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Autoantigens and autoantibodies in the pathogenesis of disease with special reference to blackwater fever
301 TRANSACTIONS TROPICAL
Vol. XxX1X.
OF THE ROYAL SOCIETY OF MEDICINE AND HYGIENE.
No. 4. February,
1946.
COMMUNICATIONS. AUTOANTIGENS GENESIS OF
AND AUTOANTIBODIES DISEASE WITH SPECIAL BLACKWATER FEVER.
IN THE PATHOREFERENCE TO
BY
Medical
JAMES GEAR, Major, Laboratory Service, S.A.M.C.
In the course of another investigation undertaken in the Laboratories of the International Health Division of the Rockefeller Foundation in 1942, it was shown that the inoculation of an emulsion of fresh normal liver obtained from a healthy rhesus monkey, into either a normal monkey or into one previously protected by vaccination against yellow fever, failed to elicit any antibody response. On the other hand it was found that an emulsion prepared from the liver of a rhesus monkey that had died of yellow fever was antigenic when inoculated into a rhesus monkey previously protected by vaccination against yellow fever. This latter monkey developed antibodies which could be demonstrated by the precipitin test not only against an emulsion of yellow fever infected liver, but also against an emulsion of normal liver. It appeared, therefore, that the presence of virus in liver cells acted as a “ schlepper ” for normal liver substance which thus became antigenic when introduced into a monkey of the same species. Assuming that a substance which was homologously antigenic would also be autoantigenic and assuming that antibodies are produced by the reticuloendothelial system, this finding may be diagrammatically presented thus :Liver cell + virus = Autoantigen. Autoantigen + R.E. cell system = Autoantibody. Autoantigen + autoantibody, = Reaction causing degeneration of affected cell. This interesting observation, as well as indicating a possible sequence of events in the evolution of the pathological process of yellow fever, suggested a hypothesis of the pathogenesis of some disease conditions, which may be summarized thus :Tissue cell + virus or + bacterial toxin = Autoantigen or + chemical or + intracellular parasite I
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Autoantigen + R.E. cell system = Autoantibody. Autoantibody + tissue cell affected or sometimes + normal tissue cell = Sensitized cell. Sensitized tissue cell + comple- = Degeneration or lysis of affected ment cell. This hypothesis has already been advanced to explain such obscure conditions as post-vaccinal encephalitis and other post infective encephalitides (SCHWENTBER and RIVERS, 1934) and glomerular nephritis (SMADEL, 1936 ; SCHWENTKER and COMPLOIER, 1939). It is to put forward and discuss a similar hypothesis of the pathogenesis of blackwater fever that is the main purpose of this paper. There is now general agreement that blackwater fever is related to malaria and is especially liable to affect those who have suffered from repeated attacks, which have been inadequately treated with quinine. It was once considered that blackwater fever was associated only with malignant tertian malaria (THOMSON, 1924), but later observations (FoY, 1938) have shown that in Salonika almost as great a proportion of cases are associated with Plasmodium vivax (33 per cent.) as with P. fahparum (47 per cent.) while mixed infections were noted in 14 per cent. of cases. Although many theories have been advanced to explain the acute intravascular haemolysis which is the fundamental event in an attack of blackwater fever, the cause, to summarize current literature and textbooks, remains a mystery. It has often been suggested, however, that a haemolysin or an anaphylactic sensitizing substance is responsible for the haemolysis, but so far no adequate explanation for their development has been put forward. To quote Stitt’s Tropical Diseases (STRONG, 1942), “ A scientific explanation of the mechanism by which haemolysis is brought about is not yet possible. It is conceivable that a haemolysin may be free in the blood stream or bound to certain cells, but it is not clear what suddenly frees it or precipitates its action.” However, it appears that in the hypothesis enunciated above a reasonable explanation is found. According to this hypothesis, the development of haemolysins may be accounted for by the fact that a red cell infected with a malaria parasite, possibly only after treatment with an antimalarial drug, especially The sequence of events may be formulated quinine, may become antigenic. as follows :Red cell + malarial parasite or Red cell + malarial parasite + antimalarial drug = Autoantigen. or I Red cell + antimalarial drug Autoantigen + R.E. cell system = Autoantibody’ (haemolysin). Red cell + haemolysin = Sensitized red cell. Sensitized cell + complement = Lysis of red cell.
