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B.C.G. AND IMMUNOLOGICAL ANERGY
989 B.C.G. AND IMMUNOLOGICAL ANERGY SiR,—B.c.G. immunotherapy has been used in cancer as a non-specific immunological adjuvant to increase the cellmediated immune response against tumour cells.l.2 Surprisingly enough, and quite the reverse of what we were expecting, we found that a certain percentage of our cancer patients undergoing B.C.G. immunotherapy became anergic after being injected subcutaneously with 1-5 mg. of B.c.G. every week, in spite of their good general condition. Because various factors, including cancer invasion, general condition, or B.C.G. itself, its frequence and route of injection could induce this anergic state, an experiment was designed to investigate in guineapigs the effect of weekly injections of B.C.G. on the cell-mediated immune response to four different skin-test antigens. For immunisation, each guineapig from our closely bred colony weighing 400-500 mg. received in the right and left hindfoot pads 2000 units of streptokinase and 1000 units of
streptodornase (’ Varidase ’), 0-1 ml. of mumps skin-test antigen (Lilly), 01 ml. of Candida albicans allergenic extract 1/100 (Hollister-Stier) in equal volume of Freund’s complete adjuvant (Difco). Thirty days after sensitisation (day 0), each guineapig was skin-tested with the immunising antigens using the following doses: 250 U.S. units Of P.P.D. (Parke-Davis); 0-ml. of mumps skin-test antigen; 0-1 ml. of C. albicans allergenic extract 1/100; 1000 units of streptokinase and 250 units of streptodornase each in 0ml. of saline. Animals having a 48 hours induration exceeding 3 mm. in diameter were considered positive. Animals having a P.P.D. positive skin test were selected and divided in 6 groups for treatment. Guineapigs of the first three groups were given, for 5 months, a weekly subcutaneous injection of 02 mg., 2 mg., and 20 mg. of B.C.G., respectively, per kg. of body-weight (lot 1590-4, Institute of Microbiology and Hygiene of Montreal). Guineapigs of group 4 were treated weekly with 1 mg. of B.c.G. by scarifications over a 2 x 2 cm. area, while those of group 5 received 1 x 10’ pertussis vaccine (i.M.H.) and those of the control group 6, 0-2 ml. of physiological saline instead of B.C.G. At 2-week intervals after day 0, each animal of each group was skin-tested for delayed hypersensitivity reaction (D.H.R.) to each antigen at the doses already mentioned. 1. 2.
Mathé, G. in Progress in Immunology; p. 959. New York, 1971. Morton, D. L., Eilber, F. R., Joseph, W. L. Ann Surg. 1970, 172, 740.
Results in the accompanying table express the percentage of animals in each treated group having a positive 48 hours D.H.R. to the immunising antigens exceeding 3 mm in diameter when skin-tested at 2-week intervals. Four weeks (4 injections) after the beginning of treatment, B.C.G.treated guineapigs had a significantly (p < 0-001) smaller positive D.H.R. to P.P.D. as well as to the other immunising antigens than group 6 guineapigs. The percentage of positive D.H.R. in the B.C.G. or pertussis-treated groups dropped rapidly to zero for all immunising antigens after 6-8 injections. The degree of anergy induced as a function of time after the beginning of the treatment was related to the dose of B.C.G. used. This state of anergy was persistent for the length of the treatment and appeared more rapidly for the non-related antigen than for P.P.D. Histological findings of the 48-hours reactions sites have shown a weak mononuclear cell infiltrate in group 1 guineapigs, a much weaker one in group 2, and a total absence of reaction in group 3. Furthermore, animals of all groups undergoing B.C.G. immunotherapy had a negative P.H.A. and M.L.C. response and a much weaker conA and pokeweed mitogens stimulation than controls. In summary, guineapigs undergoing a chronic B.C.G. therapy did not have a stimulation of their cell-mediated immune response, but had rather an abolished T-cell function measured by both the in-vivo and in-vitro tests for cell-mediated immunity. The abolished T-cell and B-cell function was not related to the immunising antigen P.P.D. but has also induced a non-specific anergy to nonrelated antigens. The reduced conA and pokeweed blastogenic response in B.c.G.-treated guineapigs suggests that the immunosuppressive effect of the B.C.G. can also influence the B-cell function. The fact that the degree of anergy in B.C.G.-treated animals was dose-related, present in groups of guineapigs treated with subcutaneous injections or scarifications and not observed, at least to the same degree, in control animals of group 6, explains similar findings obtained by Frappier and Fredette3 with old tuberculin, and show that B.C.G. and to a smaller extent pertussis vaccine can induce immuno-
3.
