- Email: [email protected]
HEPATITIS B VIRUS IN LYMPHOCYTES OF SERONEGATIVE CARRIERS
395 pretreatment levels within a week of the drug being withdrawn. Furthermore, in contrast to Thompson et al, we have found a similar effect in seven patients treated with azathioprine after renal transplantation (figure, B). One of these patients took the drug during three periods and a reversible rise in serum creatinine was recorded every time. In all patients the indication for treatment was asymptomatic urinary tract infection, and the TMP dose was 90-150 mg twice daily. Some of our patients, like Thompson’s cyclosporin-treated patients, were subjected to diagnostic and therapeutic measures such as transplant biopsy and anti-rejection therapy, which in retrospect seem unwarranted. One of our azathioprine patients (figure, B) later had a second course of treatment with co-trimoxazole, because of Pneumocystis carinii infection. Her renal function then deteriorated so much that dialysis was required. Her transplant was considered to have failed due to chronic rejection, and immunosuppressive therapy was reduced. She is still on dialysis but has residual graft function. Her graft function might have been better if the side-effect of TMP had been recognised and immunosuppression had remained unchanged. Since the renal impairment elicited by TMP is usually reversible after short periods of treatment, use of this drug can probably be considered safe in cases where monitoring of renal function is not necessary. In patients whose renal function has to be assessed, however, such as recent transplant patients, alternative drugs should be given when possible.
Department of Surgery I and Nephrology, Sahlgren’s Hospital, 41345 Gothenborg, Sweden; and Department of Medicine, Sundsvalls Hospital
GUDRUN NYBERG HÅKAN GÄBEL PETER ALTHOFF STAFFAN BJÖRK HANS HERLITZ HANS BRYNGER
FETAL RHESUS KIDNEY CELLS USED FOR HEPATITIS A VIRAL VACCINE DEVELOPMENT ARE FREE FROM POLYOMAVIRUS
SIR,-Parry et all have found, by immunofluorescence and electron microscopy, a polyomavirus-like agent in certain fetal rhesus monkey kidney (FRhK) cell cultures derived from those developed by Wallace and colleagues.2 They detected the polyomavirus in FRhK4 cells obtained from B. Flehmig(Tubingen, West Germany) at pass 76; in FRhK4 cells obtained at pass 20 from the American Type Culture Collection (ATCC) and then passed 5 times in their laboratory; and in FRhK6 cells obtained from the ATCC at pass 4 and passed 3 more times. Parry and co-workers suggested that contamination of FRhK cells by polyomavirus might be widespread. Their work indicated the agent to be of bovine origin. Since we had used FRhK6 cells in hepatitis A virus (HAV) vaccine development work, we urgently investigated the matter. The FRhK6 cells we used to isolate HAV and to propagate it3,4 were derived from two frozen ampoules (pass 2 and pass 3) received through Dr John Petricciani in 1978. They had been prepared by R. E. Wallace and co-workers under contract to the Division of Biologics Standards and deposited at the ATCC. We cultured cells from each ampoule through 2 passages and froze them. Cultures derived by direct planting from frozen ampoules were used for all our HAV propagation work. We searched for polyomavirus in these FRhK6 cells, in FRhK4 and FRhK6 cells obtained in June, 1983, from the ATCC for the purposes of this investigation, and in MRC-5 cells inoculated with HAV seed viruses which had been passed through FRhK6 cells. We also looked for antibodies to polyomavirus in recipients of an experimental live HAV vaccine.