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There is a vast collection of facts relating to blackwater fever, and these have often been analyzed and considered in the past. However, it is now desirable to re-examine and to discuss them as well as several recent observations, in relation to this hypothesis of its pathogenesis. It will become apparent in this discussion that these facts are all in keeping with, or are rationally explained, by this hypothesis. There is little direct evidence of the presence of a haemolysin in the blood of cases of blackwater fever. To quote THOMSON (1924), “ Attempts to demonstrate an autohaemolysin in the serum of blackwater fever during the attack of haemoglobinuria have failed or indefinite results have been obtained.” “ We were quite unable to detect any haemolysin in the serum of blackwater fever at any period of the disease, but as we have pointed out this by no means proves that a serum haemolysin is not present.” Indeed, THOMSON states, “ There are strong grounds for suspecting that the condition is brought about by a specific serum haemolysin elaborated from the prolonged action of Plasmodium falciparum on the red blood corpuscles.” He suggests that, “ it is an altered chemical change which produces the so-called brassy corpuscles in malignant tertian. Corpuscles so altered act as foreign bodies and are thus capable of producing a specific haemolytic amboceptor which can act in the presence of complement on corpuscles so altered and not on normal red cells.” ROSS (1932), although stating that there is no doubt as to the reality of the appearance of the altered corpuscles that THOMSON describes, states that attempts to demonstrate the presence of such altered cells were very seldom successful during his investigation, and therefore considers the hypothesis regarding the role of such cells as open to criticism. FAIRLEY and BROMFIELD (1934) came to the conclusion that the intravascular haemolysis of blackwater fever was caused by a haemolytic agent arising in chronic subtertian malaria as a result of a metabolic breakdown precipitated by quinine or plasmoquine, or possibly by other factors such as chill or exhaustion. They considered their findings were against either the action of an immunological haemolysin or a direct drug effect on the corpuscles. Although the direct evidence is so unconvincing, there is considerable indirect evidence that an antibody acting on red cells is present in cases of blackwater fever. This will now be considered. It has repeatedly been observed that red cells from cases of blackwater fever show a tendency to autoagglutination. This in itself is suggestive of the presence of antibodies, but by itself is not conclusive, for autoagglutination of the red cells has been observed in many conditions that are not associated with acute intravascular haemolysis. For example, autoagglutination of the red cells is often observed in the blood of cases of human trypanosomiasis. However, even in this condition erythrophagocytosis has been noted, a phenomenon which suggests the presence of red cell opsonins.
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Erythrophagocytosis has also been noted in cases of blackwater fever. (1924), in his observations on eight fatal cases of blackwater fever, noted that there was evidence of endothelial activity and the endothelial cells showed active phagocytosis of the red cells. YORKE, MURGATROYD and OWEN (1929) note that phagocytes containing red cells are frequently seen both in films of the peripheral blood and of the splenic pulp. Ross (1932) also reported that smears from the spleen of cases coming to autopsy showed the presence of phagocytosed red cells, and in several instances the phenomenon was also present in films of the peripheral blood. The engulfed red cells always appeared to retain their haemoglobin. For that reason he considered that the appearance indicated the presence of an increased erythrophagocytosis in the -disease. These authors all conclude that its importance in the haemolytic process is probably small, but that it may account for part of the reduction that occurs in the red cell count. However, its occurrence suggests the presence of red cell antibodies. Autoagglutination of the red cells was clearly seen in the two most recent cases of blackwater fever admitted to the Johannesburg Hospital. Both these cases in their early stages also showed a tendency for the red cells to be microcytic and hyperchromic, an observation in turn indicating that there was a tendency to spherocytosis. Spherocytosis, in cases of blackwater fever, was noted as long ago as I908 by CHRISTOPHERS and BENTLEY (1908). THOMSON (1924) confirmed this finding, noting that in stained preparations these small spherical erythrocytes assumed much darker red. than the other red cells when stained with Leishman.. More recently, in a case of blackwater fever admitted to the Children’s Hospital,. Johannesburg, FOY and KONDI (1943) studied this question in’detail and found that cells from a case of blackwater fever were more spherocytic than normal but less so than those of haemolytic jaundice. Spherocytosis of the red cells, of course, is one of the characteristic findings in familial haemolytic jaundice. DAMESHEK and SCHWARTZ (1938) suggest that this condition is caused by an autohaemolysin and has shown that one of the earliest demonstrable effects of a haemolysin’acting on a red cell is for the affected cell to become spherocytic. Accordingly, the occurrence of spherocytosis in blackwater fever favours the hypothesis that this condition is caused by a haemolysin. Two other important observations that suggest the presence of haemolysin similar in action to artificjally produced antibodies have been made by FOY and KONDI. FOY, KONDI and MOUMJIDIS (1941) showed that red cells transfused into a haemolysing case of blackwater fever from three different donors underwent haemolysis just as readily as did the blackwater fever patient’s own cells. This was interpreted as indicating that there is some circulating haemolysin that destroys all red cells that come into contact with it, irrespective of their origin. This finding is what would be anticipated if an immunological haemolysin is THOMSON
