1364
Housedust mite allergens and
IgG
colleagues (March 21, p 746) suggest that be IgG proteolysed by an extract from housedust mites. These investigators incubated IgG with such an extract, or with papain, for 7 h or 24 h at 37°C in the presence of dithiothreitol 1 mmol/1 to "activate cysteine proteinase". Subsequent SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of these incubation mixtures demonstrated bands corresponding to relative molecular weights of about 52 000 and 25 000. Dithiothreitol at a concentration of 1 mmol/l will reduce the interchain disulphide bonds of IgG in a few minutes at 37OC.1 SDS-PAGE will then produce bands corresponding to the heavy (52 000) and light (25 000) chains of IgG, as shown in lanes 3-6 of the gel presented by Colloff and colleagues. Evidently the intact IgG "negative control" (lane 2 of the gel) was not treated with dithiothreitol. It has been our experience2 that SDS-PAGE is more useful for assessing proteolytic fragmentation of IgG when the disulphide bonds are not reduced. It is possible that the material examined by Colloff and colleagues does contain the activity proposed and is responsible for the biological effects suggested. However, from the reported findings, there is no evidence that housedust mite allergenic extract contains a proteinase capable of digesting IgG. SIR,-Dr
Colloff and
can
Plasma thrombin-antithrombin-111 (TATc) and plasmin-a-2antiplasmin (PAPc) in representative patient during first day of treatment with 5 mg OKT3 for acute renal allograft
rejection. Arrow indicates first administration of OKT3. Time points are: before and at 15,30, 60,120, 240, 360 min after the first OKT3 administration. TATc was measured by ELISA (Enzygnost TATc kit, Behringwerke). PAPc was measured by radioimmunoassay.2
episode. 23 patients (14 males, 9 females; median age 48 years, range 33-72) were treated with intravenous methylprednisolone 500 mg
DONALD L. TANKERSLEY
J. S. FINLAYSON
1. Schroeder DD, Tankersley DL, Lundblad JL. A new preparation of modified immune serum globulin suitable for intravenous administration I. standardization of the reduction and alkylation reaction. Vox Sang 1991; 40: 373-82. 2. Young AM, Aronson DL, Finalyson JS. A urokinase incubation method for predicting the stability of immune globulin. J Biol Stand 1978; 6: 27-43.
6 consecutive
days. 26 (14 males, 12 females; median age 50, 25-67) were treated with OKT3, 5 mg intravenous bolus injection on 10 consecutive days; the first dose of OKT3 was always preceded by methylprednisolone 500 mg. The incidence of thromboembolic complications occurring within 3 months after treatment did not differ between both groups of patients. Thrombosis of the arteriovenous shunt occurred in 2 patients treated with methylprednisolone and in 1 patient treated with OKT3, and deep venous thrombosis of a leg developed in 1 patient on the fifth day of treatment with OKT3. Renal vascular thrombosis did not occur in any patient. Our findings indicate that a daily dose of 5 mg OKT3 is not associated with an incidence of thromboembolic complications any greater than that for treatment with methylprednisolone. This may be explained by the fact that upon the first dose of 5 mg OKT3 not only the coagulation system but also the fibrinolytic system is on
Division of Hematology, Center for Biologics Evaluation and Research, Bethesda, Maryland 20892, USA
range
immediately
activated.’ This simultaneous activation of both
probably results in a net non-procoagulant situation, thus explaining the absence of thrombotic complications. The systems
differences between the results of Abramowicz et al and our data probably indicate that thrombotic complications during treatment with OKT3 are dose related and may be avoided by treatment with the usual dose of 5 mg instead of the high dose of 10 mg daily. Renal Transplant Unit and Clinical Immunology Laboratory, Department of Internal Medicine, and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Academic Medical Centre, 1105 AZ Amsterdam, Netherlands
* Thisletter
has been shown
to
Dr
Colloff, whose reply
follows.-ED. L.
