Saturday
ADMINISTRATION OF HUMAN FIBROBLAST INTERFERON IN CHRONIC HEPATITIS-B INFECTION M. B. RAY A. F. BRADBURNE V. G. EDY
J. DESMYTER J. DE GROOTE V. J. DESMET
P. DE SOMER
A. BILLIAU
J.
MORTELMANS
Rega Institute for Medical Research and Faculty of Medicine, Leuven; and the Zoo, Antwerp, Belgium
patient with hepatitis-B surface chronic (HBsAg)-positive antigen aggressive hepatitis, and two chimpanzee carriers of HBsAg, were each given seven doses of 107 I.U. of One
Summary
human fibroblast interferon over two weeks. The main difference observed after treatment was a depression of the nucleocapsid hepatitis-B core antigen in the liver, indicating that hepatitis-B virus infection is sensitive to interferon. Except for a short febrile reaction, no undesirable effects were seen after the administration of human fibroblast interferon which has not been previously
given to man. Introduction 1_aTERFExort has become start
therapeutic
sufficiently available to by parenteral injection in man. inducers, the human leucocyte inter-
Materials and Methods was
September 1976
the double-stranded R.N.A., poly (I). poly (C), and superinduction.’ It was concentrated and purified by fractional precipitation with ammonium sulphate, titrated by inhibition of vesicular stomatitis virus replication in human diploid cells, using a dye-uptake method,8 and calibrated against the Medical Research Council 69/19 standard of human interferon. Each dose contained 10’i.u. of human interferon and was 10 ml in volume. One treatment consisted of seven doses injected intramuscularly every alternate day. Hepatitis-B surface antigen (HBsAg) was measured in
by complement fixation, passive haemagglutination (Wellcome), and radioimmunoassay (’Ausria’ n, Abbott) adapted for end-point titration. Anti-HBs was measured by passive haemagglutination and by radioimmunoassay (’Ausab’, Abbott). Antibody against the core antigen (anti-HBc) was titrated by a solid phase radioimmunoassay developed in our The e antigen was laboratory, using a liver-extracted HBcAg. determined by double immunodiffusion.99 HBsAg and HBcAg were determined in the liver by immunofluorescence on frozen sections of needle biopsy speci11 mens as previously described. to Quantitation of the fluorescent cells was found to be sufficiently reproducible by comparison of different sections from the same biopsy specimen prepared at different times. Sampling error was not a problem, as seen by the comparison of 2 or 3 biopsy cylinders which were taken during the same session, and by the absence of variable results in chimpanzees who were biopsied frequently. Variability due to subjectivity of interpretation and to technical variation was further minimised by examining the liver and serum specimens repeatedly, grouped together, and in blind serum
order.
trials
L’ntike interferon feron, which has been used up to now, did not cause obvious toxicity.1-3 Since no satisfactory therapy exists for potentially life-threatening forms of hepatitis B, attempts at therapy with interferon seem justified in these cases. The sensitivity of hepatitis-B virus to interferon is not known. Interferon has not been detected in Mtients with various stages of acute and chronic hepatitis, including hepatitis B,4-6 suggesting that hepatitis-B irus is a poor inducer of interferon and that passively dmlillstered interferon is not rendered redundant by endogenous interferon in this infection. We report our first results of the administration of human fibroblast inin chronic hepatitis-B infection.
!,.-,erferon
25
prepared in human diploid fibroblasts, using
Results The treatment was first given to two chimpanzees and then to a patient. They had no detectable interferon in their serum before treatment. Injections of interferon were well tolerated, and clinical, biochemical, and hxmatological screening showed no side-effects, with one exception: a raised temperature, 2-10 h after injection, and with a peak of up to 389°C, was observed in the patient; this was treated with aspirin. Temperature was not measured in the chimpanzees, who required general anaesthesia for handling. Two male chimpanzees each weighing 60 kg, had been chronic carriers of HBsAg since they were first examined. Their liver-function tests and liver histology were within the normal range in the year before, during, and in the six months after treatment. Their serumHBsAg titres were not influenced by treatment. They ,.