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concerned, thus donors’ cells on transference to the recipient would become sensitized by the haemolysin present in the recipient’s circulation. The ‘cells are thus rendered susceptible to the action of complement and so to haemolysis. Later, FOY et al. (1945) showed that the red cells from a case of blackwater fever, when transfused into a normal individual, were haemolyzed as rapidly as those remaining in the patient. This observation is also what would be anticipated if an immunological ,haemolysin were concerned, for the patient’s red cells are already sensitized by the autohaemolysin, so that, even if transferred to a healthy person, they remain susceptible to the action of complement and are haemolyzed. That an antigen-antibody reaction is probably concerned in the pathogenesis of blackwater fever is also suggested by a consideration of other conditions characterized by intravascular haemolysis. Two of the best known of these are paroxysmal haemoglobinuria and incompatible blood transfusion. Both are known to be due to red cell antibodies which can be demonstrated in vitro. A condition analogous to blackwater fever can be brought about experimentally by the injection of an artificially prepared haemolytic serum into experimental animals. CHRISTOPHERS and BENTLEY (1908) and DUDGEON (1920), who investigated the effects of haemolytic sera on animals, showed that the pathological appearances were the same’ as produced in blackwater fever. THOMSON (1924) remarks “ there is so marked a similarity between the action of a true specific serum haemolysin and an attack of haemoglobinuric fever that there can be little doubt that the mechanism is the same, and if we can produce such a condition by continual attacks of malaria the problem is solved.” It is known that blackwater fever rarely affects an individual who has been resident in a malarious area less than 6 months, but thereafter the chances of developing an attack increase with length of residence. The greatest number of cases occur in individuals who have lived for from 1 to 3 years in an area where malaria is hyperendemic, and who, during this time, have usually suffered from repeated attacks of malaria often inadequately treated with quinine. This time lag between the first infection with malaria and the development of blackwater fever in itself suggests a process of sensitization. As the time taken for sensitization exceeds 6 months, it is reasonable to conclude that the antigenic value of the hypothetical autoantigen is relatively low, so that repeated attacks of malaria are necessary before a state of sensitivity develops. It may well be that, in addition to malaria, treatment by antimalarial drugs is a necessary factor for the development of this state. The evidence discussed above may now be summarized :1. Autoagglutination and spherocytosis of the red cells in cases of blackwater fever have been observed.
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2. Erythrophagocytosis is commonly seen in smears made from the spleen and occasionally in films of peripheral blood. 3. Red cells from healthy donors haemolyze as rapidly in a case of blackwater fever as the patient’s own cells. Red cells, when transfused into a normal person from a case of blackwater fever, haemolyze as rapidly as the red cells remaining in the patient. 4. Blackwater fever is closely simulated by paroxysmal haemoglobinuria and incompatible blood transfusion, conditions both caused by known and demonstrable anti-red cell antibodies ; and by the effects of artificially produced haemolytic sera in experimental animals. 5. There is usually a lag period between the first attack of malaria and the onset of blackwater fever. This time interval suggests .a process of sensitization. All these facts are readily explained on the assumption that an autohaemolysin is concerned in the pathogenesis of blackwater fever. Other hypotheses put forward cannot satisfactorily account for them. Indeed, it may be concluded that blackwater fever is almost certainly caused by an autohaemolysin. One of the most puzzling features of blackwater fever has always been its sudden and dramatic onset. Sir PATRICK MANSON put this puzzle in his question to Dr. THOMSON in 1922. “ Malaria is undoubtedly one of the factors but what pulls the trigger ? ” In Stitt’s Tropical Diseases (STRONG, 1942) it is stated that it is conceivable that a haemolysin may be free in the blood stream or Bound in certain cells, but it is not clear what suddenly frees it or precipitates its action. It is considered that the hypothesis put forward in this paper also accounts for the sudden onset of blackwater fever. In this connection it is relevant to discuss the site of formation of the haemolysin. There has been a considerable amount of experimental work on the site of formation of antibodies, and an extensive literature dealing with this subject has accumulated, but it is not intended to review it here. Suffice it for the present to note that much evidence has been presented favouring the view that antibodies are produced by the reticula-endothelial system of cells. The spleen is the largest depot of this system of cells in the body. Several observations have been made which show that the spleen is intimately connected with serum immune bodies. It is known that the removal of the spleen seriously undermines the resistance of rats to an infection with Bartonella murk. An infection which previously was easily tolerated, after removal of the spleen becomes rapidly fatal. In the field of human medicine, removal of the spleen is known to benefit the clinical condition of cases.of familial haemolytic anaemia, a condition which is possibly caused by autohaemolysins (DAMESHEK and SCHWARTZ, 1938). Splenectomy also benefits many cases of chronic thrombocytopenic purpura, and some cases of chronic leucopenia. Incidentally, it is considered very likely
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that these two conditions are also caused by autoantibodies, and will be considered from this point of view of this hypothesis in more detail at a later stage. It was noted, incidentally, in a paper on cases of Rhodesian sleeping sickness (GEAR and DE MEILLON, 1939), that smears made from the spleen of experimentally infected animals showed few intact trypanosomes, although degenerating forms and remnants of degenerated forms, were numerous. In contrast, smears made from all the other organs, including the lymph glands, It seemed from this observation that showed numerous intact trypanosomes. the spleen is the principal site of the formation of trypanosomicidal antibodies. It is reasonable to expect that it is also the principal site of formation of other circulating antibodies. It is likely, then, that haemolysins responsible for the acute intravascular haemolysis of blackwater fever would also be formed principally in the spleen. It would be expected, then, that when a person had become sensitized, any factors causing a sudden contraction of the spleen so expressing its contained haemolysins into the general