SIR,-Mr Tankersley and Dr Finlayson state that dithiothreitol (DTT) 1 mmol/1 will reduce interchain disulphide bonds of IgG in a few minutes, thus accounting for the bands corresponding to heavy and light chains on our gel. Thus, our fmding may be caused by DTT and not by mite allergen. We also incubated mite allergen extracts with IgG in the absence of DTT and found the same effect; we should have mentioned this in our paper. On rechecking the concentration of DTT used I discovered a dilution error: the stock DTT concentration was 1 mmol/1 but the final concentration used was only 9 umol/1, which is too dilute to have much reducing effect on IgG (see ref 1 in above letter). Evidently the protease(s) in the mite extract do not require activation/enhancement of activity with DTT. Department of Zoology, University of Glasgow, Glasgow G12 8QQ, UK
M. J. COLLOFF
Are health-care workers really at risk of HCV infection? SIR,-Dr Jochen (Feb 1, p 304) has reported an increased risk of hepatitis C virus (HCV) in hospital personnel. He
exposure to
M. H. M. RAASVELD S. SURACHNO
C. E. HACK R. J. M. TEN BERGE
1. Raasveld MHM, Hack CE, ten Berge RJM. Activation of coagulation and fibrinolysis following OKT3 administration to renal transplant recipients: association with distinct mediators. Thromb Haemost (in press). 2. Levi M, de Boer JP, Roem D, ten Cate JW, Hack CE. Plasminogen activation in vivo upon intravenous infusion of DDAVP: quantitative assessment of plasmin-&agr;2antiplasmin complexes with a novel monoclonal antibody based radioimmunoassay. Thromb Haemost 1992; 67: 111-16. 3. Ortho Multicentre Transplant Group. A randomized clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplants. Ortho Multicenter Transplant Study Group. N Engl J Med 1985; 313: 337-42. 4. Thistlethwaite JR, Stuart JK, Mayes JT, et al. Complications and monitoring of OKT3 therapy. Am J Kidney Dis 1988; 11: 112-19.
found higher HCV antibody in 6 of 1033 hospital workers (0-58%) and 5 of 2113 volunteer blood donor controls (0-24%). In a similar study in southern Italy in 1990, we too found anti-HCV more often in hospital personnel than in blood donors (odds ratio 4’5, 95% CI
2-87-7-16; P < 0.001).1,2
However,
we
subsequently investigated
anti-HCV prevalence in another group of workers: factory employees in the same geographical area, and found antibody twice as often in this group than in hospital personnel (odds ratio 2 2, 95 % CI1.5-3.4; p
Housedust mite allergens and
IgG
colleagues (March 21, p 746) suggest that be IgG proteolysed by an extract from housedust mites. These investigators incubated IgG with such an extract, or with papain, for 7 h or 24 h at 37°C in the presence of dithiothreitol 1 mmol/1 to "activate cysteine proteinase". Subsequent SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of these incubation mixtures demonstrated bands corresponding to relative molecular weights of about 52 000 and 25 000. Dithiothreitol at a concentration of 1 mmol/l will reduce the interchain disulphide bonds of IgG in a few minutes at 37OC.1 SDS-PAGE will then produce bands corresponding to the heavy (52 000) and light (25 000) chains of IgG, as shown in lanes 3-6 of the gel presented by Colloff and colleagues. Evidently the intact IgG "negative control" (lane 2 of the gel) was not treated with dithiothreitol. It has been our experience2 that SDS-PAGE is more useful for assessing proteolytic fragmentation of IgG when the disulphide bonds are not reduced. It is possible that the material examined by Colloff and colleagues does contain the activity proposed and is responsible for the biological effects suggested. However, from the reported findings, there is no evidence that housedust mite allergenic extract contains a proteinase capable of digesting IgG. SIR,-Dr
Colloff and
can
Plasma thrombin-antithrombin-111 (TATc) and plasmin-a-2antiplasmin (PAPc) in representative patient during first day of treatment with 5 mg OKT3 for acute renal allograft
rejection. Arrow indicates first administration of OKT3. Time points are: before and at 15,30, 60,120, 240, 360 min after the first OKT3 administration. TATc was measured by ELISA (Enzygnost TATc kit, Behringwerke). PAPc was measured by radioimmunoassay.2
episode. 23 patients (14 males, 9 females; median age 48 years, range 33-72) were treated with intravenous methylprednisolone 500 mg
DONALD L. TANKERSLEY
J. S. FINLAYSON
1. Schroeder DD, Tankersley DL, Lundblad JL. A new preparation of modified immune serum globulin suitable for intravenous administration I. standardization of the reduction and alkylation reaction. Vox Sang 1991; 40: 373-82. 2. Young AM, Aronson DL, Finalyson JS. A urokinase incubation method for predicting the stability of immune globulin. J Biol Stand 1978; 6: 27-43.
