- Email: [email protected]
Cyclosporin A
779 a balanced venous and arterial effect, be prazosin,33 used in much the same doses as for hypertension. One study33 revealed sustained benefit during an average of 12 weeks, but no truly long-term formal investigation of this or any other vasodilator drug in heart-failure has been reported. So the approach, although encouraging, must still be regarded as experimental.
drug with
seems to
Cyclosporin CYCLOSPORIN A is in the
A
because it is a powerful immunosuppressive agent which, unlike cyclophosphamide or corticosteroids, does not produce leucopenia or cushingoid changes. The pharmacological basis for the highly desirable properties is obscure. CyA is a cyclic polypeptide of 11 aminoacids which can be extracted from several species of fungus.l Several of the aminoacids are N-methylated and this unusual structure probably explains why it resists digestion in the gut. During short courses of CyA mice are unable to make primary or secondary antibody responses to a wide range of antigens but respond to unrelated antigens as little as 24 h after the last dose.2 The antibody responses of nude mice to thymus-independent antigens are not inhibited by CyA, so T lymphocytes are the likely targets for the drug’s immunosuppressive actions.3 Results of in-vitro lymphocyte stimulation are consistent with this view: CyA inhibits the proliferation of mouse lymphocytes stimulated by the T-cell mitogen concanavalin A but does not prevent the response of B lymphocytes to lipopolysaccharide.4 Once T lymphocytes have started to divide they become resistant to inhibition by CyA-a property in keeping with the observation that, for maximum immunosuppression, CyA must be given concurrently with antigen. The initial animal work showed that, as well as suppressing antibody, CyA prolonged the survival of foreign skin grafts and the survival of animals with graft-versus-host disease; it also prevented the development of experimental allergic encephalomyelitis and reduced joint swelling in Freund’s adjuvant arthritis. Acute inflammatory responses were not impaired.2 Transplantation groups have been quick to explore the drug’s possibilities and impresnews
33. Awan NA, Miller RR, Miller MP, Specht K, Vera Z, Mason DT. Clinical pharmacology and therapeutic application of prazosin in acute and chronic refractory congestive heart failure. Am J Med 1978; 65: 146-54. 1. Dreyfus M, Hyerri E, Hotman H, Kobel H. Cyclosporin A and C, new metabolites from Trichoderma polysporum. Eur J Appl Microbiol 1976; 3: 125-33. 2. Borel JF, Feurer C, Gubler HV, Stahelin H. Biological effects of cyclosporin A: anew anti-lymphocytic agent. Ag Actions 1976; 6: 468-75. 3. Borel JF, Feurer C, Magnee C, Stahelin H. Effects of the new antilymphocytic peptide cyclosporin A in animals. Immunology 1977; 32: 1017-25. 4. Borel JF, Wiesinger D. Effect of cyclosporin A on murine lymphoid cells. In: Lucas D, ed. Regulatory mechanisms in lymphocyte activation. London: Academic Press. 1977.