-
646 had no demonstrable anti-HBs. Immunofluorescence on the liver (3 biopsy specimens in the three months before, one immediately before, 3 during, and 12 up to four months after treatment) showed weak peripheral cytoplasmic and membrane staining for HBsAg in 15% of the hepatocytes in both animals; this was not modified during or after treatment, with one exception: chimpanzee 1, one to two wk after treatment, had areas in which all hepatocytes showed full cytoplasmic staining for HBsAg; membrane expression was minimal at that time. The most striking changes were seen in the HBc system. In chimpanzee 1, 15% of the nuclei of the hepatocytes stained positively for HBcAg in biopsy specimens taken before treatment. After one week of treatment, the number of positive nuclei and the intensity of staining decreased progressively during five weeks; at that time less than 0.5% of the nuclei were positive. The number of positive nuclei then rose quickly again and attained 30% after one month. Serum-anti-HBc rose from a stable titre of 16 before and during treatment to 128 one week afterwards; this was maintained for six weeks and then declined. Chimpanzee 2 had no demonstrable HBcAg in the liver at any time. He had a constant, high anti-HBc titre of 20 000 before treatment. It fell surprisingly to 128 after one week’s treatment, and remained at that level during the following weeks. The patient, a 42-year-old male weighing 54 kg, had HBsAg-positive chronic aggressive hepatitis which was first diagnosed in 1969. He had been taken off immunosuppressive drugs two years before treatment with interferon. No evolution was noted in the year before treatment ; serum glutamic-oxaloacetic-transaminase and glutamic-pyruvic-transaminase values were pathological and fluctuated between 30 and 100 u/litre; the serumbilirubin levels were normal. Frequent blood-samples taken between three months before and three months after treatment did not show significant changes in these values. The titres of HBsAg and of anti-HBs (which was demonstrable in this patient only by radioimmunoassay), also did not change. However, a change was observed in HBsAg expression in the liver. A biopsy specimen taken immediately before treatment showed strong HBsAg staining of the membrane in 90% of the cells, and cytoplasmic staining in 5%. Immediately after treatment, there was 60% membrane staining, and 15% cytoplasmic staining, both of moderate intensity. Five weeks later, in the last biopsy specimen available, there was 60% membrane staining, and 35% cytoplasmic staining, both of moderate intensity. Liver histology remained unchanged during this period. Changes were again seen in the HBc system. HBcAg stained at high intensity in 30% of the hepatocyte nuclei before treatment. Immediately after treatment, 20% of the nuclei were positive at very low intensity; five weeks later, only 3%. A few hepatocytes showed cytoplasmic HBcAg staining in the pretreatment biopsy specimen, but not later. The serumanti-HBc titre of 3200 remained essentially unchanged during the observation period. Evidence that the changes were due to the treatment and not to unrelated, spontaneous fluctuations in the expression of HB antigens was as follows. No changes were seen in the different biopsy specimens taken from each chimpanzee before treatment. The changes arose quickly after interferon had been administered, and were interpretable in terms of antiviral action. In 4 con-
trol
patients, 3 with chronic aggressive hepatitis and HBsAg and HBcAg in their liver, and one apparenth normal carrier of HBsAg in whose liver HBsAg but no HBcAg was detected, repeat biopsy specimens were taken over a period of 2 years during which their histological and clinical status did not change; there were no changes in the expression of HB antigens in their livers, In contrast, five other patients with chronic, HBsAgpositive hepatitis whose histological and clinical status did evolve, also showed changing patterns of viral antigens in their livers. The e antigen was not detectable in serum of the patient and of the chimpanzees at any time of the study. Chimpanzee 1 had been the first chimpanzee in which Dane particles could be shown,12 however, this animal had lost most of its Dane particles two years before the study. At the time of the study, Dane particles could be shown in the patient and in chimpanzee 1 only with difficulty and in sporadic specimens, not allowing longitudinal comparison; they could not be shown in chimpanzee 2.
