- Email: [email protected]
KETOCONAZOLE-CYCLOSPORIN INTERACTION
1342 EFFECT OF KETOCONAZOLE ON SERUM CYCLOSPORIN
SiR.—Dr David and Dr Phillips point out that cystic fibrosis need
overdiagnosed if sweat tests are performed in centres where this test is carried out frequently and are repeated in cases of doubt. Furthermore, all the clinical criteria should be taken into account before the diagnosis is made. Surely these are minimum requirements in contemporary paediatric care. That some patients should not only have been misdiagnosed in the manner they described but also that four of the eight patients should have been sent to special schools on the basis of the wrong diagnosis is surely serious mismanagement. This is seldom if ever necessary in an established case of cystic fibrosis. This article draws attention away from the important fact of underdiagnosis of cystic fibrosis. There are no accurate data on the number of children and young adults in the U.K. with cystic fibrosis; however, P. Phelan (personal communication) has reviewed the mortality rates of cystic fibrosis for 1976-80 inclusive from Office of Population Censuses and Surveys data in the U.K., using the principal cause of death, and compared them with those of the State of Victoria in Australia where comprehensive child care and pathological services can provide accurate information. He has revealed the disturbing fact that mortality rates are two to three times higher in the U.K. in the age groups <1, 1-4, 5-9, and 9-15 years. The genetic make-up of the two populations is very similar. While it is possible that some of this difference stems from difficulties in diagnosis it is likely that inadequacies in management are also responsible. It has already been shown in the United States and Denmark as well as in Australia that reference to special units early after the onset of symptoms has a profoundly beneficial effect not
on
CONCENTRATION
be
prognosis.
There is clearly a need for regional centres in this country to help with the diagnosis and participate in the care of patients with cystic fibrosis. Early referral to such special units for accurate sweat tests and subsequent joint management is essential if our patients with cystic fibrosis are to be given opportunities similar to those that exist elsewhere. Brompton Hospital, London SW3 6HP
J. C. BATTEN D. M. GEDDES
CYCLOSPORIN INTERACTION WITH KETOCONAZOLE AND MELPHALAN
SiR,—Dr Ferguson and colleagues (Oct. 16, p. 882) report an cyclosporin and ketoconazole in a renal transplant patient, causing renal impairment and an increase in cyclosporin blood levels which they attribute to interference with the hepatic metabolism of cyclosporin. This interaction has been reported in bone marrow transplant recipients, in whom it was attributed to increased cyclosporin absorption, and we have seen a similar interaction in a marrow transplant recipient who received both agents simultaneously (see table). We therefore avoid the concomitant use of cyclosporin and ketoconazole. We have also found a potentially dangerous interaction between cyclosporin and high-dose melphalan,2adrug we have been exploring as a conditioning agent for allogeneic bone marrow transplantation for acute leukaemia.3Seventeen patients received melphalan as a single injection of 140-250 mglm2with forced diuresis, followed, 1-4 days later, by infusion of donor bone marrow and cyclosporin in standard dose (12-5mg/kg/day orally) as prophylaxis against graft-versus-host disease. Thirteen patients (76%) had severe renal failure (plasma urea >30 mmolll, plasma creatinine >400 mol/1 or both); five required peritoneal and/or interaction between
*Ketaconazole 200 mg
daily days 62-68.
haemodialysis. Only two patients had mild or no impairment of renal function (plasma urea <11 mmol/1 and creatinine <200 mol/1). This compares with seven patients who received syngeneic or repeat allogeneic transplants after melphalan (180-240 mg/m2) but without cyclosporin3where no deterioration in renal function occurred. Urea and creatinine levels remained normal in five and were static or improved in the two patients who had renal impairment at the time of transplantation, demonstrating that melphalan itself is not overtly nephrotoxic in these circumstances. In the original study of high-dose melphalan renal impairment occurred 2 only in two patients who had not received forced diuresis.2 The nature of the interaction between melphalan and cyclosporin is unclear. Serum cyclosporin levels were determined 2 h post dose by radioimmunoassay in fourteen of the patients who received cyclosporin following melphalan and in sixty patients conditioned with cyclophosphamide and total body irradiation who received similar cyclosporin prophylaxis. 8 days after transplantation the levels were 165-1450 ng/ml (median 715) for the melphalan patients and 59-2922 ng/ml (median 473) for the irradiated patients. This difference is not significant and the interaction may be directly on the kidney rather than in the absorption/metabolism of cyclosporin. Although some degree of renal impairment is seen in more than 90% ofour patients receiving cyclosporin after total body irradiation and transplantation,4 it is mild or moderate (plasma urea <20 mmol/1) in 55% of them and generally reversible when the cyclosporin dose is reduced. We are now testing lower dose schedules of cyclosporin, together with early interruption of the agent to prevent severe Leukaemia Unit, Royal Marsden Hospital, Sutton, Surrey SM2 5PT
nephrotoxicitv.