sive results have been obtained in kidney-grafted rabbits. 6 of 10 animals which for 18 or 28 days after their allografts received CyA as their only immunosuppression were alive with functioning kidneys over a year later. These animals accepted skin or further kidney grafts from their original donors but they rejected grafts from third-party donors. In pigs the survival of heart allografts was prolonged by CyA, and in some animals the graft continued to be tolerated after the drug was stopped.6 Heterotopic heart grafts in monkeys fared rather less well, in that histological evidence of rejection was present after 50-day course of CyA given daily at first and then on alternate days,7 though the grafts themselves continued to beat. There were no toxic effects clearly attributable to the CyA in these series though in another study focal liver cell necrosis, cholestasis, and jaundice developed in 5 of 34 dogs given unmatched kidney grafts.8CyA has been tried for the treatment of graft versus-host (GVH) disease.95 patients, all with GVH disease after sibling marrow grafts for the treatment of relapsed leukaemia, were given CyA, and in each case the typical rash resolved. The other features, including diarrhoea and hepatitis, persisted and 4 of the patients died. In the survivor the rash remitted several times after intermittent courses of CyA. Severe GVH disease is usually fatal so these results are not as bleak as they seem. Since the immunosuppressive effects of CyA are greatest when it is given concurrently with antigen priming, the drug might be expected to be more effective in preventing than in treating GVH disease. Work in animals seems to confirm this view. Rats rendered aplastic with busulphan were given incompatible marrow in a strain combination which, without immunosuppression, does not result in engraftment.10 CyA-treated recipients of incompatible marrow became chimseric and their survival over a 28-day follow-up equalled that of busulphan-treated controls which were grafted with syngeneic marrow. None of the CyA-treated rats acquired GVH disease, but it did develop in each of the surviving cyclophosphamide-treated rats. Further information on the action of CyA and its use in human transplantation emerged at an international symposium on immunosuppression held
5. Green CJ, Allison AC, Precious S. Induction of specific tolerance in rabbits by kidney allografting and short periods of cyclosporin A treatment. Lancet 1979; ii: 123-25. 6. Calne RY, White DJG, Rolles K, Smith DP, Herbertson BM. Prolonged survival of pig orthotopic heart grafts treated with cyclosporin A. Lancet
1978; i: 1183-85. Jamieson SW, et al. Cardiac allograft survival in primates treated with cyclosporin A. Lancet 1979; i: 545. 8. Calne RY. Immunosuppression for organ grafting—observations on cyclosporin A. Immunol Rev 1979; 46: 113-24. 9. Powles RL, et al. Cyclosporin A for the treatment of graft versus host disease 7.
in man. Lancet 1978; ii: 1327. 10. Tutschka PJ, Beschomer WE, Allison AC, Burns WH, Santos GW. Use of cyclosporin A in allogeneic bone marrow transplantation in the rat. Nature 1979; 280: 148-51.
780
in Cardiff last month. Once again CyA was shown to prolong skin and kidney’ allograft survival in rodents, although its ability to suppress GVH disease in rats with bone-marrow transplants was not so striking. Three groups described the use of CyA in experimental pancreatic islet transplantation, the results being rather poor. Segmental or wholeorgan transplants fared much better; in one experiment pancreatectomised dogs were given whole pancreas grafts, and half had normal blood-sugars 80 days after transplantation. Prof. R. Y. CALNE recounted his experience with CyA in 30 patients who had received cadaveric kidney transplants. Although mild rejection episodes occurred, no graft was rejected and 25 of the patients still have functioning transplants. 5 died from sepsis, 4 of them having been on two immunosuppressive drugs other than CyA-a combination which Professor CALNE now feels should be avoided. His present policy is to give CyA (17 mg/kg) alone and to treat rejection with a single short course of steroids. Various toxic effects have been observed, the least serious of which are an increase in body hair, gum hypertrophy, and tremors. All but 2 of the patients had abnormal liver-function tests and 16 became anuric immediately after transplantation or within a few days. This last complication may be avoided by giving fluids and mannitol to the kidney recipient at the time of transplantation. By far the most serious complication has been the development of lymphoma in three patients, 4, 6, and 9 months after the start of CyA treatment. The lymphoma was discovered post mortem in the lung and ileum of two patients who had died of sepsis, and in the stomach of a third patient who has had the tumour resected. This 10% incidence of lymphoma is very
worrying.
ENZYME IMMUNOASSAYS REVISITED
SOME three years ago a short editorial promoted the virtues of enzyme immunoassays. This prompted several leading radioimmunoassay enthusiasts to put pen to paper in defence of their treasured technique. With the
benefit of time we can now see some trends emerging more clearly. - The enzyme immunoassays are of two types-the homogeneous type without washing steps (exemplified by the EMIT system, Syva Corporation), and the heterogeneous types generally known as enzymelinked immunosorbent assays (ELISA). The homogeneous assays are very rapid but are not very sensitive and are really only suitable for the measurement of small molecules. They have been remarkably successful and are now accepted as routine methods for the assay of anticoagulants, drugs of abuse, cardioactive drugs, and thyroxine. The availability of easy-to-use reagent kits and convenient processing apparatus has done much to ensure that this method is acceptable in both large and small laboratories.