Discussion The results suggest that hepatitis-B virus infectionis sensitive to interferon, and that it is the expression of the HBc or nucleocapsid system which is primarilv affected. Suppression of HBcAg was seen upon treatment in both livers which had demonstrable antigen. Changes were also seen in the titres of circulating antiHBc, but no simple relationship was present with the changes in HBcAg. The titres of circulating HBsAg were not modified under the conditions of this study, but a shift from membrane staining to cytoplasmic staining was noted for HBsAg in two livers. It is important to note that, in chronic hepatitis in man, predominant membrane staining for HBsAg is the hallmark of the aggressive form and coincides with a high level of nuclear HBcAg, while predominant cytoplasmic staining for HBsAg is the hallmark of the clinically subdued persistent form and coincides with little or no demonstrable nuclear HBcAg.l0 1’ Interferon therefore seems to have modified the expression of HB antigens in the liver into a pattern associated with lower disease activity. The changes in viral expression appeared to be transient, although this needs to be further substantiated by additional observations. With other viruses, interferon treatment has not led to the eradication of the viral genome from a cell with established chronic infection. It is therefore not surprising that the effects observed in this study could be annulled after withdrawal of interferon. However, more prolonged or more intensive treatment might, hopefully, have a more durable influence on cell population dynamics-and eventually improve liver function and histology-by favouring the replacement of infected cells by uninfected cells, which are shielded infection by the interferon. The immune system is widely suspected to be paramount in the removal of cells infected with HB virus. In this respect, the fact that interferon exerts a complex influence on the immune system,13 albeit at doses which exceed those required fo: antiviral activity, also needs consideration. The activity of interferon in chronic hepatitis-B mfe; tion suggests that it may also have useful acting ir acute hepatitis, since acute infection by other viruses:
against
647
seneraity more readily influenced than chronic infection. Viruses which in acute infection are sensitive to interferon, have indeed been shown to be either insensitlBe14or sensitive15 to interferon in chronic infection. This study represents the first application of human fibroblast interferon to man. Fibroblast interferon has the advantage over leucocyte interferon in that it is produced from a type of cell which could be grown in unlimited amounts and which has been approved for the
preparation of substances which require stringent safety conditions, such as vaccines. However, important differences exist in the chemical and biological properties of both interferons;16 17 they have different dose-response curves and therefore their titres cannot be strictly compared,18 and their pharmacology in man could thus be different. Human fibroblast interferon was well tolera febrile reaction which was similar to that observed by others with many but not all preparations of leucocyte interferon. Comparative pyrogenicity tests in rabbits between the preparation used in this study, and fibroblast interferon presently being purified on glass,19 suggest that this hindrance has now been
ated, except for
overcome.
Requests for reprints should be addressed to J. D., Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Rega Institute,
REFERENCES
1. Strander, H., Cantell, K., Carlström, G., Jakobsson, P. A. J. natn. Cancer Inst. 1973, 51, 733. 2. Jordan, G. W., Fried, R. P., Merigan, T. C. J. infect. Dis. 1974, 130, 56. 3 Emödi, G., Just, M., Hernandez, R., Hirt, H. R. J. natn. Cancer Inst. 1975, 54, 1045. 4. Taylor, P. E., Zuckerman, A. J. J. med. Microbiol. 1968, 1, 217. 5 Wheelock, E. F., Schenker, S., Combes, B. Proc. Soc. exp. Biol. Med. 1968, 128, 251. 6. Hill, D. A., Walsh, S. H., Purcell, R. H. ibid. 1971, 136, 853. 7 Billiau, A., Joniau, M., De Somer, P. J. gen. Virol. 1973, 19, 1. 8 Finter, N B. ibid. 1969, 5, 419. 9 Magnius, L. O., Espmark, J. A. J. Immun. 1972, 109, 1017. 10 Ray, M. B., Desmet, V. J., Fevery, J., DeGroote, J., Bradburne, A. F., Desmyter, J. J. clin. Path. 1976, 29, 94. 11 Ray, M. B., Desmet, V. J., Bradburne, A. F., Desmyter, J., Fevery, J., De Groote, J. Gastroenterology, (in the press). 12 Desmyter, J., Liu, W. T., Creemers, J. Am. J. Dis. Child. 1972, 123, 315. 13. Johnson, H M., Baron, S. I.R.C.S. med. Sci. 1976, 4, 50. 14 Desmyter, J., Rawls, W. E., Melnick, J. L., Yow, M. D., Barrett, F. J. Immun. 1967, 99, 771. 15. Billiau, A., Sobis, H., De Somer, P. Int. J. Cancer 1973, 12, 646. 16 Havell, E. A., Berman, B., Ogburn, C. A., Berg, K., Paucker, K., Vilcek, J. Proc. natn. Acad. Sci. U.S.A. 1975, 72, 2185. 17 Desmyter, J., Stewart, W. E. II, Virology, 1976, 70, 451. 18 Edy, V.G, Billiau, A., De Somer, P. J. gen. Virol. 1976, 31, 251. 19 Edy, V G., Braude, I. A., De Clercq, E., Billiau, A., De Somer, P. J. gen. Virol. (in the press).