G. R. MORGENSTERN RAY POWLES BRIDGET ROBINSON T. J. McELWAIN
KETOCONAZOLE-CYCLOSPORIN INTERACTION
SIR,-The rise in cyclosporin concentrations in two patients after the start of ketoconazole therapy5,6 has been interpreted as ketoconazole-induced inhibition of hepatic metabolism.5 These patients received a ketoconazole dose of 400 mg daily (about 8 mg/kg), while in animals a much greater dose (50 mg/kg) was required to inhibit microsomal enzymes.7 In eight healthy volunteers I and my colleagues (unpublished) have examined the effect of ketoconazole, 400 mg daily for 5 days, on antipyrine kinetics, an indirect in-vivo measure of hapatic microsomal oxidative activity.88 Antipyrine clearance was 4.
F, Poirier O, Gluckman E, et al. Pharmacokinetic study of cyclosporin A: Preliminary results. In. Touraine J, Gluckman E, Griscelli C, eds. Bone marrow transplantation in Europe II. Amsterdam: Excerpta Medica, 1981: 160-64. 2. McElwain TJ, Hedley DW, Burton G, et al. Marrow autotransplantation accelerates haematological recovery in patients with malignant melanoma treated with highdose melphalan. Br J Cancer 1979; 40: 72-80. 3. Lumley HS, Powles R, Morgenstern GR, et al. Pseudosyngeneic transplantation as a treatment for recurrent leukaemia following allogeneic bone marrow transplantation. In: Touraine J, Gluckman E, Griscelli C, eds. Bone marrow transplantation in Europe II. Amsterdam: Excerpta Medica, 1981: 24-28. 1. Lokiec
Hedley D, Powles RL, Morgenstern GR. Toxicity of cyclosporin A in patients following bone marrow transplantation. In: White DJ, ed. Cyclosporin A. Amsterdam: Elsevier, 1982: 545-51 5. Ferguson RM, Sutherland DER, Simmons RL, Najarian JS. Ketoconazole, cyclosporin metabolism, and renal transplantation. Lancet 1982; ii: 882-83. 6. Dieperink H, Moller J. Ketoconazole and cyclosporin. Lancet 1982; ii. 1217. 7. Niemegeers CJE, Levron JCI, Awouters F, Janssen PAJ. Inhibition and induction of microsomal enzymes in the rat: a comparative study of four antimycotics: miconazole, econazole, clotrimazole and ketoconazole Arch Int Pharmacodyn 1981; 251: 26. 8. Vesell ES.
The antipyrine test in clinical pharmacology: Conceptions misconceptions. Clin Pharmacol Ther 1979; 26: 275-86.