The
heterogeneous enzyme immunoassays (ELISA) quite different in that they are rather slow, requiring several incubation/washing steps, but they are suitable for assaying large molecules and they are very sensitive. To date, ELISA has not become a routine method in most clinical laboratories, possibly because few reagent kits are available commercially and automated equipment is still in its infancy. However, ELISA has been the subject of several hundred scientific publications and there has been especial emphasis in the sphere of infectious diseases. These applications have dealt both with the detection of antigen (or the infectious agent) and with the antibody response to the infectious agent. As usual, hepatitis has been a focus of attention, and at first ELISA was developed for the detection of HBsAg.l,2’ Various commercial ELISA kits were produced for this purpose--e.g., ’Hepanostika’ (Organon Teknika), ’Cordia H’ (Cordis Laboratories), and ’Auszyme’ (Abbott Laboratories)-and they are regarded as third-generation tests, being slightly more sensitive than reverse passive agglutination tests but not quite as sensitive as are
the radioimmunoassays.3 Since then assays have also been developed for the "e" antigen and for anti-HBs. Hepatitis A antigen has been detected, but in this disease the ELISA for IgM antibody to the virus seems a much more important diagnostic tool. 5,6 ELISA in viral serology is progressing along two lines-general screening tests which measure antibody in all immunoglobulin classes, and tests for recent infection which are based on IgM antibody assays. Already numerous papers have been published on the ELISA screening methods for rubella, cytomegalovirus, measles, herpes, and influenza,’-9 and commercial ELISA kits exist for rubella and cytomegalovirus. The IgM detection systems have been plagued with false positives and false negatives, but new solid-phase and column serum fractionation methods should mean that IgM ELISA can be used as a routine method for early diagnosis. Very promising results have been obtained with ELISA tests for antibody to rotavirus and for detecting virus in fæces.10,11 The fact that ELISA is cheap and easily performed
1. Wolters G, Kuijpers L, Kacaki J, Schuurs A. Solid phase enzyme immunoassay for detection of hepatitis B surface antigen. J Clin Path 1976; 29: 873-79. 2. Halbert ST, Anken M. Detection of hepatitis B surface antigen (HBsAg) with use of alkaline phosphatase labelled antibody to HBsAg. J Infect Dis 1977; 136: suppl 318-23. 3. Vandervelde EM. An enzyme linked immunosorbent assay test for hepatitis B surface antigen. J Clin Path 1977; 30: 714-16. 4. Waart MVD, Snelting A, Cichy J, Wolters G, Scuurs A. Enzyme immunoassay in the diagnosis of hepatitis with emphasis on the detection of ‘e’ antigen (HBeAg). J Med Virol 1978; 3: 43-49. 5. Duermeyer W, van der Veen J. Specific detection of IgM antibodies by ELISA applied in hepatitis A. Lancet 1978; ii, 684-85. 6. Matthiesen LR, Feinstone SM, Skinhoe JP, Purcell RM. Enzyme immunoas-
say for hepatitis A. J Clin Microbiol 1978; 7: 184. 7. Bidwell DE, Bartlett A, Voller A. Enzyme immunoassays for viral diseases. J Infect Dis 1977; 136: suppl 274-78. 8. Morgan-Capner P, Pullen HJM, Pattison JR, Bidwell DE, Bartlett A, Voller A. A comparison of three tests for rubella antibody screening. J Clin Path
1979; 32: 542-45. P, Passila S. Solid phase antibody assay by means of enzyme conjugated to anti-immunoglobulin. J Clin Path 1977; 29: 1116-20. 10. Yolken RH, Kim HW, Clem T, Wyatt RG, Kalica AR, Chanock RM, Kapikian AZ. Enzyme linked immunosorbent assay (ELiSA) for detection of human reovirus-like agent of gastroenteritis. Lancet 1977; ii: 263-66. 11. Yolken RH, Wyatt RG, Kim HW, Kapikian AZ, Chanock RM. Immunologi9. Leinikki
cal response to infection with human reovirus-like agent: measurement of anti-human reovirus-like immunoglobulin G and M levels by the method
of ELISA. Infect Immunity 1978; 19: 540-46.