circulation, would result in an attack of blackwater fever. On referring to the known precipitating causes of blackwater fever it is found that they are usually given in this order, quinine, chill, exertion and violent emotion. These, the chief exciting causes of this fever, have one effect in common, they cause contraction of the spleen. Thus it becomes clear why the onset of an attack of blackwater fever is so sudden and dramatic. It is only when a sudden contraction of the spleen occurs that sufficient haemolysin is liberated to cause intravascular haemolysis. At other times it seems probable that the haemolysin is mopped up as it is formed and the number of red cells so sensitized is not too large to be removed by the reticula-endothelial cells before intravascular haemolysis takes place. In that case one would expect that cases of subacute or chronic haemolytic anaemia, not associated with intravascular haemolysis, would occur. That such cases do occur has been made clear by FAIRLEY, BROMFIELD, FOY and KONDI (1938), who described cases of haemolytic anaemia associated with chronic malaria in Macedonia. It is very interesting to note that these cases occurred in Macedonia, a region where more cases of blackwater fever are seen than anywhere else in the world. It seems possible, then, that these cases are more or less benign manifestations of the process which in more violent form results in blackwater fever. Several writers on blackwater fever have considered that in some way or other the spleen is involved in the pathogenesis of blackwater fever. BLACKLOCK and MACDONALD (1928) believed that the sudden massive haemolysis is occasioned by the over-production of sarcolactic acid, a normal constituent of the blood and tissues. This is caused by anoxaemia particularly in the spleen, where the adherence of malaria infected red cells to each other and to the vascular endothelium interferes with the circulation. They point out that the agencies chill, exertion, quinine, which precipitate an attack, all cause
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FEVER.
contraction of the spleen, and this they consider accentuates the existing anoxaemia, so that lactic acid formation and accumulation increase to haemolytic concentrations and leakage of haemoglobin into the portal circulation results. This leakage becomes increasingly apparent as circulation through the spleen becomes re-established, leading to haemoglobinaemia and haemoglobinuria. ROSS (1932) studied cases from the point of view of this hypothesis and concluded that the theory on which it was founded was fallacious. FAIRLEY (1940) suggests that a perversion of the activity of the reticuloendothelial system occurs as a result of chronic malaria, which induces a considerable hypertrophy of this system in general and of the spleen in particular. He suggests that an escape into the blood stream from a pathological reticula-endothelial system of the intracellular ly& enzyme, normally responsible for the destruction of effete red cells, occurs and is responsible for the intravascular haemolysis. VINT (1941) puts forward a hypothesis of the causation of blackwater fever based on experiments of FAHRAEUS (1939), who showed that under certain conditions red cells undergo a change he called stabilization. VINT suggests that a similar change occurring in the splenic vein is responsible for the liability of red cells in cases of blackwater fever to haemolyze. Fov,and KONDI (1943), who found that the red cell fragility to lysolecithin was increased in cases of blackwater fever, state, “ The enlargement of the spleen in so many of these haemolytic conditions is regarded by many as of considerable significance. The production of lysolecithin as a result of the separation of the cells and plasma in this organ has led to the suggestion that this powerfully haemolytic substance may play a part in the haemolyses of certain of these conditions.” Although the hypotheses put forward by these authors differ, often very considerably, in their basis, they all bring forward evidence that in some way or other the spleen is intimately concerned in the pathogenesis of blackwater fever. It is considered that the role assigned to the spleen in the hypothesis here put forward accords with the observed facts more satisfactorily than the other hypotheses of the causation of the condition. Most of these hypotheses do not explain what might be called the specific nature of blackwater fever, a specificity that is clearly explained by the assumption that a biological haemolysin is concerned. It is now opportune to suggest the probable sequence of events leading up to an attack of blackwater fever. Red cells infected with malarial parasites, possibly only after treatment with an antimalarial drug or red cells altered by or combined with an antimalarial drug, become autoantigenic, and in response to this autoantigen an antibody or haemolysin is produced by the reticula-endothelial system, particularly by the spleen. The titre of this haemolysin is boosted by each repeat attack of malaria. When the circulation of the blood through the spleen is
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free, this haemolysin is mopped up by the red cells and these sensitized red cells are removed by the reticula-endothelial cells as they are sensitized and before a demonstrable intravascular haemolysis occurs. However, when the circulation through the spleen is impeded, and the spleen becomes congested, as it does in an attack of malaria, which also provides a secondary stimulus to boost the titre, this haemolysin accumulates, Factors which cause a sudden of quinine, chill, and contraction of the spleen, such as the administration exertion, now would sudden!y express into the general circulation sufficient haemolysin to sensitize a large number of red cells and their haemolysis intravascularly would result in haemoglobinaemia and haemoglobinuria, followed by the other signs and symptoms of blackwater fever. The acceptance of this hypothesis of the pathogenesis of blackwater fever may be of practical value. If the haemolysin is produced by the spleen and expressed from it by conditions which cause that organ to contract, it is obvious that it is desirable to prevent such contraction, especially sudden contraction. Measures to ensure this would be :Placing the patient at absolute rest in bed lying flat. Placing his mind at rest. Avoidance of drugs which would cause smooth muscle contraction. It would also be desirable, if such were available, to administer a drug which would prevent the union of antibody and antigen. The commonly accepted method of treatment of cases of blackwater fever aims at achieving