and
1343 37’7± 12 -3ml/min (mean±SD) before and 40 -7± 12 -4ml/min after ketoconazole (not significant; Student’s t test for paired data). Thus, at the doses used, ketoconazole does not appear to be an inhibitor of microsomal enzymes in man. The ketoconazole-cyclosporin interaction may be due to mechanisms other than enzyme inhibition-e.g., a ketoconazoleassociated change in the apparent volume of distribution of cyclosporin, protein binding, or competition for an excretion pathway. Since systematic investigation of this important interaction may not be ethically feasible, it would be important to measureand report ketoconazole as well as cyclosporin and creatinine concentrations in future. Department of Medicine, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW
T. K. DANESHMEND
DETERMINATION OF ISLET-CELL ANTIBODIES BY IMMUNOFLUORESCENCE
SIR,-We would like to endorse the views of Dr Yagihashi and coworkers (Nov. 27, p. 1218) that, in the absence of fully characterised tissue antigens, fresh unfixed pancreas should be used for the clinical screening of patients with insulin-dependent diabetes mellitus (IDDM). Complement-fixing antibodies (CFare vital ICA) in the sera of non-diabetic siblings ofIDDM probands markers for the potential development of diabetes, 1,2 because some CF-ICA are specifically directed against the insulin-secreting betacells which bear the brunt of the insulitis leading to diabetes.3 We have found that Bouin-fixed pancreas cannot be used for the CFICA test since human complement attaches preferentially to betacells and positive results are seen with normal sera. Although our experience with Bouin-fixed pancreas is limited, the results (table) COMPARISON OF ISLET CELL ANTIBODIES
(ICA-IgG) ON UNFIXED AND
BOUIN-FIXED PANCREAS
COMPLEMENT C4 ALLOTYPES IN ALZHEIMER’S DISEASE
SIR,-Although no conclusion can be drawn about a possible association between HLA antigen and Alzheimer’s disease,I-3 we have noted that C4 B2, a rare variant of complement C4 which is encoded by genes within the HLA complex, is more common than expected in patients with senile dementia of the Alzheimer’s type
(SDAT). was established by history and exclusion of other The criteria were those of Blessed et allthey correlate significantly with pathological 5and biochemical changes characteristic of Alzheimer’s disease. Complement C4 allotyping was done in 38 patients with SDAT, in 42 patients with Parkinson’s disease, and in 59 unaffected, agematched controls of the same ethnic background. The C4 B2 variant was found in 14% (8/59) of the age-matched controls, a figure in accord with the frequency of C4 B2 in our random panel of White subjects and that of others.The frequency of the C4 B2 variant in the Parkinson’s disease group was 12% (5/42). In SDAT, however, we found a striking increase of C4 B2, with 55% (21/38) of patients positive for this variant, resulting in a relative risk of 8 -8(p<0 - 0001) for this disorder. The frequencies of all other C4 alleles at either the C4A (= Rodgers) or C4B (= Chido) locus did not differ from those of the control groups. In HLA associated diseases, a corresponding association with the HLA linked complement markers (C2, C4, and Bf) is often observed. This, however, is usually interpreted as being due to linkage disequilibria between the complement markers and HLA antigens, and regarded as a secondary effect. Alzheimer’s disease, therefore, represents the first disorder with a strong association with an HLA-linked complement component (in this case C4), in the absence of any clear association with a particular HLA antigen. Since the complement system plays a vital role in host defence against virus infection, this finding is of particular importance in Alzheimer’s disease, where a viral aetiology is suspected. It will be of considerable interest, therefore, to determine whether certain C4 variants, such as C4 B2, are less effective in virus neutralisation.
The
diagnosis
causes.
Departments of Pathology and Neurology, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032, U.S.A.
CHRISTOPH W. NERL RICHARD MAYEUX GEOFFREY J. O’NEILL
*After de-waxing, 4 nm sections were preincubated for 2 h at 37° with 0 - I% calcium chloride containing O. 1% trypsin.. - fThe number of sera with cytoplasmic membrane or antinuclear (speckled or diffuse) staining in the islets is shown. A single pancreas ofpre-tested antigenicity (PI 5) was used for cryostat sections, whereas four different pancreases, including P15, were used for the Boum fixed ICA determinations. N=no. of subjects, n=no. of sera (applies to all three antibodies).
substantiate the conclusions of Yagihashi et al. that, after fixation, less than 20% of the substrates are’ suitable for ICA-IgG determinations as compared with 80% of unfixed group 0 glands. We used sera from patients without insulin treatment, to avoid discrepancies due to insulin antibodies, which are readily detected on Bouin-fixed but not on unfixed pancreas. On fixed sections the islet staining was complex, and cytoplasmic, cell-membrane, and nuclear patterns occurred concurrently or singly. Omission of trypsin, or both trypsin and calcium chloride, from the preincubation media did not alter the intensity of cytoplasmic or membrane immunofluorescence, but considerably reduced antinuclear staining. The patterns and intensity of immunofluorescence obtained with normal controls on fixed tissue were mostly indistinguishable from those of sera scored as positive on cryostat sections. This disagrees with the initial report of Dobersen et al.5 but accords with the low percentage of fixed pancreases found suitable by Yagihashi et al. for ICA determination. In our laboratory unfixed human pancreas has been used routinely and in research projects for the past eight years and, on average, 25% of the pancreases have to be discarded because of weak antigenicity. On cryostat sections, normal sera invariably show a clear negative reaction. A diffuse cytoplasmic staining covering all the cells of the
9. Daneshmend
TK, Warnock DW, Turner A, Robert CJC. Pharmacokinetics of ketoconazole in normal subjects. J Antimicrob Chemother 1981; 8: 299-304. 1. Henschke PJ, Bell DA, Cape RDT. Alzheimer’s disease and HLA. Tissue Antigens
1978; 12: 132-35. R, Koskimies S, Wikström J, Palo J HLA antigens in Alzheimer’s disease. Tissue Antigens 1980; 16: 191-94. 3. Walford RL, Hodge SE. HLA distribution in Alzheimer’s disease. In: Teraski PI, ed. Histocompatibility testing 1980: Los Angeles, UCLA 1980; 727-29. 4 Blessed G, Tomlinson BE, Roth M. The association between quantitative measures of dementia and of senile change in the cerebral grey matter of elderly subjects. Br J Psychiatry 1968; 114: 797-881. 5. Perry EK. The cholinergic system in old age and Alzheimer’s disease. Age Aging 1980; 2. Sulkava
9: 1-8. 6. Awdeh ZL,
Alper CA. Inherited structural polymorphism of the fourth component of complement. Proc Natl Acad Sci (USA) 1980; 77: 3576-80.