Our FRhK6 cell cultures used for HAV cultivation have never shown cytopathic effects and they have been maintained, without splitting, for as long as 63 days. For this investigation we planted a frozen ampoule of FRhK6 cells and used indirect immunofluorescence microscopy to examine acetone-fixed cell sheets maintained without splitting over a 28-day incubation period. The antibody, raised in rabbits against disrupted SV40 virus, can detect antigens common to the SV40-polyoma group5 and was kindly provided by P. Howley (National Cancer Institute) and by K. Shah (Johns Hopkins University). No polyomavirus immunofluorescence was detected. Acetone-fixed coverslips infected with known polyomaviruses invariably produced brilliant nuclear fluorescence. These control materials were coverslips of calf kidney cells infected with the polyomavirus described by Parry et al (Dr Sylvia Gardner, London) or with bovine polyomavirus6 (William Coackley, Perth, Australia) and monkey kidney cell coverslips infected with stump-tailed macaque virus,an agent possibly identical to bovine polyomavirus (K. Shah). Electron microscopy of 30-fold concentrated extracts of two frozen-thawed FRhK6 cultures by B. Wolanski of our laboratories revealed no evidence of polyomavirus. Indirect immunofluorescence studies of the FRhK4 and FRhK6 cells acquired from the ATCC, similar to those examined by Parry et al, were also negative for polyomavirus. Because we had adapted HAV to grow in MRC-5 cells after prior passage in FRhK6 cells and prepared experimental vaccines in MRC-5 cells,s,9 we also examined MRC-5 monolayers inoculated with three different HAV seed viruses and held for 28-69 days. Again, we saw no evidence of polyomavirus antigen by indirect immunofluorescence. For serological studies on six volunteers given an experimental HAV vaccine we used indirect immunofluorescence on coverslips infected with polyomaviruses (W. Coackley, S. Gardner) and with HAV. Sera before inoculation and 3-4 months and 5-6 months afterwards were all negative for antibody to polyomavirus. On the other hand post-inoculation sera were positive for anti-HAV. We confirmed that the polyomavirus-infected coverslips were indeed positive for polyomavirus antigen by indirect immunofluorescence assays with rabbit antibody to disrupted SV40 virus. Thus there is no evidence for a polyomavirus contaminant in the FRhK6 or MRC-5 cells used by us for HAV vaccine development, and vaccine recipients have not acquired polyomavirus antibody. This agent is not a factor in our programme. Why should FRhK cells obtained recently from the ATCC be found positive for polyomavirus after cultivation in Parry and co-workers’ laboratory but not after cultivation in ours? Parry and co-workers have suggested that the polyomavirus may have come from occasional lots of contaminated fetal calf serum. We have had the good fortune not to have used such contaminated sera.
Virus & Cell Biology Research, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486, USA
SIR,-We read with interest Professor Lie-Injo’s letter (Jan 7, 54) on hepatitis B virus DNA in the white blood cells of hepatoma patients. We have examined the peripheral blood lymphocytes of chronic hepatitis B carriers, of a donor responsible for three episodes of non-A, non-B transfusion hepatitis, and of p
Ghazey HM, et al. Common structural antigen of papovaviruses of the simian virus 40-polyoma subgroup. J Viral 1977; 21: 179-86. Coackley W, Maker D, Smith VW. A possible bovine polyomavirus. Arch Virol 1980; 66: 161-66. Reissig M, Kelly TJ Jr, Daniel RW, et al. Identification of the stumptailed macaque virus as a new papovavirus. Infect Immun 1976; 14: 225-31. Provost PJ, Banker FS, Giesa PA, et al. Progress toward a live, attenuated human hepatitis A vaccine. Proc Soc Exp Biol Med 1982; 170: 8-14. Provost PJ, Conti PA, Giesa PA, et al. Studies in chimpanzees of live, attenuated hepatitis A vaccine candidates. Proc Soc Exp Biol Med 1983; 172: 357-63.
5. Shah KV, Ozer HL,
167: 201-06.
P. E. A. E.
HEPATITIS B VIRUS IN LYMPHOCYTES OF SERONEGATIVE CARRIERS
1 Parry JV, Richmond JE, Gardner SD. Polyomavirus in fetal rhesus monkey kidney cell lines used to grow hepatitis A virus. Lancet 1983; i: 994. 2. Wallace RE, Vasington PJ, Petricciani JC, et al. Development and characterization of cell lines from subhuman primates. In Vitro 1973; 8: 333-41. 3. Provost PJ, Hilleman MR. Propagation of human hepatitis A virus in cell culture in vitro. Proc Soc Exp Biol Med 1979; 160: 213-21. 4 Provost PJ, Giesa PA, McAleer WJ, Hilleman MR. Isolation of hepatitis A virus in vitro in cell culture directly from human specimens. Proc Soc Exp Biol Med 1981;
PROVOST A. GIESA B. BUYNAK A. MCLEAN M. SCOLNICK
P. J.
6 7.