Addendum Since this paper was printed Greenberg et al.* have reported that human leucocyte interferon depressed hepatitis-B core expression in the blood of 3 patients ,,’.ith chronic aggressive hepatitis. Liver specimens were not examined; and Greenberg et al. were unable to demonstrate an effect in a patient who did not have sufficient core in the serum. The two papers thus show that interferon can depress hepatitis-B core in two different ’.’,ays in serum and in liver). Our methods are applicable the majority of cases of chronic hepatitis B which are r. accompanied by large amounts of core antigen or !jMe particles in the blood. * Greenberg, H B., Pollard, W. S, Merigan, T C.
R. B., Lutwick, L. I., Gregory, P. B., New Engl. J. Med. 1976, 295, 517.
Robinson,
IDENTIFICATION OF HIGH-RISK PATIENTS WITH APLASTIC ANÆMIA IN SELECTION FOR
ALLOGENEIC BONE-MARROW TRANSPLANTATION HANS-PETER LOHRMANN DIETRICH NIETHAMMER
PETER KERN HERMANN HEIMPEL
Division of Hœmatology, Department of Internal Medicine and Division of Hœmatology, Department of Pœdiatrics, University of Ulm, D-7900 Ulm, Federal Republic of Germany
Of 75 patients with aplastic anæmia treated between 1968 and 1975, 33 were retrospectively considered as potential candidates for allogeneic bone-marrow transplantation on the basis of their age and severity of marrow failure. The prognosis of these patients with conservative treatment was assessed from parameters obtained at the time of the initial diagnosis. Initial peripheral-blood granulocyte or platelet concentrations were not of prognostic value. In contrast, initial reticulocyte concentrations, allowed separation of the patients into two groups with poor and good prognosis. Low initial reticulocyte concentrations (less than 10 000/µl) indicated those patients at extremely high risk of succumbing to their marrow aplasia (there were no survivors 36 months after diagnosis). In contrast, 75% of those patients with more than 10 000 reticulocytes per µl at diagnosis survived for 3 years. Initial peripheral-blood reticulocyte concentrations thus appear to indicate the extent of the marrow failure in aplastic anæmia more accurately than granulocytes or platelets. Low initial reticulocyte concentrations may indicate, among patients with severe aplastic anæmia, those for whom allogeneic bone-marrow transplanation should be seriously considered; patients with higher initial reticulocyte concentrations may benefit from conservative treatment.
Summary
Introduction
improved supportive care, patients with aplastic anaemia continue to have a high mortarate. 1- The therapeutic value of androgenic steroids lity remains controversial.46 Allogeneic bone-marrow transplantation has been introduced as a therapeutic alternaDESPITE
severe
tive to conservative treatment, and recent results have been encouraging.7 11 Although conservatively treated controls were not available in these studies, clinical experience suggests the superiority of the transplantation
approach. The uncertainty about the individual spontaneous prognosis remains a major problem in marrow transplantation for aplastic anaemia. Patients who might have otherwise succumbed to their disease, may survive after marrow transplantation, but patients who might have achieved spontaneous remission of their marrow failure may die. Parameters to indicate patients at high risk early in the course of their disease are needed to help in the decision for or against a marrow transplantation. In the present study we have attempted to define the prognosis of potential bone-marrow transplant recipients with severe aplastic ansemia from criteria available at the time of the initial diagnosis.