Bottazzo GF, Dean BM, Gorsuch AN, Cudworth AG, Doniach D. Complement-fixing islet-cell antibodies in type I diabetes: Possible monitors of active beta-cell damage. Lancet 1980; i: 668-72. 2. Gorsuch AN, Lister J, Dean BM, Spencer KM, Mc Nally JM, Bottazzo GF, Cudworth AG. Evidence for a long pre-diabetic period in type I (insulin-dependent) diabetes mellitus. Lancet 1981; ii: 1363-65. 3 Doniach D, Bottazzo GF, Cudworth AG. Etiology of Type I diabetes mellitus: heterogeneity and immunological events leading to clinical onset. Annu Rev Med 1983; 34 (in press). 4. Huang SN, Minassian H, Morey TD. Application of immunofluorescent staining on paraffin sections improved by trypsin digestion. Lab Invest 1976; 35: 383-90. 5. Dobersen MJ, Bell AM, Jenson AB, Notkins AL, Ginsberg-Fellner F. Detection of antibodies to islet cells and insulin with paraffin embedded pancreas as antigen. Lancet 1979; ii: 1078. 1.
SiR.—Dr David and Dr Phillips point out that cystic fibrosis need
overdiagnosed if sweat tests are performed in centres where this test is carried out frequently and are repeated in cases of doubt. Furthermore, all the clinical criteria should be taken into account before the diagnosis is made. Surely these are minimum requirements in contemporary paediatric care. That some patients should not only have been misdiagnosed in the manner they described but also that four of the eight patients should have been sent to special schools on the basis of the wrong diagnosis is surely serious mismanagement. This is seldom if ever necessary in an established case of cystic fibrosis. This article draws attention away from the important fact of underdiagnosis of cystic fibrosis. There are no accurate data on the number of children and young adults in the U.K. with cystic fibrosis; however, P. Phelan (personal communication) has reviewed the mortality rates of cystic fibrosis for 1976-80 inclusive from Office of Population Censuses and Surveys data in the U.K., using the principal cause of death, and compared them with those of the State of Victoria in Australia where comprehensive child care and pathological services can provide accurate information. He has revealed the disturbing fact that mortality rates are two to three times higher in the U.K. in the age groups <1, 1-4, 5-9, and 9-15 years. The genetic make-up of the two populations is very similar. While it is possible that some of this difference stems from difficulties in diagnosis it is likely that inadequacies in management are also responsible. It has already been shown in the United States and Denmark as well as in Australia that reference to special units early after the onset of symptoms has a profoundly beneficial effect not
on
CONCENTRATION
be
prognosis.