8. 9.
396
hepatocellular
carcinoma
patients (HCC)
who were also HBeAg isolated and purified2,3 and assessed for integrity and viability by trypan-blue dye exclusion tests. They were then homogenised and fractionated by ultracentrifugation. Hepatitis B markers were measured by ’Auszyme II’, Ausab EIA’, ’Corzyme’, and HBe EIA (Abbott Laboratories). We found the HBeAg consistently in the lymphocytes of chronic hepatitis B carriers and HCC patients who were HBeAg seronegative. Two patients were of special interest. The first was a 34-year-old man who had been associated with three episodes of transfusion-related hepatitis. His sera were repeatedly negative for HBsAg, anti-HBc, anti-HBs, HBeAg, and IgM HAV. The second was a 66-year-old patient with HCC who was repeatedly seronegative for all HBV markers. Both these patients had HBsAg and HBeAg in their lymphocytes. Intralymphocytic HBeAg was found consistently in six chronic HBV carriers who were also seronegative for HBeAg. None of these HBV markers were found in healthy adult controls. In all these patients, HBeAg was present in the cytosol and particulate fractions of the lymphocytes. The detection of HBsAg and HBeAg in the.lymphocytes may indicate intracellular synthesis, or destruction of viral coat protein and genetic material. The work of Lie-Injo and colleagues on HBV DNA in leucocytes confirms our view that HBV may be sequestered in leucocytes and remain undetected serologically. These findings have important implications. They may explain the failure of immunological attack on HBV in chronic carriers and HCC patients and they raise the question of whether tests for HBV in blood donors need to be improved. They also provide another hypothesis for the mechanism of hepatic malignant transformation, since leucocytes containing integrated HBV DNA can fuse with normal hepatocytes. Such HBV DNA translocated en-bloc from infected leucocytes could become integrated into hepatocyte genome. This would be 4 analogous to the liver cell/cell fusion theory proposed by Shafritz.4
seronegative. Lymphocytes
were
National Centre for Reference and Research on Hepatitis and Related Diseases, University Department of Medicine I, National University of Singapore,
Singapore 0316
OON CHONG-JIN DING JEAK-LING
IMMUNE STATUS OF ANTI-HBc POSITIVE
INDIVIDUALS
Gerety (Jan 21, p 172) suggest that antibody directed to the core antigen of the hepatitis B virus (antiHBc) might be protective. Chimpanzees immunised with purified core antigen (HBcAg) were found to be resistant to virus challenge. This observation may throw light on the hepatitis B immune status of individuals who are anti-HBc positive alone. We had some puzzling results when immunising anti-HBc positive volunteers during a clinical trial of hepatitis B vaccine in hospital personnel. 14 staff members who were anti-HBc positive alone received three doses of a 5 jg HBsAg alum vaccine (’Hevac B’; Institut Pasteur Production) one month apart. The anti-HBs seroconversion rate was 85%, significantly lower than the 96% SIR,-Dr Tabor and
Dr
observed in seronegative staff in the same settings.5 A true anamnestic response (rapid increase in anti-HBs after the first injection) was observed in only 1 subject, others having a primary type of response. There was no obvious relation between this relatively low response and either increasing (2 cases), decreasing (3 cases), or stable anti-HBc titres (’Corzyme’; Abbott) or the presence of anti-HBc IgM (6 cases) (’Corzyme-M’). These results suggested that a degree of anergy to HBsAg may be one characteristic of individuals who do not acquire anti-HBs after a Oon CJ. Detection of HBeAg in lymphocytes of sero-eAg-negative patients with chronic hepatitis B and primary hepatocellular carcinoma Cytobios (in press). 2. Boyum A. Separation of leucocytes from blood and bone marrow. Scand J Lab Clin Invest 1968; 21: (suppl): 97. 3. Territo M, Cline MJ. Monocyte function in man. J Immunol 1977; 118: 187-92. 4. Shafritz D. Hepatitis B virus DNA molecules in the liver of HBsAg carriers: mechanistic considerations in the pathogenesis of hepatocellular carcinoma. Hepatology 1982; 2: (suppl): 5S-41S. 5. Goudeau A, Dubois F, Barin F, Dubois MC, Coursaget P. Hepatitis B vaccine. clinical trials in high-risk settings in France (September 1975-September 1982). Develop Biol Standard 1983; 54: 267-84. 1.