Frappier, A., Fredette, C. C.
r.
Séanc. Soc. Biol.
PERCENTAGE OF ANIMALS IN EACH GROUP HAVING A POSITIVE D.H.R. TO DIFFERENT ANTIGENS OVER A 70-DAY PERIOD
1939, 131, 491.
(10 INJECTIONS)
990
suppression and even complete anergy when chronically injected. This was particularly true with a high dose Of B.C.G. These results suggest that for being beneficial B.C.G. immunotherapy in cancer patients should perhaps be given at weaker doses, at longer intervals than those usually used, and manipulated only in centres equipped to supervise adequately the immunological status of patients. Whether or not the immunological anergy in B.C.G. and pertussis treated guineapigs was the result of an over-dose of antigens, antigenic competition, or a direct action on T and B cells remains to be investigated. Institut de
Microbiologie d’Hygiène de Montréal,
Unité d’Oncologie, Hôpital St.-Luc, Montréal.
R. POISSON.
approval. A mixed respiratory and metabolic alkalosis of the extracellular fluids is usual in these patients, although in some with renal failure or hypothermia the metabolic change is an acidosis, which corrects or exceeds the respiratory component. These pH effects are only part of the complicated metabolic changes in hepatic failure, and their isolated correction by desperate therapy may not benefit the patient. Indeed, a few patients have improved for a few hours after increasing their alkalosis by the rapid infusion of sodium bicarbonate.1 The severity of the alkalosis is an uncertain guide to prognosis, although extreme changes are most common in terminal patients. If correction of the alkalosis is desirable, then the use of phosphate buffers seems more logical, because the metabolic component of the pH shift is related to changes in plasma-phosphate concentration.2 As the alkalosis develops, the plasma-phosphate falls to very low levels. On two occasions I have used sodium phosphate solutions, pH 7-0, at a concentration of 50 mg. phosphorus per 100 ml. ; injecting slowly 50 ml. into a fast-running 5% dextrose drip. In both cases the alkalosis was improved and the plasma-phosphate concentration increased, but there was no clinical benefit. Warneford Hospital,
A. J. KNELL.
SiR,—Your editorial (April 20, p. 720) on the use of hydrochloric acid for metabolic alkalosis emphasises the advantages of such therapy, and notes the paucity of reported complications. We have been using a solution made up from 1N hydrochloric acid in distilled water, adding 100ml. of a solution containing 95 to 105 meq. of H+ to 1000ml. of 5% dextrose to give an approximately 0-09N solution. The solution referred to, using 150 ml. of IN hydrochloric, would contain 150 meq. of H+ and 150 meq. Cl-, not 300 meq. of each ion, but would be approximately isotonic, as stated. It is important to emphasise the absolute necessity to use a well-placed central venous catheter as the intravenous access route. We treated a patient in whom the hydro1. 2.
Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Massachusetts 02115,
U.S.A.
James, I. M., Sampson, D., Nashat, S., Williams, H. S., Garassini, M. Lancet, 1969, ii, 1106. Knell, A. J., Pratt, O. E., Curzon, G., Williams, R. S. Eighth Symposium on Advances in Medicine, Royal College of Physicians of London; p. 156. London, 1974.
MURRAY F. BRENNAN
THYMUS TRANSPLANTATION IN LEUKÆMIA AND MALIGNANT LYMPHOGRANULOMATOSIS
G. LAMOUREUX.
TREATMENT OF METABOLIC ALKALOSIS SIR,-I was concerned by your leading article (April 20, p. 720) in which the use of intravenous hydrochloric acid to correct the alkalosis of liver failure received guarded
Warwickshire.
small-calibre low-flow vessels.
et
C.P. 100, Ville de Laval, P.Q., Canada.