There is clearly a need for regional centres in this country to help with the diagnosis and participate in the care of patients with cystic fibrosis. Early referral to such special units for accurate sweat tests and subsequent joint management is essential if our patients with cystic fibrosis are to be given opportunities similar to those that exist elsewhere. Brompton Hospital, London SW3 6HP
J. C. BATTEN D. M. GEDDES
CYCLOSPORIN INTERACTION WITH KETOCONAZOLE AND MELPHALAN
SiR,—Dr Ferguson and colleagues (Oct. 16, p. 882) report an cyclosporin and ketoconazole in a renal transplant patient, causing renal impairment and an increase in cyclosporin blood levels which they attribute to interference with the hepatic metabolism of cyclosporin. This interaction has been reported in bone marrow transplant recipients, in whom it was attributed to increased cyclosporin absorption, and we have seen a similar interaction in a marrow transplant recipient who received both agents simultaneously (see table). We therefore avoid the concomitant use of cyclosporin and ketoconazole. We have also found a potentially dangerous interaction between cyclosporin and high-dose melphalan,2adrug we have been exploring as a conditioning agent for allogeneic bone marrow transplantation for acute leukaemia.3Seventeen patients received melphalan as a single injection of 140-250 mglm2with forced diuresis, followed, 1-4 days later, by infusion of donor bone marrow and cyclosporin in standard dose (12-5mg/kg/day orally) as prophylaxis against graft-versus-host disease. Thirteen patients (76%) had severe renal failure (plasma urea >30 mmolll, plasma creatinine >400 mol/1 or both); five required peritoneal and/or interaction between
*Ketaconazole 200 mg
daily days 62-68.
haemodialysis. Only two patients had mild or no impairment of renal function (plasma urea <11 mmol/1 and creatinine <200 mol/1). This compares with seven patients who received syngeneic or repeat allogeneic transplants after melphalan (180-240 mg/m2) but without cyclosporin3where no deterioration in renal function occurred. Urea and creatinine levels remained normal in five and were static or improved in the two patients who had renal impairment at the time of transplantation, demonstrating that melphalan itself is not overtly nephrotoxic in these circumstances. In the original study of high-dose melphalan renal impairment occurred 2 only in two patients who had not received forced diuresis.2 The nature of the interaction between melphalan and cyclosporin is unclear. Serum cyclosporin levels were determined 2 h post dose by radioimmunoassay in fourteen of the patients who received cyclosporin following melphalan and in sixty patients conditioned with cyclophosphamide and total body irradiation who received similar cyclosporin prophylaxis. 8 days after transplantation the levels were 165-1450 ng/ml (median 715) for the melphalan patients and 59-2922 ng/ml (median 473) for the irradiated patients. This difference is not significant and the interaction may be directly on the kidney rather than in the absorption/metabolism of cyclosporin. Although some degree of renal impairment is seen in more than 90% ofour patients receiving cyclosporin after total body irradiation and transplantation,4 it is mild or moderate (plasma urea <20 mmol/1) in 55% of them and generally reversible when the cyclosporin dose is reduced. We are now testing lower dose schedules of cyclosporin, together with early interruption of the agent to prevent severe Leukaemia Unit, Royal Marsden Hospital, Sutton, Surrey SM2 5PT
nephrotoxicitv.
G. R. MORGENSTERN RAY POWLES BRIDGET ROBINSON T. J. McELWAIN
KETOCONAZOLE-CYCLOSPORIN INTERACTION
SIR,-The rise in cyclosporin concentrations in two patients after the start of ketoconazole therapy5,6 has been interpreted as ketoconazole-induced inhibition of hepatic metabolism.5 These patients received a ketoconazole dose of 400 mg daily (about 8 mg/kg), while in animals a much greater dose (50 mg/kg) was required to inhibit microsomal enzymes.7 In eight healthy volunteers I and my colleagues (unpublished) have examined the effect of ketoconazole, 400 mg daily for 5 days, on antipyrine kinetics, an indirect in-vivo measure of hapatic microsomal oxidative activity.88 Antipyrine clearance was 4.
F, Poirier O, Gluckman E, et al. Pharmacokinetic study of cyclosporin A: Preliminary results. In. Touraine J, Gluckman E, Griscelli C, eds. Bone marrow transplantation in Europe II. Amsterdam: Excerpta Medica, 1981: 160-64. 2. McElwain TJ, Hedley DW, Burton G, et al. Marrow autotransplantation accelerates haematological recovery in patients with malignant melanoma treated with highdose melphalan. Br J Cancer 1979; 40: 72-80. 3. Lumley HS, Powles R, Morgenstern GR, et al. Pseudosyngeneic transplantation as a treatment for recurrent leukaemia following allogeneic bone marrow transplantation. In: Touraine J, Gluckman E, Griscelli C, eds. Bone marrow transplantation in Europe II. Amsterdam: Excerpta Medica, 1981: 24-28. 1. Lokiec
Hedley D, Powles RL, Morgenstern GR. Toxicity of cyclosporin A in patients following bone marrow transplantation. In: White DJ, ed. Cyclosporin A. Amsterdam: Elsevier, 1982: 545-51 5. Ferguson RM, Sutherland DER, Simmons RL, Najarian JS. Ketoconazole, cyclosporin metabolism, and renal transplantation. Lancet 1982; ii: 882-83. 6. Dieperink H, Moller J. Ketoconazole and cyclosporin. Lancet 1982; ii. 1217. 7. Niemegeers CJE, Levron JCI, Awouters F, Janssen PAJ. Inhibition and induction of microsomal enzymes in the rat: a comparative study of four antimycotics: miconazole, econazole, clotrimazole and ketoconazole Arch Int Pharmacodyn 1981; 251: 26. 8. Vesell ES.