Ding JL,
hepatitis B virus infection, or lose it quickly. Some of them remain chronically infected, with low-level synthesis of HBsAg that is undetectable by the usual methods. 6, Such patients may experience a reactivation of their infection when immunosuppressed and some However, most may be implicated in post-transfusion hepatitis. individuals who are anti-HBc positive alone are protected against
,10
further manifestation of HBV infection or reinfection. This suggests that an immune mechanism distinct from the usual HBsAg/anti-HBs humoral response may be operating and that further studies of the role of specific immunity to HBcAg are warranted. Institute of Virology, Faculty of Medicine and Pharmacy, 37032
Tours, France
A. GOUDEAU F. DUBOIS
MYASTHENIA GRAVIS AND THE ANTI-IDIOTYPE THEORY OF AUTOIMMUNITY
SIR,-Plotz’s theory of autoimmune disease’ is based on the autoantibodies could be anti-idiotype antibodies to
possibility that
antiviral antibodies. If the virus binds to a cell receptor then antibodies to the binding site on the virus might look like the receptor and anti-idiotype to these antibodies might bind to the receptor. A viral infection in a susceptible individual might result in an autoimmune disease against the receptor for virus. I wish to add a possibly supporting fact to this hypothesis which is relevant to myasthenia gravis. There is evidence to suggest that the nicotinic acetylcholine receptor at the neuromuscular junction is the receptor for rabies virus.2Specific inhibitors of this receptor, such as a-bungarotoxin and curare, reduce the activity of rabies virus in cultured chick myotubes. Perhaps a virus (or viruses) exists that binds to human acetylcholine receptor and induces the production of anti-idiotype antibodies which also binds to the receptor and could initiate the autoimmune disease myasthenia gravis. Presumably, anti-receptor antibodies in these patients would block the virus binding to the receptor. Anti-idiotypic antibodies to antireceptor antibodies can be measured in myasthenia gravisand these might be directed against the virus itself A search for viruses which bind to acetylcholine receptor and a study of their interaction with myasthenic antibodies might shed some light on this
interesting hypothesis. Neurosciences Program, School of Medicine, University of Alabama in Birmingham, Birmingham, Alabama 35294, USA
RONALD J. BRADLEY
INFECTIOUS MONONUCLEOSIS LATE IN LIFE
SIR,-Your Jan 21 editorial rightly emphasises the importance of considering Epstein-Barr virus (EBV) infection in patients late in life. This virus causes a spectrum of illness: asymptomatic, classical infectious mononucleosis (IM) with good recovery, IM with recovery over years, and fatal IM.4 You compared classical IM with IM late in life, and implied that liver involvement and negative heterophil serology were features of IM late in life. This is misleading. In classical IM, over 80% of cases will have liver 6. Katchaki JN, Brouwer R, Siem TH. Anti-HBc and blood. N Engl J Med 1978; 298: 1421-22. 7. Brechot Ch, Hadchouel M, Scotto J, Fonck M, Potet F, Vyas GN, Tiollais P. State of hepatitis B virus DNA in hepatocytes of patients with hepatitis B surface antigenpositive and -negative liver diseases. Proc Natl Acad Sci USA 1981; 78: 3906-10 8 Nagington J, Cossart YE, Cohen BJ. Reactivation of hepatitis B after transplantation operations. Lancet 1977; i: 558-62. 9. Katchaki JN, Siem TH, Brouwer R, Brandt KH, van der Waart M. Detection and significance of anti-HBc in the blood bank: preliminary results of a controlled prospective study. J Virol Methods 1980; 2: 119-25. 10. Lander JJ, Gitnick GL, Gelb LH, Aach RD. Anticore antibody screening of transfused blood. Vox Sang 1978; 34: 77-80. 1. Plotz PH. Autoantibodies are anti-idiotype antibodies to antiviral antibodies. Lancet 1983; ii: 824. 2 Lentz TL, Burrage TG, Smith AL, Crick J, Tignor GH. Is the acetylcholine receptor a rabies virus receptor? Science 1982; 215: 182-84. 3. Dwyer DS, Bradley RJ, Urquhart CK, Kearney JF. Naturally occurring anti-idiotype antibodies in myasthenia gravis patients. Nature 1983; 301: 611-14. 4. Ho-Yen DO. The significance of Downey lymphocytes and a serum inhibitor in the control of infectious mononucleosis. MD thesis, Dundee University, 1983.
