /20 Virus and NRG... 4, 6, and 8 days after infection 15 3/ /20 Virus only ATG only No treatment / / / /20 0 0/ /20 hepatitis, mortality was 30% among those receiving MLD50 of reo followed by sequential injections of ATG, in contrast to a mortality of 5% or less among mice in the other groups (P < 0.01). Bilirubinuria was found in 90% of the ATG-treated virus-infected mice but not in any mice in the other groups. The A TG-treated infected mice showed all of the clinical manifestations of reo infection while the untreated infected mice appeared normal. Experimental chronic hepatitis. Chronically ill infected mice did not differ grossly during the course of these studies whether they received ATG, normal rabbit globulin, or no treatment; growth retardation, ruffled hair, and bilirubinuria were observed in each of the groups. No virus was isolated from the livers of mice dying during the course of the study or from the livers of mice killed at the end of the study. Although 15% of the animals receiving ATG died compared with none of the mice in the other groups, this difference was not significant (P = 0.2). Hepatocellular necrosis, mononuclear cell infiltration, and fibrosis were present to approximately equivalent degrees among mice in each of the. treatment and appropriate control groups. Although fibrosis was seen in the livers of mice throughout the chronic phase, cirrhosis was not found. Discussion ATG has been thought by some to suppress allograft rejection without suppression of the defense mechanisms against infectious agents. 6 However, severe cytomegalovirus infections among renal homo- transplantation patients treated with ALS, corticosteroids, and azathioprine have been reported,21 although the contribution of ALS had not been isolated. The present studies have demonstrated in an in vivo system that ATG significantly altered the course of experimental acute viral hepatitis. Mortality was increased and the degree of hepatic histopathological change was more severe in the ATG-treated virus-infected animals, both in mice pretreated with ATG and in those treated with ATG after virus inoculation. Since a minimum of two injections of A TG was necessary to alter the mortality in reo-infected mice, it might be questioned whether the increased mortality after two or three injections could be ascribed to anaphylaxis after challenge of an animal previously sensitized to rabbit serum protein. However, if the increased mortality were due to anaphylaxis, it would have been expected that uninfected mice receiving two or three A TG inj ections would have died, but there was no mortality in these control groups. A more plausible explanation is that ATG suppressed the immune response of the mice to the virus. The mechanism of this effect of ATG on acute hepatitis is difficult to explain because, despite many theories, little is known of the mode of action of ALS. ALS does not decrease immunoglobulin levels in monkeys.24 Although the injection of ALS usually produces a lymphopenia,25 it has not been possible to correlate the degree of lymphopenia with the degree of immunosuppressive activity in vivo ALS has

7 522 GITNICK ET AL. Vol. 58, 4 been found to transform human lymphocytes to lymphoblasts in vitro.28 When used for the cultivation of virus, these transformed cells were shown to produce up to 1000 times more vesicular stomatitis virus particles than did their lymphocyte precursors.29 This mechanism may have been operative in vivo in our experimental model. However, our findings do not contribute directly to the proof of this concept and further data to substantiate the hypothesis have not been reported from elsewhere. Whatever the mechanism of action of A TO in modifying viral infection may be, the demonstration that an inapparent viral infectious process can be transformed to an evident clinical disease could provide a useful tool in the study of other viral processes, particularly when the virus cannot be cultivated or can be cultivated only with difficulty. The administration of viral isolation specimens to ATO-treated animals might enhance the clinical expression of the virus and so indicate the presence of an infectious agent. Stanley and co-workers2 described the development of chronic hepatitis in mice inoculated with reo. The present studies confirm these observations and further demonstrate that the administration of A TO in the dosage schedule used had no demonstrable beneficial or harmful effect on the course of this chronic hepatitis in mice. Since autoimmune mechanisms have been thought to be active in the chronic phase of reo hepatitis? the finding of a lack of beneficial effect resulting from the administration of ATO would suggest either that autoimmune mechanisms in fact do not play a prominent role in its perpetuation or that ATO is not effective in the dosage used against the autoimmune mechanisms which may be operative in this disease. Although the current studies do not resolve this question, the previously documented inability to transfer the disease by serum or by cells from affected to normal animals,2 together with the present data demonstrating that treatment with a potent immunosuppressive agent fails to affect the course of the disease, might support a conclusion that autoimmunity does not play a prominent role in the perpetuation of this experimental chronic hepatitis. The inability to cultivate infectious virus from the liver during the chronic phase of this process suggests either that virus is present at levels or in a form not amenable to current isolation procedures, that virus