ADMINISTRATION OF HUMAN FIBROBLAST INTERFERON IN CHRONIC HEPATITIS-B INFECTION M. B. RAY A. F. BRADBURNE V. G. EDY
J. DESMYTER J. DE GROOTE V. J. DESMET
P. DE SOMER
A. BILLIAU
J.
MORTELMANS
Rega Institute for Medical Research and Faculty of Medicine, Leuven; and the Zoo, Antwerp, Belgium
patient with hepatitis-B surface chronic (HBsAg)-positive antigen aggressive hepatitis, and two chimpanzee carriers of HBsAg, were each given seven doses of 107 I.U. of One
Summary
human fibroblast interferon over two weeks. The main difference observed after treatment was a depression of the nucleocapsid hepatitis-B core antigen in the liver, indicating that hepatitis-B virus infection is sensitive to interferon. Except for a short febrile reaction, no undesirable effects were seen after the administration of human fibroblast interferon which has not been previously
given to man. Introduction 1_aTERFExort has become start
therapeutic
sufficiently available to by parenteral injection in man. inducers, the human leucocyte inter-
Materials and Methods was
September 1976
the double-stranded R.N.A., poly (I). poly (C), and superinduction.’ It was concentrated and purified by fractional precipitation with ammonium sulphate, titrated by inhibition of vesicular stomatitis virus replication in human diploid cells, using a dye-uptake method,8 and calibrated against the Medical Research Council 69/19 standard of human interferon. Each dose contained 10’i.u. of human interferon and was 10 ml in volume. One treatment consisted of seven doses injected intramuscularly every alternate day. Hepatitis-B surface antigen (HBsAg) was measured in
by complement fixation, passive haemagglutination (Wellcome), and radioimmunoassay (’Ausria’ n, Abbott) adapted for end-point titration. Anti-HBs was measured by passive haemagglutination and by radioimmunoassay (’Ausab’, Abbott). Antibody against the core antigen (anti-HBc) was titrated by a solid phase radioimmunoassay developed in our The e antigen was laboratory, using a liver-extracted HBcAg. determined by double immunodiffusion.99 HBsAg and HBcAg were determined in the liver by immunofluorescence on frozen sections of needle biopsy speci11 mens as previously described. to Quantitation of the fluorescent cells was found to be sufficiently reproducible by comparison of different sections from the same biopsy specimen prepared at different times. Sampling error was not a problem, as seen by the comparison of 2 or 3 biopsy cylinders which were taken during the same session, and by the absence of variable results in chimpanzees who were biopsied frequently. Variability due to subjectivity of interpretation and to technical variation was further minimised by examining the liver and serum specimens repeatedly, grouped together, and in blind serum
order.
trials
L’ntike interferon feron, which has been used up to now, did not cause obvious toxicity.1-3 Since no satisfactory therapy exists for potentially life-threatening forms of hepatitis B, attempts at therapy with interferon seem justified in these cases. The sensitivity of hepatitis-B virus to interferon is not known. Interferon has not been detected in Mtients with various stages of acute and chronic hepatitis, including hepatitis B,4-6 suggesting that hepatitis-B irus is a poor inducer of interferon and that passively dmlillstered interferon is not rendered redundant by endogenous interferon in this infection. We report our first results of the administration of human fibroblast inin chronic hepatitis-B infection.
!,.-,erferon
25
prepared in human diploid fibroblasts, using
Results The treatment was first given to two chimpanzees and then to a patient. They had no detectable interferon in their serum before treatment. Injections of interferon were well tolerated, and clinical, biochemical, and hxmatological screening showed no side-effects, with one exception: a raised temperature, 2-10 h after injection, and with a peak of up to 389°C, was observed in the patient; this was treated with aspirin. Temperature was not measured in the chimpanzees, who required general anaesthesia for handling. Two male chimpanzees each weighing 60 kg, had been chronic carriers of HBsAg since they were first examined. Their liver-function tests and liver histology were within the normal range in the year before, during, and in the six months after treatment. Their serumHBsAg titres were not influenced by treatment. They ,.