drug with
seems to
Cyclosporin CYCLOSPORIN A is in the
A
because it is a powerful immunosuppressive agent which, unlike cyclophosphamide or corticosteroids, does not produce leucopenia or cushingoid changes. The pharmacological basis for the highly desirable properties is obscure. CyA is a cyclic polypeptide of 11 aminoacids which can be extracted from several species of fungus.l Several of the aminoacids are N-methylated and this unusual structure probably explains why it resists digestion in the gut. During short courses of CyA mice are unable to make primary or secondary antibody responses to a wide range of antigens but respond to unrelated antigens as little as 24 h after the last dose.2 The antibody responses of nude mice to thymus-independent antigens are not inhibited by CyA, so T lymphocytes are the likely targets for the drug’s immunosuppressive actions.3 Results of in-vitro lymphocyte stimulation are consistent with this view: CyA inhibits the proliferation of mouse lymphocytes stimulated by the T-cell mitogen concanavalin A but does not prevent the response of B lymphocytes to lipopolysaccharide.4 Once T lymphocytes have started to divide they become resistant to inhibition by CyA-a property in keeping with the observation that, for maximum immunosuppression, CyA must be given concurrently with antigen. The initial animal work showed that, as well as suppressing antibody, CyA prolonged the survival of foreign skin grafts and the survival of animals with graft-versus-host disease; it also prevented the development of experimental allergic encephalomyelitis and reduced joint swelling in Freund’s adjuvant arthritis. Acute inflammatory responses were not impaired.2 Transplantation groups have been quick to explore the drug’s possibilities and impresnews
33. Awan NA, Miller RR, Miller MP, Specht K, Vera Z, Mason DT. Clinical pharmacology and therapeutic application of prazosin in acute and chronic refractory congestive heart failure. Am J Med 1978; 65: 146-54. 1. Dreyfus M, Hyerri E, Hotman H, Kobel H. Cyclosporin A and C, new metabolites from Trichoderma polysporum. Eur J Appl Microbiol 1976; 3: 125-33. 2. Borel JF, Feurer C, Gubler HV, Stahelin H. Biological effects of cyclosporin A: anew anti-lymphocytic agent. Ag Actions 1976; 6: 468-75. 3. Borel JF, Feurer C, Magnee C, Stahelin H. Effects of the new antilymphocytic peptide cyclosporin A in animals. Immunology 1977; 32: 1017-25. 4. Borel JF, Wiesinger D. Effect of cyclosporin A on murine lymphoid cells. In: Lucas D, ed. Regulatory mechanisms in lymphocyte activation. London: Academic Press. 1977.
sive results have been obtained in kidney-grafted rabbits. 6 of 10 animals which for 18 or 28 days after their allografts received CyA as their only immunosuppression were alive with functioning kidneys over a year later. These animals accepted skin or further kidney grafts from their original donors but they rejected grafts from third-party donors. In pigs the survival of heart allografts was prolonged by CyA, and in some animals the graft continued to be tolerated after the drug was stopped.6 Heterotopic heart grafts in monkeys fared rather less well, in that histological evidence of rejection was present after 50-day course of CyA given daily at first and then on alternate days,7 though the grafts themselves continued to beat. There were no toxic effects clearly attributable to the CyA in these series though in another study focal liver cell necrosis, cholestasis, and jaundice developed in 5 of 34 dogs given unmatched kidney grafts.8CyA has been tried for the treatment of graft versus-host (GVH) disease.95 patients, all with GVH disease after sibling marrow grafts for the treatment of relapsed leukaemia, were given CyA, and in each case the typical rash resolved. The other features, including diarrhoea and hepatitis, persisted and 4 of the patients died. In the survivor the rash remitted several times after intermittent courses of CyA. Severe GVH disease is usually fatal so these results are not as bleak as they seem. Since the immunosuppressive effects of CyA are greatest when it is given concurrently with antigen priming, the drug might be expected to be more effective in preventing than in treating GVH disease. Work in animals seems to