Leamington Spa,
chloric acid was inadvertently given via a flow-directed balloon-tipped pulmonary-artery catheter. The resulting lobar pulmonary infarction and haemorrhage emphasised the vessel-damaging potential of 0-1N HCI infused into
SIR,-Last year we reportedour trials of non-specific immunostimulation in patients with leukaemia and Hodgkin’s disease by transplantation of fragments of thymus removed from patients with myasthenia gravis. In reply to the letter of Dr A. E. Papatestas and his colleagues,22 we should like to add the following remarks. Thymus-mediated immune surveillance has been indicated as an important mechanism in protecting the host from cancers arising from the reticuloendothelial system. Several workers, using the concept of Hodgkin’s disease as a syndrome of hypolymphocytic hypothymism,4 have transplanted fetal thymus to patients with this disease. An increase in immunological reactivity and improvement in clinical condition have not been noted.s-’ When transplanting myasthenic thymuses, we were guided by indications that they may have a higher content of immunologically active substances (lymphocytosis-promoting factor, thymosin) than fetal thymuses, as well as by the availability of unpreserved graft material, selected and histologically verified to eliminate thymuses showing signs of neoplasia. The existence of lymphoid germinal centres and increased numbers of lymphocytes and plasmacytes in the medullary zone of myasthenic thymuses seems to be an expression of an autoimmune reaction (thymitis), which releases a humoral substance causing the characteristic myasthenic neuromuscular block,8 as well as an " opsonising " factor, which endows blood-lymphocytes with the capacity to recognise antigens.99 Studies on the reactivity of lymphocytes from myasthenia-gravis patients 48 hours after thymectomy have shown profound suppression of transformation after exposure in vitro to P.P.D. and thymus extract which is evidence for the important and enduring role of the thymus in the antigen-lymphocyte interaction in adults. We assumed that such thymuses can mobilise the depressed immunological barrier in patients with proliferative hsemocytopathies. This assumption was confirmed by the observation of conversion of the tuberculin reaction after grafting in 5 of 20 patients and raised immunoglobulin levels in 8 of 20 patients, accompanied by clinical and haxmatological improvement. Tuberculosis Institute, Thoracic Surgery Clinic, Zakopane, Poland.
W. M. RZEPECKI M. LUKASIEWICZ.
J. ALEKSANDROWICZ Hæmatological Clinic, Institute of Internal Medicine, Medical Academy of Krakow, Poland. 1.
2. 3. 4. 5. 6. 7. 8. 9. 10.
Z. SZMIGIEL A. SKOTNICKI J. LISIEWICZ.
Rzepecki, W. M., Lukasiewicz, M., Aleksandrowicz, J., Szmigiel, Z., Skotnicki, A., Lisiewicz, J. Lancet, 1973, ii, 508. Papatestas, A. E., Genkins, G., Kark, A. E. ibid. p. 795. Burnet, F. M. Transplant. Rev. 1971, 7, 3. Svet-Moldavsky, G. J. Nature, 1966, 209, 932. Ringenbach, U. G. Bordeaux med. 1970, 4, 1009. Stutzman, L., Mittelman, A., Ohkochi, T., Ambrus, J. Proc. Am. Ass. Cancer Res. 1971, 12, 101. Marcolongo, R., Di Paolo, N. Blood, 1973, 41, 625. Goldstein, G. Lancet, 1966, ii, 1164. Bach, J. C. ibid. 1973, i, 1320. Field, E. J. ibid. 1973, ii, 675.
streptodornase (’ Varidase ’), 0-1 ml. of mumps skin-test antigen (Lilly), 01 ml. of Candida albicans allergenic extract 1/100 (Hollister-Stier) in equal volume of Freund’s complete adjuvant (Difco). Thirty days after sensitisation (day 0), each guineapig was skin-tested with the immunising antigens using the following doses: 250 U.S. units Of P.P.D. (Parke-Davis); 0-ml. of mumps skin-test antigen; 0-1 ml. of C. albicans allergenic extract 1/100; 1000 units of streptokinase and 250 units of streptodornase each in 0ml. of saline. Animals having a 48 hours induration exceeding 3 mm. in diameter were considered positive. Animals having a P.P.D. positive skin test were selected and divided in 6 groups for treatment. Guineapigs of the first three groups were given, for 5 months, a weekly subcutaneous injection of 02 mg., 2 mg., and 20 mg. of B.C.G., respectively, per kg. of body-weight (lot 1590-4, Institute of Microbiology and Hygiene of Montreal). Guineapigs of group 4 were treated weekly with 1 mg. of B.c.G. by scarifications over a 2 x 2 cm. area, while those of group 5 received 1 x 10’ pertussis vaccine (i.M.H.) and those of the control group 6, 0-2 ml. of physiological saline instead of B.C.G. At 2-week intervals after day 0, each animal of each group was skin-tested for delayed hypersensitivity reaction (D.H.R.) to each antigen at the doses already mentioned. 1. 2.
Mathé, G. in Progress in Immunology; p. 959. New York, 1971. Morton, D. L., Eilber, F. R., Joseph, W. L. Ann Surg. 1970, 172, 740.