-
646 had no demonstrable anti-HBs. Immunofluorescence on the liver (3 biopsy specimens in the three months before, one immediately before, 3 during, and 12 up to four months after treatment) showed weak peripheral cytoplasmic and membrane staining for HBsAg in 15% of the hepatocytes in both animals; this was not modified during or after treatment, with one exception: chimpanzee 1, one to two wk after treatment, had areas in which all hepatocytes showed full cytoplasmic staining for HBsAg; membrane expression was minimal at that time. The most striking changes were seen in the HBc system. In chimpanzee 1, 15% of the nuclei of the hepatocytes stained positively for HBcAg in biopsy specimens taken before treatment. After one week of treatment, the number of positive nuclei and the intensity of staining decreased progressively during five weeks; at that time less than 0.5% of the nuclei were positive. The number of positive nuclei then rose quickly again and attained 30% after one month. Serum-anti-HBc rose from a stable titre of 16 before and during treatment to 128 one week afterwards; this was maintained for six weeks and then declined. Chimpanzee 2 had no demonstrable HBcAg in the liver at any time. He had a constant, high anti-HBc titre of 20 000 before treatment. It fell surprisingly to 128 after one week’s treatment, and remained at that level during the following weeks. The patient, a 42-year-old male weighing 54 kg, had HBsAg-positive chronic aggressive hepatitis which was first diagnosed in 1969. He had been taken off immunosuppressive drugs two years before treatment with interferon. No evolution was noted in the year before treatment ; serum glutamic-oxaloacetic-transaminase and glutamic-pyruvic-transaminase values were pathological and fluctuated between 30 and 100 u/litre; the serumbilirubin levels were normal. Frequent blood-samples taken between three months before and three months after treatment did not show significant changes in these values. The titres of HBsAg and of anti-HBs (which was demonstrable in this patient only by radioimmunoassay), also did not change. However, a change was observed in HBsAg expression in the liver. A biopsy specimen taken immediately before treatment showed strong HBsAg staining of the membrane in 90% of the cells, and cytoplasmic staining in 5%. Immediately after treatment, there was 60% membrane staining, and 15% cytoplasmic staining, both of moderate intensity. Five weeks later, in the last biopsy specimen available, there was 60% membrane staining, and 35% cytoplasmic staining, both of moderate intensity. Liver histology remained unchanged during this period. Changes were again seen in the HBc system. HBcAg stained at high intensity in 30% of the hepatocyte nuclei before treatment. Immediately after treatment, 20% of the nuclei were positive at very low intensity; five weeks later, only 3%. A few hepatocytes showed cytoplasmic HBcAg staining in the pretreatment biopsy specimen, but not later. The serumanti-HBc titre of 3200 remained essentially unchanged during the observation period. Evidence that the changes were due to the treatment and not to unrelated, spontaneous fluctuations in the expression of HB antigens was as follows. No changes were seen in the different biopsy specimens taken from each chimpanzee before treatment. The changes arose quickly after interferon had been administered, and were interpretable in terms of antiviral action. In 4 con-
trol
patients, 3 with chronic aggressive hepatitis and HBsAg and HBcAg in their liver, and one apparenth normal carrier of HBsAg in whose liver HBsAg but no HBcAg was detected, repeat biopsy specimens were taken over a period of 2 years during which their histological and clinical status did not change; there were no changes in the expression of HB antigens in their livers, In contrast, five other patients with chronic, HBsAgpositive hepatitis whose histological and clinical status did evolve, also showed changing patterns of viral antigens in their livers. The e antigen was not detectable in serum of the patient and of the chimpanzees at any time of the study. Chimpanzee 1 had been the first chimpanzee in which Dane particles could be shown,12 however, this animal had lost most of its Dane particles two years before the study. At the time of the study, Dane particles could be shown in the patient and in chimpanzee 1 only with difficulty and in sporadic specimens, not allowing longitudinal comparison; they could not be shown in chimpanzee 2.