Department of Surgery I and Nephrology, Sahlgren’s Hospital, 41345 Gothenborg, Sweden; and Department of Medicine, Sundsvalls Hospital
GUDRUN NYBERG HÅKAN GÄBEL PETER ALTHOFF STAFFAN BJÖRK HANS HERLITZ HANS BRYNGER
FETAL RHESUS KIDNEY CELLS USED FOR HEPATITIS A VIRAL VACCINE DEVELOPMENT ARE FREE FROM POLYOMAVIRUS
SIR,-Parry et all have found, by immunofluorescence and electron microscopy, a polyomavirus-like agent in certain fetal rhesus monkey kidney (FRhK) cell cultures derived from those developed by Wallace and colleagues.2 They detected the polyomavirus in FRhK4 cells obtained from B. Flehmig(Tubingen, West Germany) at pass 76; in FRhK4 cells obtained at pass 20 from the American Type Culture Collection (ATCC) and then passed 5 times in their laboratory; and in FRhK6 cells obtained from the ATCC at pass 4 and passed 3 more times. Parry and co-workers suggested that contamination of FRhK cells by polyomavirus might be widespread. Their work indicated the agent to be of bovine origin. Since we had used FRhK6 cells in hepatitis A virus (HAV) vaccine development work, we urgently investigated the matter. The FRhK6 cells we used to isolate HAV and to propagate it3,4 were derived from two frozen ampoules (pass 2 and pass 3) received through Dr John Petricciani in 1978. They had been prepared by R. E. Wallace and co-workers under contract to the Division of Biologics Standards and deposited at the ATCC. We cultured cells from each ampoule through 2 passages and froze them. Cultures derived by direct planting from frozen ampoules were used for all our HAV propagation work. We searched for polyomavirus in these FRhK6 cells, in FRhK4 and FRhK6 cells obtained in June, 1983, from the ATCC for the purposes of this investigation, and in MRC-5 cells inoculated with HAV seed viruses which had been passed through FRhK6 cells. We also looked for antibodies to polyomavirus in recipients of an experimental live HAV vaccine.
Our FRhK6 cell cultures used for HAV cultivation have never shown cytopathic effects and they have been maintained, without splitting, for as long as 63 days. For this investigation we planted a frozen ampoule of FRhK6 cells and used indirect immunofluorescence microscopy to examine acetone-fixed cell sheets maintained without splitting over a 28-day incubation period. The antibody, raised in rabbits against disrupted SV40 virus, can detect antigens common to the SV40-polyoma group5 and was kindly provided by P. Howley (National Cancer Institute) and by K. Shah (Johns Hopkins University). No polyomavirus immunofluorescence was detected. Acetone-fixed coverslips infected with known polyomaviruses invariably produced brilliant nuclear fluorescence. These control materials were coverslips of calf kidney cells infected with the polyomavirus described by Parry et al (Dr Sylvia Gardner, London) or with bovine polyomavirus6 (William Coackley, Perth, Australia) and monkey kidney cell coverslips infected with stump-tailed macaque virus,an agent possibly identical to bovine polyomavirus (K. Shah). Electron microscopy of 30-fold concentrated extracts of two frozen-thawed FRhK6 cultures by B. Wolanski of our laboratories revealed no evidence of polyomavirus. Indirect immunofluorescence studies of the FRhK4 and FRhK6 cells acquired from the ATCC, similar to those examined by Parry et al, were also negative for polyomavirus. Because we had adapted HAV to grow in MRC-5 cells after prior passage in FRhK6 cells and prepared experimental vaccines in MRC-5 cells,s,9 we also examined MRC-5 monolayers inoculated with three different HAV seed viruses and held for 28-69 days. Again, we saw no evidence of polyomavirus antigen by indirect immunofluorescence. For serological studies on six volunteers given an experimental HAV vaccine we used indirect immunofluorescence on coverslips infected with polyomaviruses (W. Coackley, S. Gardner) and with HAV. Sera before inoculation and 3-4 months and 5-6 months afterwards were all negative for antibody to polyomavirus. On the other hand post-inoculation sera were positive for anti-HAV. We confirmed that the polyomavirus-infected coverslips were indeed positive for polyomavirus antigen by indirect immunofluorescence assays with rabbit antibody to disrupted SV40 virus. Thus there is no evidence for a polyomavirus contaminant in the FRhK6 or MRC-5 cells used by us for HAV vaccine development, and vaccine recipients have not acquired polyomavirus antibody. This agent is not a factor in our programme. Why should FRhK cells obtained recently from the ATCC be found positive for polyomavirus after cultivation in Parry and co-workers’ laboratory but not after cultivation in ours? Parry and co-workers have suggested that the polyomavirus may have come from occasional lots of contaminated fetal calf serum. We have had the good fortune not to have used such contaminated sera.
Virus & Cell Biology Research, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486, USA
SIR,-We read with interest Professor Lie-Injo’s letter (Jan 7, 54) on hepatitis B virus DNA in the white blood cells of hepatoma patients. We have examined the peripheral blood lymphocytes of chronic hepatitis B carriers, of a donor responsible for three episodes of non-A, non-B transfusion hepatitis, and of p
Ghazey HM, et al. Common structural antigen of papovaviruses of the simian virus 40-polyoma subgroup. J Viral 1977; 21: 179-86. Coackley W, Maker D, Smith VW. A possible bovine polyomavirus. Arch Virol 1980; 66: 161-66. Reissig M, Kelly TJ Jr, Daniel RW, et al. Identification of the stumptailed macaque virus as a new papovavirus. Infect Immun 1976; 14: 225-31. Provost PJ, Banker FS, Giesa PA, et al. Progress toward a live, attenuated human hepatitis A vaccine. Proc Soc Exp Biol Med 1982; 170: 8-14. Provost PJ, Conti PA, Giesa PA, et al. Studies in chimpanzees of live, attenuated hepatitis A vaccine candidates. Proc Soc Exp Biol Med 1983; 172: 357-63.
5. Shah KV, Ozer HL,
167: 201-06.
P. E. A. E.
HEPATITIS B VIRUS IN LYMPHOCYTES OF SERONEGATIVE CARRIERS
1 Parry JV, Richmond JE, Gardner SD. Polyomavirus in fetal rhesus monkey kidney cell lines used to grow hepatitis A virus. Lancet 1983; i: 994. 2. Wallace RE, Vasington PJ, Petricciani JC, et al. Development and characterization of cell lines from subhuman primates. In Vitro 1973; 8: 333-41. 3. Provost PJ, Hilleman MR. Propagation of human hepatitis A virus in cell culture in vitro. Proc Soc Exp Biol Med 1979; 160: 213-21. 4 Provost PJ, Giesa PA, McAleer WJ, Hilleman MR. Isolation of hepatitis A virus in vitro in cell culture directly from human specimens. Proc Soc Exp Biol Med 1981;
PROVOST A. GIESA B. BUYNAK A. MCLEAN M. SCOLNICK
P. J.
6 7.
8. 9.
396
hepatocellular
carcinoma
patients (HCC)
who were also HBeAg isolated and purified2,3 and assessed for integrity and viability by trypan-blue dye exclusion tests. They were then homogenised and fractionated by ultracentrifugation. Hepatitis B markers were measured by ’Auszyme II’, Ausab EIA’, ’Corzyme’, and HBe EIA (Abbott Laboratories). We found the HBeAg consistently in the lymphocytes of chronic hepatitis B carriers and HCC patients who were HBeAg seronegative. Two patients were of special interest. The first was a 34-year-old man who had been associated with three episodes of transfusion-related hepatitis. His sera were repeatedly negative for HBsAg, anti-HBc, anti-HBs, HBeAg, and IgM HAV. The second was a 66-year-old patient with HCC who was repeatedly seronegative for all HBV markers. Both these patients had HBsAg and HBeAg in their lymphocytes. Intralymphocytic HBeAg was found consistently in six chronic HBV carriers who were also seronegative for HBeAg. None of these HBV markers were found in healthy adult controls. In all these patients, HBeAg was present in the cytosol and particulate fractions of the lymphocytes. The detection of HBsAg and HBeAg in the.lymphocytes may indicate intracellular synthesis, or destruction of viral coat protein and genetic material. The work of Lie-Injo and colleagues on HBV DNA in leucocytes confirms our view that HBV may be sequestered in leucocytes and remain undetected serologically. These findings have important implications. They may explain the failure of immunological attack on HBV in chronic carriers and HCC patients and they raise the question of whether tests for HBV in blood donors need to be improved. They also provide another hypothesis for the mechanism of hepatic malignant transformation, since leucocytes containing integrated HBV DNA can fuse with normal hepatocytes. Such HBV DNA translocated en-bloc from infected leucocytes could become integrated into hepatocyte genome. This would be 4 analogous to the liver cell/cell fusion theory proposed by Shafritz.4
seronegative. Lymphocytes
were
National Centre for Reference and Research on Hepatitis and Related Diseases, University Department of Medicine I, National University of Singapore,
Singapore 0316
OON CHONG-JIN DING JEAK-LING
IMMUNE STATUS OF ANTI-HBc POSITIVE
INDIVIDUALS
Gerety (Jan 21, p 172) suggest that antibody directed to the core antigen of the hepatitis B virus (antiHBc) might be protective. Chimpanzees immunised with purified core antigen (HBcAg) were found to be resistant to virus challenge. This observation may throw light on the hepatitis B immune status of individuals who are anti-HBc positive alone. We had some puzzling results when immunising anti-HBc positive volunteers during a clinical trial of hepatitis B vaccine in hospital personnel. 14 staff members who were anti-HBc positive alone received three doses of a 5 jg HBsAg alum vaccine (’Hevac B’; Institut Pasteur Production) one month apart. The anti-HBs seroconversion rate was 85%, significantly lower than the 96% SIR,-Dr Tabor and
Dr
observed in seronegative staff in the same settings.5 A true anamnestic response (rapid increase in anti-HBs after the first injection) was observed in only 1 subject, others having a primary type of response. There was no obvious relation between this relatively low response and either increasing (2 cases), decreasing (3 cases), or stable anti-HBc titres (’Corzyme’; Abbott) or the presence of anti-HBc IgM (6 cases) (’Corzyme-M’). These results suggested that a degree of anergy to HBsAg may be one characteristic of individuals who do not acquire anti-HBs after a Oon CJ. Detection of HBeAg in lymphocytes of sero-eAg-negative patients with chronic hepatitis B and primary hepatocellular carcinoma Cytobios (in press). 2. Boyum A. Separation of leucocytes from blood and bone marrow. Scand J Lab Clin Invest 1968; 21: (suppl): 97. 3. Territo M, Cline MJ. Monocyte function in man. J Immunol 1977; 118: 187-92. 4. Shafritz D. Hepatitis B virus DNA molecules in the liver of HBsAg carriers: mechanistic considerations in the pathogenesis of hepatocellular carcinoma. Hepatology 1982; 2: (suppl): 5S-41S. 5. Goudeau A, Dubois F, Barin F, Dubois MC, Coursaget P. Hepatitis B vaccine. clinical trials in high-risk settings in France (September 1975-September 1982). Develop Biol Standard 1983; 54: 267-84. 1.