confirm this view. Rats rendered aplastic with busulphan were given incompatible marrow in a strain combination which, without immunosuppression, does not result in engraftment.10 CyA-treated recipients of incompatible marrow became chimseric and their survival over a 28-day follow-up equalled that of busulphan-treated controls which were grafted with syngeneic marrow. None of the CyA-treated rats acquired GVH disease, but it did develop in each of the surviving cyclophosphamide-treated rats. Further information on the action of CyA and its use in human transplantation emerged at an international symposium on immunosuppression held
5. Green CJ, Allison AC, Precious S. Induction of specific tolerance in rabbits by kidney allografting and short periods of cyclosporin A treatment. Lancet 1979; ii: 123-25. 6. Calne RY, White DJG, Rolles K, Smith DP, Herbertson BM. Prolonged survival of pig orthotopic heart grafts treated with cyclosporin A. Lancet
1978; i: 1183-85. Jamieson SW, et al. Cardiac allograft survival in primates treated with cyclosporin A. Lancet 1979; i: 545. 8. Calne RY. Immunosuppression for organ grafting—observations on cyclosporin A. Immunol Rev 1979; 46: 113-24. 9. Powles RL, et al. Cyclosporin A for the treatment of graft versus host disease 7.
in man. Lancet 1978; ii: 1327. 10. Tutschka PJ, Beschomer WE, Allison AC, Burns WH, Santos GW. Use of cyclosporin A in allogeneic bone marrow transplantation in the rat. Nature 1979; 280: 148-51.
780
in Cardiff last month. Once again CyA was shown to prolong skin and kidney’ allograft survival in rodents, although its ability to suppress GVH disease in rats with bone-marrow transplants was not so striking. Three groups described the use of CyA in experimental pancreatic islet transplantation, the results being rather poor. Segmental or wholeorgan transplants fared much better; in one experiment pancreatectomised dogs were given whole pancreas grafts, and half had normal blood-sugars 80 days after transplantation. Prof. R. Y. CALNE recounted his experience with CyA in 30 patients who had received cadaveric kidney transplants. Although mild rejection episodes occurred, no graft was rejected and 25 of the patients still have functioning transplants. 5 died from sepsis, 4 of them having been on two immunosuppressive drugs other than CyA-a combination which Professor CALNE now feels should be avoided. His present policy is to give CyA (17 mg/kg) alone and to treat rejection with a single short course of steroids. Various toxic effects have been observed, the least serious of which are an increase in body hair, gum hypertrophy, and tremors. All but 2 of the patients had abnormal liver-function tests and 16 became anuric immediately after transplantation or within a few days. This last complication may be avoided by giving fluids and mannitol to the kidney recipient at the time of transplantation. By far the most serious complication has been the development of lymphoma in three patients, 4, 6, and 9 months after the start of CyA treatment. The lymphoma was discovered post mortem in the lung and ileum of two patients who had died of sepsis, and in the stomach of a third patient who has had the tumour resected. This 10% incidence of lymphoma is very
worrying.
ENZYME IMMUNOASSAYS REVISITED
SOME three years ago a short editorial promoted the virtues of enzyme immunoassays. This prompted several leading radioimmunoassay enthusiasts to put pen to paper in defence of their treasured technique. With the
benefit of time we can now see some trends emerging more clearly. - The enzyme immunoassays are of two types-the homogeneous type without washing steps (exemplified by the EMIT system, Syva Corporation), and the heterogeneous types generally known as enzymelinked immunosorbent assays (ELISA). The homogeneous assays are very rapid but are not very sensitive and are really only suitable for the measurement of small molecules. They have been remarkably successful and are now accepted as routine methods for the assay of anticoagulants, drugs of abuse, cardioactive drugs, and thyroxine. The availability of easy-to-use reagent kits and convenient processing apparatus has done much to ensure that this method is acceptable in both large and small laboratories.