Discussion The results suggest that hepatitis-B virus infectionis sensitive to interferon, and that it is the expression of the HBc or nucleocapsid system which is primarilv affected. Suppression of HBcAg was seen upon treatment in both livers which had demonstrable antigen. Changes were also seen in the titres of circulating antiHBc, but no simple relationship was present with the changes in HBcAg. The titres of circulating HBsAg were not modified under the conditions of this study, but a shift from membrane staining to cytoplasmic staining was noted for HBsAg in two livers. It is important to note that, in chronic hepatitis in man, predominant membrane staining for HBsAg is the hallmark of the aggressive form and coincides with a high level of nuclear HBcAg, while predominant cytoplasmic staining for HBsAg is the hallmark of the clinically subdued persistent form and coincides with little or no demonstrable nuclear HBcAg.l0 1’ Interferon therefore seems to have modified the expression of HB antigens in the liver into a pattern associated with lower disease activity. The changes in viral expression appeared to be transient, although this needs to be further substantiated by additional observations. With other viruses, interferon treatment has not led to the eradication of the viral genome from a cell with established chronic infection. It is therefore not surprising that the effects observed in this study could be annulled after withdrawal of interferon. However, more prolonged or more intensive treatment might, hopefully, have a more durable influence on cell population dynamics-and eventually improve liver function and histology-by favouring the replacement of infected cells by uninfected cells, which are shielded infection by the interferon. The immune system is widely suspected to be paramount in the removal of cells infected with HB virus. In this respect, the fact that interferon exerts a complex influence on the immune system,13 albeit at doses which exceed those required fo: antiviral activity, also needs consideration. The activity of interferon in chronic hepatitis-B mfe; tion suggests that it may also have useful acting ir acute hepatitis, since acute infection by other viruses:
against
647
seneraity more readily influenced than chronic infection. Viruses which in acute infection are sensitive to interferon, have indeed been shown to be either insensitlBe14or sensitive15 to interferon in chronic infection. This study represents the first application of human fibroblast interferon to man. Fibroblast interferon has the advantage over leucocyte interferon in that it is produced from a type of cell which could be grown in unlimited amounts and which has been approved for the
preparation of substances which require stringent safety conditions, such as vaccines. However, important differences exist in the chemical and biological properties of both interferons;16 17 they have different dose-response curves and therefore their titres cannot be strictly compared,18 and their pharmacology in man could thus be different. Human fibroblast interferon was well tolera febrile reaction which was similar to that observed by others with many but not all preparations of leucocyte interferon. Comparative pyrogenicity tests in rabbits between the preparation used in this study, and fibroblast interferon presently being purified on glass,19 suggest that this hindrance has now been
ated, except for
overcome.
Requests for reprints should be addressed to J. D., Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Rega Institute,
REFERENCES
1. Strander, H., Cantell, K., Carlström, G., Jakobsson, P. A. J. natn. Cancer Inst. 1973, 51, 733. 2. Jordan, G. W., Fried, R. P., Merigan, T. C. J. infect. Dis. 1974, 130, 56. 3 Emödi, G., Just, M., Hernandez, R., Hirt, H. R. J. natn. Cancer Inst. 1975, 54, 1045. 4. Taylor, P. E., Zuckerman, A. J. J. med. Microbiol. 1968, 1, 217. 5 Wheelock, E. F., Schenker, S., Combes, B. Proc. Soc. exp. Biol. Med. 1968, 128, 251. 6. Hill, D. A., Walsh, S. H., Purcell, R. H. ibid. 1971, 136, 853. 7 Billiau, A., Joniau, M., De Somer, P. J. gen. Virol. 1973, 19, 1. 8 Finter, N B. ibid. 1969, 5, 419. 9 Magnius, L. O., Espmark, J. A. J. Immun. 1972, 109, 1017. 10 Ray, M. B., Desmet, V. J., Fevery, J., DeGroote, J., Bradburne, A. F., Desmyter, J. J. clin. Path. 1976, 29, 94. 11 Ray, M. B., Desmet, V. J., Bradburne, A. F., Desmyter, J., Fevery, J., De Groote, J. Gastroenterology, (in the press). 12 Desmyter, J., Liu, W. T., Creemers, J. Am. J. Dis. Child. 1972, 123, 315. 13. Johnson, H M., Baron, S. I.R.C.S. med. Sci. 1976, 4, 50. 14 Desmyter, J., Rawls, W. E., Melnick, J. L., Yow, M. D., Barrett, F. J. Immun. 1967, 99, 771. 15. Billiau, A., Sobis, H., De Somer, P. Int. J. Cancer 1973, 12, 646. 16 Havell, E. A., Berman, B., Ogburn, C. A., Berg, K., Paucker, K., Vilcek, J. Proc. natn. Acad. Sci. U.S.A. 1975, 72, 2185. 17 Desmyter, J., Stewart, W. E. II, Virology, 1976, 70, 451. 18 Edy, V.G, Billiau, A., De Somer, P. J. gen. Virol. 1976, 31, 251. 19 Edy, V G., Braude, I. A., De Clercq, E., Billiau, A., De Somer, P. J. gen. Virol. (in the press).