these objects, thus to quote Stitt’s Tropical Diseases (STRONG, 1942), “ Absolute rest in bed, avoidance of chilling, and good nursing are prime considerations in treatment.” Recently BURKITT (1943) wrote that, when treating cases of blackwater fever in East Africa, he was struck by the great restlessness of mind and body characteristic of that disease, and to counteract this gave 8 to 10 grains of sodium luminal intramuscularly. In eight cases so treated in the evening, all traces of blackwater had disappeared by the following morning. He also reported that Dr. J. K. GREGORY, of Nairobi, has had a series of thirty cases all treated by intravenous injection of 15 grains of phenobarbitone and all, “ cured at once.” He remarks that he has used the unscientific expression “ cured at once ” because it gives the picture exactly, A reasonable explanation of the success of this sedative treatment has been given in this discussion. The advisability of ihe administration of quinine to cases of blackwater fever is still a vexed question. Arguing on the basis of the hypothesis here put forward, it would seem that, once the spleen had contracted and expressed its haemolysin, there would be little danger in treatment with quinine unless it was withheld for a sufficient time to once again allow a considerable amount of haemolysin to dam up in the spleen. However, as then it would be a real danger it would seem that treatment with quinine should not be given for the concomitant malaria unless mepacrine is not available. Although the
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evidence is not altogether convincing, it is generally accepted that the regular taking of prophylactic quinine decreases the liability to blackwater fever. It is known that the adequate treatment of malaria by quinine likewise diminishes the chances of blackwater fever. Yet the irregular taking of prophylactic quinine, and the inadequate treatment of malaria by quinine, are recognized as most important factors in rendering a patient prone to blackwater fever. A reason for even his paradoxical state of affairs emerges from this discussion. It has already been noted that the antigenic value of the autoantigen is probably low, and that it usually requires a prolonged exposure to its action before a state of sensitivity develops. Such a prolonged action would be ensured by irregular prophylactic quinine, and by inadequate treatment, both of which would allow a latent infection to persist and to progress, but which would also prevent such an infection from frankly asserting itself, thereby lessening the body’s natural development of immunity. Also because of the inadequate treatment relapses are frequent and provide the secondary stimuli which result in boosting the titre of an autohaemolysin. Accordingly, from the point of view of prophylaxis, it is important that, if a person is taking prophylactic quinine, he should do so regularly, but more important still should he contract malaria it is essential that his treatment should be thorough. It seems that thorough treatment, by eliminating the potential autoantigen, guards against the development of the autohaemolysin. In cases of chronic malaria that have already suffered from blackwater fever, it is essential that they should be thoroughly treated, because such thorough treatment eliminates the autoantigen and it appears that once this is eliminated the titre of the autohaemolysin rapidly diminishes. This was clearly demonstrated in a case of recurrent blackwater fever induced by quinine described by FAIRLEY and MURGATROYD (1940). The capacity of quinine to produce blackwater fever appeared in this case to be directly or indirectly related to persisting malarial infection, which was associated with a demonstrably enlarged spleen. Following effective treatment by atebrin, and disappearance of the splenomegaly, the administration of quinine was no longer followed by haemoglobinuria. Regarding the prevention of the union of antigen and antibody, it is relevant to note that such properties have been ascribed to acetyl salicylic acid. This therapeutic effect no doubt accounts for its great value in relieving the symptoms of acute rheumatic fever, a condition which it seems very likely will also be found to be caused by an autoantigen-antibody reaction taking place in tissues rendered autoantigenic by union with haemolytic streptococcus toxin. As far as can be ascertained, aspirin has not been credited with any therapeutic effect in cases of blackwater fever. It would be interesting to know the results of a trial of this drug over a large series of cases, though no striking effect could be anticipated, because, once haemoglobinuria has occurred, the
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However, its administration may process has in most cases expended itself. be of value in the remittent type of case. The other lines of treatment such as blood transfusion for combating the effects of the intravascular haemolysis, although they are of the utmost importance and often life-saving, will not be considered in detail as they do not immediately concern our theme. It is, however, pertinent to discuss briefly other conditions characterized by intravascular haemolysis, noting whether any feature of them has any bearing on this hypothesis. Of these conditions attention has already been drawn to the effects of an incompatible blood transfusion and to paroxysmal haemoglobinuria, both of which are caused by demonstrable antibodies. Of other conditions, one of the most common is acute haemolytic anaemia following the administration of certain drugs. Some of these, such as phenylhydrazine, act directly on the corpuscles. In others the reaction appears to result from an undue sensitivity. In recent years, the drugs most commonly concerned in the latter group are those or the “ sulpha ” group. We have seen two cases of acute haemolytic anaemia followThese will not now be described ing the administration of sulphapyridine. in detail, suffice it to note that in each case, towards the end of a course of treatment, acute haemolytic anaemia suddenly developed, characterized by intravascular haemolysis as shown by haemoglobinaemia and haemoglobinuria. It is recognized that these accidents are rare and usually they are attributed to an idiosyncrasy of the patient. This idiosyncrasy is usually considered to be of an allergic nature. In the hypothesis here discussed a rational explanation for their development is found which can be formulated as follows :Red cell + sulpha compound or derivative Red cell + antibody (haemolysin) Sensitized red cell + complement
= Autoantigen. = Sensitized red cell. = Haemolysis.