The antipyrine test in clinical pharmacology: Conceptions misconceptions. Clin Pharmacol Ther 1979; 26: 275-86.
and
1343 37’7± 12 -3ml/min (mean±SD) before and 40 -7± 12 -4ml/min after ketoconazole (not significant; Student’s t test for paired data). Thus, at the doses used, ketoconazole does not appear to be an inhibitor of microsomal enzymes in man. The ketoconazole-cyclosporin interaction may be due to mechanisms other than enzyme inhibition-e.g., a ketoconazoleassociated change in the apparent volume of distribution of cyclosporin, protein binding, or competition for an excretion pathway. Since systematic investigation of this important interaction may not be ethically feasible, it would be important to measureand report ketoconazole as well as cyclosporin and creatinine concentrations in future. Department of Medicine, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW
T. K. DANESHMEND
DETERMINATION OF ISLET-CELL ANTIBODIES BY IMMUNOFLUORESCENCE
SIR,-We would like to endorse the views of Dr Yagihashi and coworkers (Nov. 27, p. 1218) that, in the absence of fully characterised tissue antigens, fresh unfixed pancreas should be used for the clinical screening of patients with insulin-dependent diabetes mellitus (IDDM). Complement-fixing antibodies (CFare vital ICA) in the sera of non-diabetic siblings ofIDDM probands markers for the potential development of diabetes, 1,2 because some CF-ICA are specifically directed against the insulin-secreting betacells which bear the brunt of the insulitis leading to diabetes.3 We have found that Bouin-fixed pancreas cannot be used for the CFICA test since human complement attaches preferentially to betacells and positive results are seen with normal sera. Although our experience with Bouin-fixed pancreas is limited, the results (table) COMPARISON OF ISLET CELL ANTIBODIES
(ICA-IgG) ON UNFIXED AND
BOUIN-FIXED PANCREAS
COMPLEMENT C4 ALLOTYPES IN ALZHEIMER’S DISEASE
SIR,-Although no conclusion can be drawn about a possible association between HLA antigen and Alzheimer’s disease,I-3 we have noted that C4 B2, a rare variant of complement C4 which is encoded by genes within the HLA complex, is more common than expected in patients with senile dementia of the Alzheimer’s type
(SDAT). was established by history and exclusion of other The criteria were those of Blessed et allthey correlate significantly with pathological 5and biochemical changes characteristic of Alzheimer’s disease. Complement C4 allotyping was done in 38 patients with SDAT, in 42 patients with Parkinson’s disease, and in 59 unaffected, agematched controls of the same ethnic background. The C4 B2 variant was found in 14% (8/59) of the age-matched controls, a figure in accord with the frequency of C4 B2 in our random panel of White subjects and that of others.The frequency of the C4 B2 variant in the Parkinson’s disease group was 12% (5/42). In SDAT, however, we found a striking increase of C4 B2, with 55% (21/38) of patients positive for this variant, resulting in a relative risk of 8 -8(p<0 - 0001) for this disorder. The frequencies of all other C4 alleles at either the C4A (= Rodgers) or C4B (= Chido) locus did not differ from those of the control groups. In HLA associated diseases, a corresponding association with the HLA linked complement markers (C2, C4, and Bf) is often observed. This, however, is usually interpreted as being due to linkage disequilibria between the complement markers and HLA antigens, and regarded as a secondary effect. Alzheimer’s disease, therefore, represents the first disorder with a strong association with an HLA-linked complement component (in this case C4), in the absence of any clear association with a particular HLA antigen. Since the complement system plays a vital role in host defence against virus infection, this finding is of particular importance in Alzheimer’s disease, where a viral aetiology is suspected. It will be of considerable interest, therefore, to determine whether certain C4 variants, such as C4 B2, are less effective in virus neutralisation.