Results in the accompanying table express the percentage of animals in each treated group having a positive 48 hours D.H.R. to the immunising antigens exceeding 3 mm in diameter when skin-tested at 2-week intervals. Four weeks (4 injections) after the beginning of treatment, B.C.G.treated guineapigs had a significantly (p < 0-001) smaller positive D.H.R. to P.P.D. as well as to the other immunising antigens than group 6 guineapigs. The percentage of positive D.H.R. in the B.C.G. or pertussis-treated groups dropped rapidly to zero for all immunising antigens after 6-8 injections. The degree of anergy induced as a function of time after the beginning of the treatment was related to the dose of B.C.G. used. This state of anergy was persistent for the length of the treatment and appeared more rapidly for the non-related antigen than for P.P.D. Histological findings of the 48-hours reactions sites have shown a weak mononuclear cell infiltrate in group 1 guineapigs, a much weaker one in group 2, and a total absence of reaction in group 3. Furthermore, animals of all groups undergoing B.C.G. immunotherapy had a negative P.H.A. and M.L.C. response and a much weaker conA and pokeweed mitogens stimulation than controls. In summary, guineapigs undergoing a chronic B.C.G. therapy did not have a stimulation of their cell-mediated immune response, but had rather an abolished T-cell function measured by both the in-vivo and in-vitro tests for cell-mediated immunity. The abolished T-cell and B-cell function was not related to the immunising antigen P.P.D. but has also induced a non-specific anergy to nonrelated antigens. The reduced conA and pokeweed blastogenic response in B.c.G.-treated guineapigs suggests that the immunosuppressive effect of the B.C.G. can also influence the B-cell function. The fact that the degree of anergy in B.C.G.-treated animals was dose-related, present in groups of guineapigs treated with subcutaneous injections or scarifications and not observed, at least to the same degree, in control animals of group 6, explains similar findings obtained by Frappier and Fredette3 with old tuberculin, and show that B.C.G. and to a smaller extent pertussis vaccine can induce immuno-
3.
Frappier, A., Fredette, C. C.
r.
Séanc. Soc. Biol.
PERCENTAGE OF ANIMALS IN EACH GROUP HAVING A POSITIVE D.H.R. TO DIFFERENT ANTIGENS OVER A 70-DAY PERIOD
1939, 131, 491.
(10 INJECTIONS)
990
suppression and even complete anergy when chronically injected. This was particularly true with a high dose Of B.C.G. These results suggest that for being beneficial B.C.G. immunotherapy in cancer patients should perhaps be given at weaker doses, at longer intervals than those usually used, and manipulated only in centres equipped to supervise adequately the immunological status of patients. Whether or not the immunological anergy in B.C.G. and pertussis treated guineapigs was the result of an over-dose of antigens, antigenic competition, or a direct action on T and B cells remains to be investigated. Institut de
Microbiologie d’Hygiène de Montréal,
Unité d’Oncologie, Hôpital St.-Luc, Montréal.
R. POISSON.
approval. A mixed respiratory and metabolic alkalosis of the extracellular fluids is usual in these patients, although in some with renal failure or hypothermia the metabolic change is an acidosis, which corrects or exceeds the respiratory component. These pH effects are only part of the complicated metabolic changes in hepatic failure, and their isolated correction by desperate therapy may not benefit the patient. Indeed, a few patients have improved for a few hours after increasing their alkalosis by the rapid infusion of sodium bicarbonate.1 The severity of the alkalosis is an uncertain guide to prognosis, although extreme changes are most common in terminal patients. If correction of the alkalosis is desirable, then the use of phosphate buffers seems more logical, because the metabolic component of the pH shift is related to changes in plasma-phosphate concentration.2 As the alkalosis develops, the plasma-phosphate falls to very low levels. On two occasions I have used sodium phosphate solutions, pH 7-0, at a concentration of 50 mg. phosphorus per 100 ml. ; injecting slowly 50 ml. into a fast-running 5% dextrose drip. In both cases the alkalosis was improved and the plasma-phosphate concentration increased, but there was no clinical benefit. Warneford Hospital,
A. J. KNELL.
SiR,—Your editorial (April 20, p. 720) on the use of hydrochloric acid for metabolic alkalosis emphasises the advantages of such therapy, and notes the paucity of reported complications. We have been using a solution made up from 1N hydrochloric acid in distilled water, adding 100ml. of a solution containing 95 to 105 meq. of H+ to 1000ml. of 5% dextrose to give an approximately 0-09N solution. The solution referred to, using 150 ml. of IN hydrochloric, would contain 150 meq. of H+ and 150 meq. Cl-, not 300 meq. of each ion, but would be approximately isotonic, as stated. It is important to emphasise the absolute necessity to use a well-placed central venous catheter as the intravenous access route. We treated a patient in whom the hydro1. 2.
Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Massachusetts 02115,
U.S.A.
James, I. M., Sampson, D., Nashat, S., Williams, H. S., Garassini, M. Lancet, 1969, ii, 1106. Knell, A. J., Pratt, O. E., Curzon, G., Williams, R. S. Eighth Symposium on Advances in Medicine, Royal College of Physicians of London; p. 156. London, 1974.
MURRAY F. BRENNAN
THYMUS TRANSPLANTATION IN LEUKÆMIA AND MALIGNANT LYMPHOGRANULOMATOSIS
G. LAMOUREUX.
TREATMENT OF METABOLIC ALKALOSIS SIR,-I was concerned by your leading article (April 20, p. 720) in which the use of intravenous hydrochloric acid to correct the alkalosis of liver failure received guarded
Warwickshire.
small-calibre low-flow vessels.
et
C.P. 100, Ville de Laval, P.Q., Canada.
Leamington Spa,
chloric acid was inadvertently given via a flow-directed balloon-tipped pulmonary-artery catheter. The resulting lobar pulmonary infarction and haemorrhage emphasised the vessel-damaging potential of 0-1N HCI infused into
SIR,-Last year we reportedour trials of non-specific immunostimulation in patients with leukaemia and Hodgkin’s disease by transplantation of fragments of thymus removed from patients with myasthenia gravis. In reply to the letter of Dr A. E. Papatestas and his colleagues,22 we should like to add the following remarks. Thymus-mediated immune surveillance has been indicated as an important mechanism in protecting the host from cancers arising from the reticuloendothelial system. Several workers, using the concept of Hodgkin’s disease as a syndrome of hypolymphocytic hypothymism,4 have transplanted fetal thymus to patients with this disease. An increase in immunological reactivity and improvement in clinical condition have not been noted.s-’ When transplanting myasthenic thymuses, we were guided by indications that they may have a higher content of immunologically active substances (lymphocytosis-promoting factor, thymosin) than fetal thymuses, as well as by the availability of unpreserved graft material, selected and histologically verified to eliminate thymuses showing signs of neoplasia. The existence of lymphoid germinal centres and increased numbers of lymphocytes and plasmacytes in the medullary zone of myasthenic thymuses seems to be an expression of an autoimmune reaction (thymitis), which releases a humoral substance causing the characteristic myasthenic neuromuscular block,8 as well as an " opsonising " factor, which endows blood-lymphocytes with the capacity to recognise antigens.99 Studies on the reactivity of lymphocytes from myasthenia-gravis patients 48 hours after thymectomy have shown profound suppression of transformation after exposure in vitro to P.P.D. and thymus extract which is evidence for the important and enduring role of the thymus in the antigen-lymphocyte interaction in adults. We assumed that such thymuses can mobilise the depressed immunological barrier in patients with proliferative hsemocytopathies. This assumption was confirmed by the observation of conversion of the tuberculin reaction after grafting in 5 of 20 patients and raised immunoglobulin levels in 8 of 20 patients, accompanied by clinical and haxmatological improvement. Tuberculosis Institute, Thoracic Surgery Clinic, Zakopane, Poland.
W. M. RZEPECKI M. LUKASIEWICZ.
J. ALEKSANDROWICZ Hæmatological Clinic, Institute of Internal Medicine, Medical Academy of Krakow, Poland. 1.
2. 3. 4. 5. 6. 7. 8. 9. 10.
Z. SZMIGIEL A. SKOTNICKI J. LISIEWICZ.
Rzepecki, W. M., Lukasiewicz, M., Aleksandrowicz, J., Szmigiel, Z., Skotnicki, A., Lisiewicz, J. Lancet, 1973, ii, 508. Papatestas, A. E., Genkins, G., Kark, A. E. ibid. p. 795. Burnet, F. M. Transplant. Rev. 1971, 7, 3. Svet-Moldavsky, G. J. Nature, 1966, 209, 932. Ringenbach, U. G. Bordeaux med. 1970, 4, 1009. Stutzman, L., Mittelman, A., Ohkochi, T., Ambrus, J. Proc. Am. Ass. Cancer Res. 1971, 12, 101. Marcolongo, R., Di Paolo, N. Blood, 1973, 41, 625. Goldstein, G. Lancet, 1966, ii, 1164. Bach, J. C. ibid. 1973, i, 1320. Field, E. J. ibid. 1973, ii, 675.