In the cases noted above, one was fatal, dying of uraemia following anuria, the other rapidly recovered after withdrawing the drug. As the condition developed soon after exposure to the drug, it would appear that red cells linked to the “sulpha” drugs have a high antigenic value,which accounts for the relatively rapid development of haemolysin, but as this antibody, unlike the haemolysin of blackwater fever, apparently acted only on red cells that had come into contact with the drug, recovery was rapid and complete. We have also seen two cases of malaria in their primary attack, which towards the end of a course of treatment with atebrin, suddenly developed an acute haemolytic anaemia, characterized by haemoglobinaemia and haemoglobinuria. Although these cases being associated with malaria were considered to be cases of true blackwater fever, it is our opinion that they were more directly comparable with the type of haemolytic anaemia in the two cases noted above, resulting from an acquired sensitivity to the drug. It seems, too, that the cases of quinine haemoglobinuria of which many have been recorded also belong
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to this group. The acute haemolytic anaemia of favism likewise appears to belong to this group. Possibly similar in pathogenesis were two cases of aplastic anaemia, recently seen in the Johannesburg Hospital. One followed a full course of treatment with sulphapyridine for pneumonia, but it is of interest to note that the sikns of aplastic anaemia did not become evident until 3 months later. The other similarly followed a course of treatment with sulphapyridine and sulphanilamide for tonsillitis. It is also of interest to note that we have under observation one case of chronic agranulocytosis that first manifested symptoms 10 months after the second full course of treatment with sulphapyridine for gonorrhoea. The blood film of this case in which the leucocyte count was usually in the neighbourhood of 2,000, was characterized by a considerable proportion ( f 20 per cent.) of disintegrating neutrophil leucocytes. A similar blood picture can be artificially produced in experimental animals by the injection of an antileucocyte antiserum. Another apparently analigous case at present under observation is that of a patient who, following a full course of treatment for pneumonia with sulphapyridine, developed thrombocytopenia. This first manifested itself 1 month after recovery from pneumonia and now has persisted for 2 years. It is relevant to note that thrombocytopenia can be produced experimentally by the injection These interesting cases will be described and discussed of antiplatelet serum. They are mentioned now because it seems that a in detail in another paper. rational explanation of their pathogenesis is to be found in the hypothesis set out in this paper. The more fundamental problems of the constitution of antibodies and of complement have not been discussed. It seems probable that such a study may show that it is possible to correlate some of the, at present, apparently diverse theories of the pathogenesis of blackwater fever. SUMMARY
A hypothesis of the pathogenesis of blackwater fever, suggested by the observation that liver emulsion from a rhesus monkey dead of yellow fever, stimulates the formation of antiliver antibodies when injected into another rhesus monkey, is put forward and discussed. This hypothesis may be formulated thus :Red cell + malarial parasite or Red cell + antimalarial drug \ = Autoantigen or i Red cell -+-malarial parasite i + antimalarial drug I
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Autoantigen + R.E. cell system Red cell + haemolysin Sensitized red cell + complement
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= Autoantibody haemolysin. = Sensitized red cell. = Haemolysis.
It is noted that, in cases of blackwater fever, the red cells show autoagglutination and spherocytosis and that erythrophagocytosis is commonly seen in smears of the spleen, and, occasionally, in films of peripheral blood: Further, it has been observed, red cells from healthy donors haemolyze as rapidly in a case of blackwater fever as the patient’s own cells and, on the other hand, red cells from .a case of blackwater fever, transfused into a healthy recipient, haemolyze as rapidly as the red cells remaining in the patient. There is usually a considerable interval between the first attack of malaria and the onset of blackwater fever, during which the patient suffers from repeated attacks of malaria, often inadequately treated with quinine. It is noted, too, that blackwater fever is closely simulated by the effects of an incompatible blood transfusion by paroxysmal haemoglobinuria and by the effects of the injection of an artificially produced haemolytic serum into experimental animals. All these observations are readily explained on the assumption that a biological autohaemolysin is concerned in the pathogenesis of blackwater fever. Other hypotheses put forward cannot satisfactorily account for them, especially for the specific association of blackwater fever with malaria. It is concluded, therefore, that an autohaemolysin is almost certainly concerned in the production of blackwater fever. The site of formation of this autohaemolysin is discussed, and it is considered that it is mostly produced in the spleen. The boosting of the titre of the autohaemolysin resulting from repeated attacks of malaria often inadequately treated, and factors producing a sudden contraction of a congested spleen, in which autohaemolysin has accumulated, are considered likely to precipitate an attack and to account for the sudden dramatic onset of blackwater fever. The treatment of the condition in the light of this hypothesis is discussed, and it is considered important to prevent such sudden contractions of the spleen. It is also important, in order to avoid the development of a state of sensitivity, to treat malarial infections adequately. Other conditions characterized by intravascular haemolysis are briefly discussed. It is noted that apart from incompatible blood transfusion, and paroxysmal haemoglobinuria which are known to be caused by biological antibodies, such conditions as the acute haemolytic anaemias following certain drugs and of favism can also be reasonably explained. Cases of aplastic anaemia, of agranulocytosis, and of thrombocytopenia, are briefly described, and it is noted that an explanation for their development is possibly to be found in the hypothesis here enunciated. D
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BLACKWATER
FEVER.
REFERENCES. BLACKLOCK, D. B. & MACDONALD, G. (1928). The mechanism of blackwater fever and certain allied conditions. Bvit. med. J., ii, 145. BURKITT, R. W. (1943). Treatment of blackwater fever. Ibid., i; 737. CHRISTOPHERS, S. R. & BENTLEY, C. A. (1908). Blackwater fever. Sci. mem. med. s&t. Dep&India, No. 35, p. 51 (quoted from THOMSON (1924)). (1908). Note on the phagocytosis of red blood corpuscles in the spleen of a case of blackwater fever. Ind. med. Guz., p. 81 (quoted from Thomson (1924)). DAMESHEK, W. & SCHWARTZ, S. 0. (1938). H emolysins as the cause of clinical and experimental hemolytic anemias ; with particular reference to the nature of spherocytosis and increased fragility. Amer. J. med. Sci., 196, 769. DUDGEON, L. S. (1920). Blackwater fever. J. Hyg., 19, 208. FAHRAEUS, R. (1939). The erythrocytes-plasma interface and the consequences of its diminution. Lancet, ii, 630. FAIRLEY, N. H. (1940). Peculiar haemolytic hypochromic anaemia associated with postmalarial splenomegaly of Banti’s type. Trans. R. Sot. hop. &led. Hyg., 34, 173. & BROMFIELD, R. J. (1934). Laboratory studies in malaria and blackwater fever. Part II. Blackwater fever. Haemoglobinaemia. Ibid., 28, 141. -, ----, FOY, H. & KONDI, A. (1938). Nutritional macrocytic anaemia in Macedonia. A preliminary report. Ibid., 32, 132. & MURGATROYD, F. (1940). R ecurrent blackwater fever induced by quinine. Ibid., 34, 187. FoY,~:.~~&~ KONDI, A. (1938). Spl een puncture findings in blackwater fever. Ibid.,
f -, -> -,
.
-, a normal -
Ibi$.,
37,
& MOUMJIDIS, individual during (1943). Lyso-lecithin 1. REBELO, A., & fever circulation
A. (1941). Transfusion haemolytic crisis. Ibid., fragility in blackwater
of
black.water
fever
blood
into
35, 119. fever
and
haemolytic
jaundice.
-, SOEIRO, A. (1945). Survival of transfused red cells in blackwater and of blackwater red cells in normal circulation. Ibid., 38, 271. GEAR, J. H. S. & DE MEILLON, B. (1939). Laboratory investigations of two cases of trypanosomiasis contracted in Ngamiland, Bechuanaland. S. Afr. med J., 13, 233. Ross, G. R. (1932). Researches on blackwater fever in Southern Rhodesia. London School of Hygiene and Tropical Medicine, &fern. Ser., No. 6. SCHWENTKER, F. F. & COMPLOIER, F. C. (1939). The production of kidney antibodies by injection of homologous kidney plus bacterial toxins. J. exp. Med., 70, 223. & RIVERS, T. M. (1934). The antibody response of rabbits to injections of emulsions and extracts of homologous brain. Ibid., 60, 559. SMADEL, J. E. (1936). Experimental nephritis in rats induced by injection of anti-kidney serum ; preparation and immunological studies of nephrotoxin. Ibid., 64, 921. STRONG, R. P. (1942). Stitt’s Diagnosis, Prevention and Treatment of Tropical Diseases. 6th ed. London : H. K. Lewis. THOMSON, J. G. (1924). Researches on blackwater fever in Southern Rhodesia. London School of Tropical Medicine, Res. Mem. Ser., 6, 75. VINT, F. W. (1941). Some recent researches on the spleen and their possible relationship to blackwater fever. East Afr. med. J., 18, 162. YORKE, W., MURGATROYD, F. & OWEN, D. U. (1929). Observations on five cases of blackwater fever. Trans. a. Sot. trop. Med. Hyg., 23, 335.