Addendum Since this paper was printed Greenberg et al.* have reported that human leucocyte interferon depressed hepatitis-B core expression in the blood of 3 patients ,,’.ith chronic aggressive hepatitis. Liver specimens were not examined; and Greenberg et al. were unable to demonstrate an effect in a patient who did not have sufficient core in the serum. The two papers thus show that interferon can depress hepatitis-B core in two different ’.’,ays in serum and in liver). Our methods are applicable the majority of cases of chronic hepatitis B which are r. accompanied by large amounts of core antigen or !jMe particles in the blood. * Greenberg, H B., Pollard, W. S, Merigan, T C.
R. B., Lutwick, L. I., Gregory, P. B., New Engl. J. Med. 1976, 295, 517.
Robinson,
IDENTIFICATION OF HIGH-RISK PATIENTS WITH APLASTIC ANÆMIA IN SELECTION FOR
ALLOGENEIC BONE-MARROW TRANSPLANTATION HANS-PETER LOHRMANN DIETRICH NIETHAMMER
PETER KERN HERMANN HEIMPEL
Division of Hœmatology, Department of Internal Medicine and Division of Hœmatology, Department of Pœdiatrics, University of Ulm, D-7900 Ulm, Federal Republic of Germany
Of 75 patients with aplastic anæmia treated between 1968 and 1975, 33 were retrospectively considered as potential candidates for allogeneic bone-marrow transplantation on the basis of their age and severity of marrow failure. The prognosis of these patients with conservative treatment was assessed from parameters obtained at the time of the initial diagnosis. Initial peripheral-blood granulocyte or platelet concentrations were not of prognostic value. In contrast, initial reticulocyte concentrations, allowed separation of the patients into two groups with poor and good prognosis. Low initial reticulocyte concentrations (less than 10 000/µl) indicated those patients at extremely high risk of succumbing to their marrow aplasia (there were no survivors 36 months after diagnosis). In contrast, 75% of those patients with more than 10 000 reticulocytes per µl at diagnosis survived for 3 years. Initial peripheral-blood reticulocyte concentrations thus appear to indicate the extent of the marrow failure in aplastic anæmia more accurately than granulocytes or platelets. Low initial reticulocyte concentrations may indicate, among patients with severe aplastic anæmia, those for whom allogeneic bone-marrow transplanation should be seriously considered; patients with higher initial reticulocyte concentrations may benefit from conservative treatment.
Summary
Introduction
improved supportive care, patients with aplastic anaemia continue to have a high mortarate. 1- The therapeutic value of androgenic steroids lity remains controversial.46 Allogeneic bone-marrow transplantation has been introduced as a therapeutic alternaDESPITE
severe
tive to conservative treatment, and recent results have been encouraging.7 11 Although conservatively treated controls were not available in these studies, clinical experience suggests the superiority of the transplantation
approach. The uncertainty about the individual spontaneous prognosis remains a major problem in marrow transplantation for aplastic anaemia. Patients who might have otherwise succumbed to their disease, may survive after marrow transplantation, but patients who might have achieved spontaneous remission of their marrow failure may die. Parameters to indicate patients at high risk early in the course of their disease are needed to help in the decision for or against a marrow transplantation. In the present study we have attempted to define the prognosis of potential bone-marrow transplant recipients with severe aplastic ansemia from criteria available at the time of the initial diagnosis.