is present as a subunit antigen, or that active viral infection is in fact not present in the chronic phase. The demonstration that the administration of A TO to animals inoculated with as little as MLD50 of reo could convert a subclinical hepatic infection to an overt illness with significant mortality suggests that, if virus were present during the chronic hepatic process, the administration of ATO would unmask its presence either clinically or by cultural techniques. Since this did not occur, it could be concluded that reovirusinduced chronic hepatitis in mice is not perpetuated by active viral infection. Evidence for the presence of circulating autoantibody has not been provided, but the presence of viral subunit particles or cellbound antibody has not been excluded. The relationship of this model to human disease remains to be determined. However, the findings suggest that, if hepatitis develops in a patient receiving an organ transplant and multiple blood transfusions, augmentation of acute hepatitis may be a complication of administration of ATO. REFERENCES 1. Walters, M. N.-I., R. A. Joske, P. J. Leak, and N. F. Stanley Murine infection with reovirus. I. Pathology of the acute phase. Brit. J. Exp. Path. 44: Stanley, N. F., P. J. Leak, M. N.-I. Walters, and R. A. Joske Murine infection with reovirus. II. The chronic disease following reovirus type 3 infection. Brit. J. Exp. Path. 45: Mackay, I. R., S. Weiden, and J. Hasker Autoimmune hepatitis. Ann. N. Y. Acad. Sci. 124: Starzl, T. E., T. L. Marchioro, K. A. Porter, Y. Iwasaki, and G. J. Cerilli The use of heterologous antilymphoid agents in canine renal and liver homotransplantation and in human renal homotransplantation. Su.rg. Gynec. Obstet. 124: Abaza, H. M., B. Nolan, J. G. Watt, and M. F. A. Woodruff Effect of anti-

8 April 1970 ANTITHYMOCYTE GLOBULIN IN HEPATITIS 523 lymphocyte serum on the survival of renal homotransplants in dogs. Transplantation 4: Russell, P. S Antilymphocyte serum as an immunosuppressive agent (editorial). Ann. Intern. M ed. 68: Allison, A. C., and L. W. Law Effects of antilymphocyte serum on virus oncogenesis. Proc. Soc. Exp. Biol. M ed. 127: 207-2' Allison, A. C., L. D. Berman, and R H. Levey Increased tumour induction by adenovirus type 12 in thymectomized mice and mice treated with anti-lymphocyte serum. Nature (London) 215: Hirsch, M. S., and F. A. Murphy Effects of anti-thymocyte. serum on rauscher virus infection of mice. Nature (London) 218: Woodruff, M. F. A Discussion. In G. E. W. Wolstenholme and M. O'Connor, [eds.], Antilymphocytic serum. Ciba Foundation Study Group 29, p Little, Brown & Company, Boston. 11. Van Bekkum, D. W Discussion. In G. E. W. Wolstenholme, and M. O'Connor, [eds.], Antilymphocytic serum. Ciba Foundation Study Group 29, p Little, Brown & Company, Boston. 12. Hirsch, M. S., and F. A. Murphy Effects on anti-thymocyte serum on 17-D yellow fever infection in adult mice. Nature (London) 216: Hirsch, M. S., and F. A. Murphy Effects of anti-lymphoid sera on viral infections. Lancet 2: Hirsch, M. S., F. A. Murphy, H. P. Russe, and M. D. Hicklin Effects of antithymocyte serum on lymphocytic choriomeningitis (LCM) virus infection in mice. Proc. Soc. Exp. Biol. Med. 125: Volkert, M., and C. Lundstedt The provocation of latent lymphocytic choriomeningitis virus infections in mice by treatment with antilymphocytic serum. J. Exp. M ed. 127: Kubista, T. P., R G. Shorter, and G. A. Hallenbeck Acceptance of Walker 256 carcinosarcoma by C57BLj6 mice treated with rabbit anti-mouse-thymus serum. Cancer Res. 27: Shutkin, M. W., and D. Caine The clinical value of bilirubin determinations in routine urinalysis with an improved method. Amer. J. Gastroent. 23: Doniach, D.,!. M. Roitt, J. G. Walker, and S. Sherlock Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other liver diseases and their clinical implications. Clin. Exp. Immun. 1: Sever, J. L Application of a microtechnique to viral serological investigations. J. Immun. 88: Snedecor, G. W., and W. G. Cochran Statistical methods, Ed. 6, p. 20. Iowa State University Press, Ames, Iowa. 21. Fulginiti, V. A., R. Scribner, C. G. Groth, C. W. Putnam, L. Brettschneider, S. Gilbert, K. A. Porter, and T. E. Starz! Infections in recipients of liver homografts. New Eng. J. Med. 279: Levey, R H., and P. B. Medawar Nature and mode of action of antilymphocytic antiserum. Proc. Nat. Acad. Sci. U. S. A. 56: '3. Russell, P. S., and A. P. Monaco Heterologous antilymphocyte sera and some of their effects. Transplantation 5: Bainer, H Discussion. In G. E. W. Wolstenholme, and M. O'Connor, [eds.], Antilymphocytic Serum. Ciba Foundation Study Group 29, p Little, Brown & Company, Boston. 25. Monaco, A. P., M. L. Wood, B. A. van der Werf, and P. S. Russell Effects of antilymphocyte serum in mice, dogs and man. In G. E. W. Wolstenholme, and M. O'Connor, [eds.], Antilymphocytic Serum. Ciba Foundation Study Group 29, p Little, Brown & Company, Boston. 26. Levey, R. H., and P. B. Medawar Some experiments on the action of antilymphoid antisera. Ann. N. Y. Acad. Sci. 129: Jeejeebhoy, H. F The relationship of lymphopenia production and lymphocyte agglutinating and cytotoxic antibody titers to the immunosuppressive potency of heterologous antilymphocyte plasma. Transplantation 5: Grasbeck, R., C. Nordman, and A. De La Chapelle Mitogenic action of antileucocyte immune serum on peripheral leucocytes in vitro. Lancet 2: Edelman, R, and E. F. Wheelock Enhancement of replication of vesicular stomatitis virus in human lymphocyte cultures treated with heterologous anti-lymphocyte serum. Lancet 1: