- Email: [email protected]
ANTIMITOCHONDRIAL-POSITIVE BUT ANTI-M2-NEGATIVE PRIMARY BILIARY CIRRHOSIS
1464
CJD, scrapie, and like agents justified.
to
heat inactivation. This
caveat was
Whole brains and 50 mg fragments from mice terminally affected by the 22A strain of scrapie agent were subjected to porous-load autoclaving at 134°C for 18 min. Before autoclaving tissue samples either fixed in formol saline or held in sterile saline solution for at room tempeiziz-ace. After autoclaving, 10% homogenates were prepared in saline, and 0.02 ml amounts were injected intracerebrally into VM mice. The results are shown in the table. 50 mg macerates of 22A-infected mouse brain can be decontaminated by the UK autoclaving standard for CJD, and our latest findings (table) show that this is also true for intact 50mg fragments and for whole mouse brains. This is reassuring because the 22A strain of scrapie agent is highly thermostable. However, in tissue fixed in 10% formol saline before autoclaving there was little reduction in infectivity (table), the loss of infectivity being only 1-5
were
48 h
the pyruvate dehydrogenase (PDH) complex. Using both a commercially available preparation of PDH (Sigma) from bovine heart and beef heart mitochondria as source of antigen in immunoblotting experiments, we confirmed that the 70 kD band of the PDH preparation was identical to that at the same relative molecular weight (mg) in beef heart mitochondria (figure, left and middle).
log or so. EFFECT OF FORMOL-FIXATION AND OR AUTOCLAVING ON MOUSE BRAIN INFECTED WITH
22A
STRAIN SCRAPIE
AGENT*
Western immunoblotting of beef heart mitochondria and PDH-(B) with three AMA-positive PBC sera.
Arrowsm,of74, 55, 52,1, and 43 kD.’Left 74 and 51 kD positive senm both reactivities present in both preparations; middle = 74, 52, 51, and 43 kD positive serum with beef heart mitochondria-52 kD reactivity absent in PDH; and right 52 kD positive serum with beef heart mitochondria-this serum is negative with PDH. =
=
*6
log ID tNeganve to date 365 days post-injection). <;0
The risk of occupational exposure to CJD agent used to be thought of as small, but to one atypical case in a neurosurgeon9 must now be added three cases in histopathology technicians.1O-12 We
advise against relying on gravity-displacement autoclaving procedures for making formol-fixed tissues safe;2A nor is the extended porous-load procedure used in the UK6 effective. We
must
know of no autoclaving circumstances.
procedure
AFRC & MRC Neuropathogenesis Unit, Institute for Animal Health, Edinburgh EH9 3JF
that is effective in these
D. M. TAYLOR I. MCCONNELL
1. Brown P, Rohwer RG, Green E, Gajdusek DC. Effect of chemicals, heat and
histopathologic processing on high infectivity hamster-adapted scrapie virus. J Infect Dis 1982, 145: 683-87 2. Masters CL, Jacobsen P, Kakulas BA. J Neuropathol Exp Neurol 1985; 44: 304-07 3 Taylor DM, McBride PA. Autoclaved, formol-fixed scrapie mouse brain is suitable for histopathological examination, but may still be infective. Acta Neuropathol 1987, 74: 194-96. 4 Masters CL, Jacobsen PF, Kakulas BA J Neuropathol Exp Neurol 1986, 45: 760-61 5. Rosenberg RN, White CL, Brown P, et al. Precautions in handling tissues, fluids and other contaminated materials from patients with documented or suspected Creutzfeldt-Jakob disease Ann Neurol 1986; 19: 75-77. 6. Department of Health and Social Security Management of patients with spongiform encephalopathy Creutzfeldt-Jakob disease (CJD)) DHSS circular DA (84) 16 1984. 7
Taylor DM Autoclaving standards for Creutzfeldt-Jakob disease agent Ann Neurol
Immunoblotting has been extensively used to assess the mr of the mitochondrial proteins reacting with AMA-positive sera and there is good agreement that the M2 antigen has a molecular weight of 70-74 kD, when beef heart mitochondria are used as source of antigen. Using the same technique, we have reported that 16% of PBC sera positive for AMA immunofluorescence react with a 52 kD polypeptide, but are consistently negative for the 70-74 kD reactivity. When tested with the PDH preparation the same sera were unreactive (figure, right), thus confirming the non-identity of the 52 kD protein with any of the PDH complex. Our data support evidence that a proportion of AMA-positive PBC sera are negative for M2 reactivity: PBC is thus a heterogeneous entity in terms of immunoserology. The site and nature of the 52 kD mitochondrial protein and the clinical significance of its corresponding antibody in patients with chronic liver disease has t L clarified. Cattedra di Semeiotica Medica,
MARCO FUSCONI
Bologna
Biochemistry Department, King’s College, London
HAROLD BAUM
Cattedra di Semeiotica Medica, Istituto di Clinica Medica II, Policlinico S Orsola Via Massarenti 9 40138 Bologna, Italy
FABIO CASSANI MARCO LENZI GIORGIO BALLARDINI FRANCESCO B. BIANCHI
1987; 22: 557-58. 8 Kimberlin RH, Walker CA, Millson GC, et al Disinfection studies with two strains of mouse-passaged scrapie agent J Neurol Sci 1983; 59: 355-69. 9 Schoene WC, Masters CL, Gibbs CJ, et al Transmissible spongiform encephalopathy (Creutzfeldt-Jakob disease). atypical clinical and pathological findings Arch Neurol 1981, 38: 473-77 10. Brown P, Cathala F, Raubertas RF, et al. The epidemiology of Creutzfeldt-Jakob disease conclusion of a 15 year investigation in France and review of the world literature Neurology 1987, 37: 895-904 11. Miller DC Creutzfeldt-Jakob disease in hislopathology technicians. N Engl J Med 1988, 318: 853-54. 12 Sitwell L, Lach B, Atack E, et al. Creutzfeldt-Jakob disease in histopathology technicians. N Engl J Med 1988, 318: 854
ANTIMITOCHONDRIAL-POSITIVE BUT ANTI-M2-NEGATIVE PRIMARY BILIARY CIRRHOSIS
SIR,-Dr Yeaman and colleagues (May 14, p 1067) identify the mitochondrial antigen reacting with antimitochondrial antibody (AMA) of patients with primary biliary cirrhosis as part of M2
1. Fusconi M,
Ghadiminejad I, Bianchi FB, Baum H, Bottazzo GF, Pisi E. Heterogeneity of antimitochondrial antibodies with the M2-M4 pasttern by immunofluorescence as assessed by western immunoblotting and enzyme linked immunosorbent assay. Gut 1988; 29: 440-47.
OVERUSE SYNDROME
Fry (April 23, p 905) is attempting to provide credibility for a disease concept which is, if not unique, certainly singularly prevalent in Australia, especially in the medicolegal arena. He contents!.2 that a specific lesion develops in muscles or associated soft tissues when they are subjected to "overuse", whether in athletes, assembly workers, or musicians. There is considerable opposition in Australia and elsewhere3--5 to this contention. There is litle if anything to find in these patients, in terms of physical findings, although a common thread appears to be SiR,—In
essence, Mr
scientific
CJD, scrapie, and like agents justified.
to
heat inactivation. This
caveat was
Whole brains and 50 mg fragments from mice terminally affected by the 22A strain of scrapie agent were subjected to porous-load autoclaving at 134°C for 18 min. Before autoclaving tissue samples either fixed in formol saline or held in sterile saline solution for at room tempeiziz-ace. After autoclaving, 10% homogenates were prepared in saline, and 0.02 ml amounts were injected intracerebrally into VM mice. The results are shown in the table. 50 mg macerates of 22A-infected mouse brain can be decontaminated by the UK autoclaving standard for CJD, and our latest findings (table) show that this is also true for intact 50mg fragments and for whole mouse brains. This is reassuring because the 22A strain of scrapie agent is highly thermostable. However, in tissue fixed in 10% formol saline before autoclaving there was little reduction in infectivity (table), the loss of infectivity being only 1-5
were
48 h
the pyruvate dehydrogenase (PDH) complex. Using both a commercially available preparation of PDH (Sigma) from bovine heart and beef heart mitochondria as source of antigen in immunoblotting experiments, we confirmed that the 70 kD band of the PDH preparation was identical to that at the same relative molecular weight (mg) in beef heart mitochondria (figure, left and middle).
log or so. EFFECT OF FORMOL-FIXATION AND OR AUTOCLAVING ON MOUSE BRAIN INFECTED WITH
22A
STRAIN SCRAPIE
AGENT*
Western immunoblotting of beef heart mitochondria and PDH-(B) with three AMA-positive PBC sera.
Arrowsm,of74, 55, 52,1, and 43 kD.’Left 74 and 51 kD positive senm both reactivities present in both preparations; middle = 74, 52, 51, and 43 kD positive serum with beef heart mitochondria-52 kD reactivity absent in PDH; and right 52 kD positive serum with beef heart mitochondria-this serum is negative with PDH. =
=
*6
log ID tNeganve to date 365 days post-injection). <;0
The risk of occupational exposure to CJD agent used to be thought of as small, but to one atypical case in a neurosurgeon9 must now be added three cases in histopathology technicians.1O-12 We
advise against relying on gravity-displacement autoclaving procedures for making formol-fixed tissues safe;2A nor is the extended porous-load procedure used in the UK6 effective. We
must
know of no autoclaving circumstances.
procedure
AFRC & MRC Neuropathogenesis Unit, Institute for Animal Health, Edinburgh EH9 3JF
that is effective in these
D. M. TAYLOR I. MCCONNELL
1. Brown P, Rohwer RG, Green E, Gajdusek DC. Effect of chemicals, heat and
histopathologic processing on high infectivity hamster-adapted scrapie virus. J Infect Dis 1982, 145: 683-87 2. Masters CL, Jacobsen P, Kakulas BA. J Neuropathol Exp Neurol 1985; 44: 304-07 3 Taylor DM, McBride PA. Autoclaved, formol-fixed scrapie mouse brain is suitable for histopathological examination, but may still be infective. Acta Neuropathol 1987, 74: 194-96. 4 Masters CL, Jacobsen PF, Kakulas BA J Neuropathol Exp Neurol 1986, 45: 760-61 5. Rosenberg RN, White CL, Brown P, et al. Precautions in handling tissues, fluids and other contaminated materials from patients with documented or suspected Creutzfeldt-Jakob disease Ann Neurol 1986; 19: 75-77. 6. Department of Health and Social Security Management of patients with spongiform encephalopathy Creutzfeldt-Jakob disease (CJD)) DHSS circular DA (84) 16 1984. 7
Taylor DM Autoclaving standards for Creutzfeldt-Jakob disease agent Ann Neurol
Immunoblotting has been extensively used to assess the mr of the mitochondrial proteins reacting with AMA-positive sera and there is good agreement that the M2 antigen has a molecular weight of 70-74 kD, when beef heart mitochondria are used as source of antigen. Using the same technique, we have reported that 16% of PBC sera positive for AMA immunofluorescence react with a 52 kD polypeptide, but are consistently negative for the 70-74 kD reactivity. When tested with the PDH preparation the same sera were unreactive (figure, right), thus confirming the non-identity of the 52 kD protein with any of the PDH complex. Our data support evidence that a proportion of AMA-positive PBC sera are negative for M2 reactivity: PBC is thus a heterogeneous entity in terms of immunoserology. The site and nature of the 52 kD mitochondrial protein and the clinical significance of its corresponding antibody in patients with chronic liver disease has t L clarified. Cattedra di Semeiotica Medica,
MARCO FUSCONI
Bologna
Biochemistry Department, King’s College, London
HAROLD BAUM
Cattedra di Semeiotica Medica, Istituto di Clinica Medica II, Policlinico S Orsola Via Massarenti 9 40138 Bologna, Italy
FABIO CASSANI MARCO LENZI GIORGIO BALLARDINI FRANCESCO B. BIANCHI
1987; 22: 557-58. 8 Kimberlin RH, Walker CA, Millson GC, et al Disinfection studies with two strains of mouse-passaged scrapie agent J Neurol Sci 1983; 59: 355-69. 9 Schoene WC, Masters CL, Gibbs CJ, et al Transmissible spongiform encephalopathy (Creutzfeldt-Jakob disease). atypical clinical and pathological findings Arch Neurol 1981, 38: 473-77 10. Brown P, Cathala F, Raubertas RF, et al. The epidemiology of Creutzfeldt-Jakob disease conclusion of a 15 year investigation in France and review of the world literature Neurology 1987, 37: 895-904 11. Miller DC Creutzfeldt-Jakob disease in hislopathology technicians. N Engl J Med 1988, 318: 853-54. 12 Sitwell L, Lach B, Atack E, et al. Creutzfeldt-Jakob disease in histopathology technicians. N Engl J Med 1988, 318: 854
ANTIMITOCHONDRIAL-POSITIVE BUT ANTI-M2-NEGATIVE PRIMARY BILIARY CIRRHOSIS
SIR,-Dr Yeaman and colleagues (May 14, p 1067) identify the mitochondrial antigen reacting with antimitochondrial antibody (AMA) of patients with primary biliary cirrhosis as part of M2
1. Fusconi M,
Ghadiminejad I, Bianchi FB, Baum H, Bottazzo GF, Pisi E. Heterogeneity of antimitochondrial antibodies with the M2-M4 pasttern by immunofluorescence as assessed by western immunoblotting and enzyme linked immunosorbent assay. Gut 1988; 29: 440-47.
OVERUSE SYNDROME
Fry (April 23, p 905) is attempting to provide credibility for a disease concept which is, if not unique, certainly singularly prevalent in Australia, especially in the medicolegal arena. He contents!.2 that a specific lesion develops in muscles or associated soft tissues when they are subjected to "overuse", whether in athletes, assembly workers, or musicians. There is considerable opposition in Australia and elsewhere3--5 to this contention. There is litle if anything to find in these patients, in terms of physical findings, although a common thread appears to be SiR,—In
essence, Mr
scientific