Ding JL,
hepatitis B virus infection, or lose it quickly. Some of them remain chronically infected, with low-level synthesis of HBsAg that is undetectable by the usual methods. 6, Such patients may experience a reactivation of their infection when immunosuppressed and some However, most may be implicated in post-transfusion hepatitis. individuals who are anti-HBc positive alone are protected against
,10
further manifestation of HBV infection or reinfection. This suggests that an immune mechanism distinct from the usual HBsAg/anti-HBs humoral response may be operating and that further studies of the role of specific immunity to HBcAg are warranted. Institute of Virology, Faculty of Medicine and Pharmacy, 37032
Tours, France
A. GOUDEAU F. DUBOIS
MYASTHENIA GRAVIS AND THE ANTI-IDIOTYPE THEORY OF AUTOIMMUNITY
SIR,-Plotz’s theory of autoimmune disease’ is based on the autoantibodies could be anti-idiotype antibodies to
possibility that
antiviral antibodies. If the virus binds to a cell receptor then antibodies to the binding site on the virus might look like the receptor and anti-idiotype to these antibodies might bind to the receptor. A viral infection in a susceptible individual might result in an autoimmune disease against the receptor for virus. I wish to add a possibly supporting fact to this hypothesis which is relevant to myasthenia gravis. There is evidence to suggest that the nicotinic acetylcholine receptor at the neuromuscular junction is the receptor for rabies virus.2Specific inhibitors of this receptor, such as a-bungarotoxin and curare, reduce the activity of rabies virus in cultured chick myotubes. Perhaps a virus (or viruses) exists that binds to human acetylcholine receptor and induces the production of anti-idiotype antibodies which also binds to the receptor and could initiate the autoimmune disease myasthenia gravis. Presumably, anti-receptor antibodies in these patients would block the virus binding to the receptor. Anti-idiotypic antibodies to antireceptor antibodies can be measured in myasthenia gravisand these might be directed against the virus itself A search for viruses which bind to acetylcholine receptor and a study of their interaction with myasthenic antibodies might shed some light on this
interesting hypothesis. Neurosciences Program, School of Medicine, University of Alabama in Birmingham, Birmingham, Alabama 35294, USA
RONALD J. BRADLEY
INFECTIOUS MONONUCLEOSIS LATE IN LIFE
SIR,-Your Jan 21 editorial rightly emphasises the importance of considering Epstein-Barr virus (EBV) infection in patients late in life. This virus causes a spectrum of illness: asymptomatic, classical infectious mononucleosis (IM) with good recovery, IM with recovery over years, and fatal IM.4 You compared classical IM with IM late in life, and implied that liver involvement and negative heterophil serology were features of IM late in life. This is misleading. In classical IM, over 80% of cases will have liver 6. Katchaki JN, Brouwer R, Siem TH. Anti-HBc and blood. N Engl J Med 1978; 298: 1421-22. 7. Brechot Ch, Hadchouel M, Scotto J, Fonck M, Potet F, Vyas GN, Tiollais P. State of hepatitis B virus DNA in hepatocytes of patients with hepatitis B surface antigenpositive and -negative liver diseases. Proc Natl Acad Sci USA 1981; 78: 3906-10 8 Nagington J, Cossart YE, Cohen BJ. Reactivation of hepatitis B after transplantation operations. Lancet 1977; i: 558-62. 9. Katchaki JN, Siem TH, Brouwer R, Brandt KH, van der Waart M. Detection and significance of anti-HBc in the blood bank: preliminary results of a controlled prospective study. J Virol Methods 1980; 2: 119-25. 10. Lander JJ, Gitnick GL, Gelb LH, Aach RD. Anticore antibody screening of transfused blood. Vox Sang 1978; 34: 77-80. 1. Plotz PH. Autoantibodies are anti-idiotype antibodies to antiviral antibodies. Lancet 1983; ii: 824. 2 Lentz TL, Burrage TG, Smith AL, Crick J, Tignor GH. Is the acetylcholine receptor a rabies virus receptor? Science 1982; 215: 182-84. 3. Dwyer DS, Bradley RJ, Urquhart CK, Kearney JF. Naturally occurring anti-idiotype antibodies in myasthenia gravis patients. Nature 1983; 301: 611-14. 4. Ho-Yen DO. The significance of Downey lymphocytes and a serum inhibitor in the control of infectious mononucleosis. MD thesis, Dundee University, 1983.