The
diagnosis
causes.
Departments of Pathology and Neurology, College of Physicians and Surgeons, Columbia University, New York, N.Y. 10032, U.S.A.
CHRISTOPH W. NERL RICHARD MAYEUX GEOFFREY J. O’NEILL
*After de-waxing, 4 nm sections were preincubated for 2 h at 37° with 0 - I% calcium chloride containing O. 1% trypsin.. - fThe number of sera with cytoplasmic membrane or antinuclear (speckled or diffuse) staining in the islets is shown. A single pancreas ofpre-tested antigenicity (PI 5) was used for cryostat sections, whereas four different pancreases, including P15, were used for the Boum fixed ICA determinations. N=no. of subjects, n=no. of sera (applies to all three antibodies).
substantiate the conclusions of Yagihashi et al. that, after fixation, less than 20% of the substrates are’ suitable for ICA-IgG determinations as compared with 80% of unfixed group 0 glands. We used sera from patients without insulin treatment, to avoid discrepancies due to insulin antibodies, which are readily detected on Bouin-fixed but not on unfixed pancreas. On fixed sections the islet staining was complex, and cytoplasmic, cell-membrane, and nuclear patterns occurred concurrently or singly. Omission of trypsin, or both trypsin and calcium chloride, from the preincubation media did not alter the intensity of cytoplasmic or membrane immunofluorescence, but considerably reduced antinuclear staining. The patterns and intensity of immunofluorescence obtained with normal controls on fixed tissue were mostly indistinguishable from those of sera scored as positive on cryostat sections. This disagrees with the initial report of Dobersen et al.5 but accords with the low percentage of fixed pancreases found suitable by Yagihashi et al. for ICA determination. In our laboratory unfixed human pancreas has been used routinely and in research projects for the past eight years and, on average, 25% of the pancreases have to be discarded because of weak antigenicity. On cryostat sections, normal sera invariably show a clear negative reaction. A diffuse cytoplasmic staining covering all the cells of the
9. Daneshmend
TK, Warnock DW, Turner A, Robert CJC. Pharmacokinetics of ketoconazole in normal subjects. J Antimicrob Chemother 1981; 8: 299-304. 1. Henschke PJ, Bell DA, Cape RDT. Alzheimer’s disease and HLA. Tissue Antigens
1978; 12: 132-35. R, Koskimies S, Wikström J, Palo J HLA antigens in Alzheimer’s disease. Tissue Antigens 1980; 16: 191-94. 3. Walford RL, Hodge SE. HLA distribution in Alzheimer’s disease. In: Teraski PI, ed. Histocompatibility testing 1980: Los Angeles, UCLA 1980; 727-29. 4 Blessed G, Tomlinson BE, Roth M. The association between quantitative measures of dementia and of senile change in the cerebral grey matter of elderly subjects. Br J Psychiatry 1968; 114: 797-881. 5. Perry EK. The cholinergic system in old age and Alzheimer’s disease. Age Aging 1980; 2. Sulkava
9: 1-8. 6. Awdeh ZL,
Alper CA. Inherited structural polymorphism of the fourth component of complement. Proc Natl Acad Sci (USA) 1980; 77: 3576-80.
Bottazzo GF, Dean BM, Gorsuch AN, Cudworth AG, Doniach D. Complement-fixing islet-cell antibodies in type I diabetes: Possible monitors of active beta-cell damage. Lancet 1980; i: 668-72. 2. Gorsuch AN, Lister J, Dean BM, Spencer KM, Mc Nally JM, Bottazzo GF, Cudworth AG. Evidence for a long pre-diabetic period in type I (insulin-dependent) diabetes mellitus. Lancet 1981; ii: 1363-65. 3 Doniach D, Bottazzo GF, Cudworth AG. Etiology of Type I diabetes mellitus: heterogeneity and immunological events leading to clinical onset. Annu Rev Med 1983; 34 (in press). 4. Huang SN, Minassian H, Morey TD. Application of immunofluorescent staining on paraffin sections improved by trypsin digestion. Lab Invest 1976; 35: 383-90. 5. Dobersen MJ, Bell AM, Jenson AB, Notkins AL, Ginsberg-Fellner F. Detection of antibodies to islet cells and insulin with paraffin embedded pancreas as antigen. Lancet 1979; ii: 1078. 1.