The
heterogeneous enzyme immunoassays (ELISA) quite different in that they are rather slow, requiring several incubation/washing steps, but they are suitable for assaying large molecules and they are very sensitive. To date, ELISA has not become a routine method in most clinical laboratories, possibly because few reagent kits are available commercially and automated equipment is still in its infancy. However, ELISA has been the subject of several hundred scientific publications and there has been especial emphasis in the sphere of infectious diseases. These applications have dealt both with the detection of antigen (or the infectious agent) and with the antibody response to the infectious agent. As usual, hepatitis has been a focus of attention, and at first ELISA was developed for the detection of HBsAg.l,2’ Various commercial ELISA kits were produced for this purpose--e.g., ’Hepanostika’ (Organon Teknika), ’Cordia H’ (Cordis Laboratories), and ’Auszyme’ (Abbott Laboratories)-and they are regarded as third-generation tests, being slightly more sensitive than reverse passive agglutination tests but not quite as sensitive as are
the radioimmunoassays.3 Since then assays have also been developed for the "e" antigen and for anti-HBs. Hepatitis A antigen has been detected, but in this disease the ELISA for IgM antibody to the virus seems a much more important diagnostic tool. 5,6 ELISA in viral serology is progressing along two lines-general screening tests which measure antibody in all immunoglobulin classes, and tests for recent infection which are based on IgM antibody assays. Already numerous papers have been published on the ELISA screening methods for rubella, cytomegalovirus, measles, herpes, and influenza,’-9 and commercial ELISA kits exist for rubella and cytomegalovirus. The IgM detection systems have been plagued with false positives and false negatives, but new solid-phase and column serum fractionation methods should mean that IgM ELISA can be used as a routine method for early diagnosis. Very promising results have been obtained with ELISA tests for antibody to rotavirus and for detecting virus in fæces.10,11 The fact that ELISA is cheap and easily performed
1. Wolters G, Kuijpers L, Kacaki J, Schuurs A. Solid phase enzyme immunoassay for detection of hepatitis B surface antigen. J Clin Path 1976; 29: 873-79. 2. Halbert ST, Anken M. Detection of hepatitis B surface antigen (HBsAg) with use of alkaline phosphatase labelled antibody to HBsAg. J Infect Dis 1977; 136: suppl 318-23. 3. Vandervelde EM. An enzyme linked immunosorbent assay test for hepatitis B surface antigen. J Clin Path 1977; 30: 714-16. 4. Waart MVD, Snelting A, Cichy J, Wolters G, Scuurs A. Enzyme immunoassay in the diagnosis of hepatitis with emphasis on the detection of ‘e’ antigen (HBeAg). J Med Virol 1978; 3: 43-49. 5. Duermeyer W, van der Veen J. Specific detection of IgM antibodies by ELISA applied in hepatitis A. Lancet 1978; ii, 684-85. 6. Matthiesen LR, Feinstone SM, Skinhoe JP, Purcell RM. Enzyme immunoas-
say for hepatitis A. J Clin Microbiol 1978; 7: 184. 7. Bidwell DE, Bartlett A, Voller A. Enzyme immunoassays for viral diseases. J Infect Dis 1977; 136: suppl 274-78. 8. Morgan-Capner P, Pullen HJM, Pattison JR, Bidwell DE, Bartlett A, Voller A. A comparison of three tests for rubella antibody screening. J Clin Path
1979; 32: 542-45. P, Passila S. Solid phase antibody assay by means of enzyme conjugated to anti-immunoglobulin. J Clin Path 1977; 29: 1116-20. 10. Yolken RH, Kim HW, Clem T, Wyatt RG, Kalica AR, Chanock RM, Kapikian AZ. Enzyme linked immunosorbent assay (ELiSA) for detection of human reovirus-like agent of gastroenteritis. Lancet 1977; ii: 263-66. 11. Yolken RH, Wyatt RG, Kim HW, Kapikian AZ, Chanock RM. Immunologi9. Leinikki
cal response to infection with human reovirus-like agent: measurement of anti-human reovirus-like immunoglobulin G and M levels by the method
of ELISA. Infect Immunity 1978; 19: 540-46.