6 consecutive
days. 26 (14 males, 12 females; median age 50, 25-67) were treated with OKT3, 5 mg intravenous bolus injection on 10 consecutive days; the first dose of OKT3 was always preceded by methylprednisolone 500 mg. The incidence of thromboembolic complications occurring within 3 months after treatment did not differ between both groups of patients. Thrombosis of the arteriovenous shunt occurred in 2 patients treated with methylprednisolone and in 1 patient treated with OKT3, and deep venous thrombosis of a leg developed in 1 patient on the fifth day of treatment with OKT3. Renal vascular thrombosis did not occur in any patient. Our findings indicate that a daily dose of 5 mg OKT3 is not associated with an incidence of thromboembolic complications any greater than that for treatment with methylprednisolone. This may be explained by the fact that upon the first dose of 5 mg OKT3 not only the coagulation system but also the fibrinolytic system is on
Division of Hematology, Center for Biologics Evaluation and Research, Bethesda, Maryland 20892, USA
range
immediately
activated.’ This simultaneous activation of both
probably results in a net non-procoagulant situation, thus explaining the absence of thrombotic complications. The systems
differences between the results of Abramowicz et al and our data probably indicate that thrombotic complications during treatment with OKT3 are dose related and may be avoided by treatment with the usual dose of 5 mg instead of the high dose of 10 mg daily. Renal Transplant Unit and Clinical Immunology Laboratory, Department of Internal Medicine, and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Academic Medical Centre, 1105 AZ Amsterdam, Netherlands
* Thisletter
has been shown
to
Dr
Colloff, whose reply
follows.-ED. L.
SIR,-Mr Tankersley and Dr Finlayson state that dithiothreitol (DTT) 1 mmol/1 will reduce interchain disulphide bonds of IgG in a few minutes, thus accounting for the bands corresponding to heavy and light chains on our gel. Thus, our fmding may be caused by DTT and not by mite allergen. We also incubated mite allergen extracts with IgG in the absence of DTT and found the same effect; we should have mentioned this in our paper. On rechecking the concentration of DTT used I discovered a dilution error: the stock DTT concentration was 1 mmol/1 but the final concentration used was only 9 umol/1, which is too dilute to have much reducing effect on IgG (see ref 1 in above letter). Evidently the protease(s) in the mite extract do not require activation/enhancement of activity with DTT. Department of Zoology, University of Glasgow, Glasgow G12 8QQ, UK
M. J. COLLOFF
Are health-care workers really at risk of HCV infection? SIR,-Dr Jochen (Feb 1, p 304) has reported an increased risk of hepatitis C virus (HCV) in hospital personnel. He
exposure to
M. H. M. RAASVELD S. SURACHNO
C. E. HACK R. J. M. TEN BERGE
1. Raasveld MHM, Hack CE, ten Berge RJM. Activation of coagulation and fibrinolysis following OKT3 administration to renal transplant recipients: association with distinct mediators. Thromb Haemost (in press). 2. Levi M, de Boer JP, Roem D, ten Cate JW, Hack CE. Plasminogen activation in vivo upon intravenous infusion of DDAVP: quantitative assessment of plasmin-&agr;2antiplasmin complexes with a novel monoclonal antibody based radioimmunoassay. Thromb Haemost 1992; 67: 111-16. 3. Ortho Multicentre Transplant Group. A randomized clinical trial of OKT3 monoclonal antibody for acute rejection of cadaveric renal transplants. Ortho Multicenter Transplant Study Group. N Engl J Med 1985; 313: 337-42. 4. Thistlethwaite JR, Stuart JK, Mayes JT, et al. Complications and monitoring of OKT3 therapy. Am J Kidney Dis 1988; 11: 112-19.
found higher HCV antibody in 6 of 1033 hospital workers (0-58%) and 5 of 2113 volunteer blood donor controls (0-24%). In a similar study in southern Italy in 1990, we too found anti-HCV more often in hospital personnel than in blood donors (odds ratio 4’5, 95% CI
2-87-7-16; P < 0.001).1,2
However,
we
subsequently investigated
anti-HCV prevalence in another group of workers: factory employees in the same geographical area, and found antibody twice as often in this group than in hospital personnel (odds ratio 2 2, 95 % CI1.5-3.4; p