Vol. XxX1X.
OF THE ROYAL SOCIETY OF MEDICINE AND HYGIENE.
No. 4. February,
1946.
COMMUNICATIONS. AUTOANTIGENS GENESIS OF
AND AUTOANTIBODIES DISEASE WITH SPECIAL BLACKWATER FEVER.
IN THE PATHOREFERENCE TO
BY
Medical
JAMES GEAR, Major, Laboratory Service, S.A.M.C.
In the course of another investigation undertaken in the Laboratories of the International Health Division of the Rockefeller Foundation in 1942, it was shown that the inoculation of an emulsion of fresh normal liver obtained from a healthy rhesus monkey, into either a normal monkey or into one previously protected by vaccination against yellow fever, failed to elicit any antibody response. On the other hand it was found that an emulsion prepared from the liver of a rhesus monkey that had died of yellow fever was antigenic when inoculated into a rhesus monkey previously protected by vaccination against yellow fever. This latter monkey developed antibodies which could be demonstrated by the precipitin test not only against an emulsion of yellow fever infected liver, but also against an emulsion of normal liver. It appeared, therefore, that the presence of virus in liver cells acted as a “ schlepper ” for normal liver substance which thus became antigenic when introduced into a monkey of the same species. Assuming that a substance which was homologously antigenic would also be autoantigenic and assuming that antibodies are produced by the reticuloendothelial system, this finding may be diagrammatically presented thus :Liver cell + virus = Autoantigen. Autoantigen + R.E. cell system = Autoantibody. Autoantigen + autoantibody, = Reaction causing degeneration of affected cell. This interesting observation, as well as indicating a possible sequence of events in the evolution of the pathological process of yellow fever, suggested a hypothesis of the pathogenesis of some disease conditions, which may be summarized thus :Tissue cell + virus or + bacterial toxin = Autoantigen or + chemical or + intracellular parasite I
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Autoantigen + R.E. cell system = Autoantibody. Autoantibody + tissue cell affected or sometimes + normal tissue cell = Sensitized cell. Sensitized tissue cell + comple- = Degeneration or lysis of affected ment cell. This hypothesis has already been advanced to explain such obscure conditions as post-vaccinal encephalitis and other post infective encephalitides (SCHWENTBER and RIVERS, 1934) and glomerular nephritis (SMADEL, 1936 ; SCHWENTKER and COMPLOIER, 1939). It is to put forward and discuss a similar hypothesis of the pathogenesis of blackwater fever that is the main purpose of this paper. There is now general agreement that blackwater fever is related to malaria and is especially liable to affect those who have suffered from repeated attacks, which have been inadequately treated with quinine. It was once considered that blackwater fever was associated only with malignant tertian malaria (THOMSON, 1924), but later observations (FoY, 1938) have shown that in Salonika almost as great a proportion of cases are associated with Plasmodium vivax (33 per cent.) as with P. fahparum (47 per cent.) while mixed infections were noted in 14 per cent. of cases. Although many theories have been advanced to explain the acute intravascular haemolysis which is the fundamental event in an attack of blackwater fever, the cause, to summarize current literature and textbooks, remains a mystery. It has often been suggested, however, that a haemolysin or an anaphylactic sensitizing substance is responsible for the haemolysis, but so far no adequate explanation for their development has been put forward. To quote Stitt’s Tropical Diseases (STRONG, 1942), “ A scientific explanation of the mechanism by which haemolysis is brought about is not yet possible. It is conceivable that a haemolysin may be free in the blood stream or bound to certain cells, but it is not clear what suddenly frees it or precipitates its action.” However, it appears that in the hypothesis enunciated above a reasonable explanation is found. According to this hypothesis, the development of haemolysins may be accounted for by the fact that a red cell infected with a malaria parasite, possibly only after treatment with an antimalarial drug, especially The sequence of events may be formulated quinine, may become antigenic. as follows :Red cell + malarial parasite or Red cell + malarial parasite + antimalarial drug = Autoantigen. or I Red cell + antimalarial drug Autoantigen + R.E. cell system = Autoantibody’ (haemolysin). Red cell + haemolysin = Sensitized red cell. Sensitized cell + complement = Lysis of red cell.
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There is a vast collection of facts relating to blackwater fever, and these have often been analyzed and considered in the past. However, it is now desirable to re-examine and to discuss them as well as several recent observations, in relation to this hypothesis of its pathogenesis. It will become apparent in this discussion that these facts are all in keeping with, or are rationally explained, by this hypothesis. There is little direct evidence of the presence of a haemolysin in the blood of cases of blackwater fever. To quote THOMSON (1924), “ Attempts to demonstrate an autohaemolysin in the serum of blackwater fever during the attack of haemoglobinuria have failed or indefinite results have been obtained.” “ We were quite unable to detect any haemolysin in the serum of blackwater fever at any period of the disease, but as we have pointed out this by no means proves that a serum haemolysin is not present.” Indeed, THOMSON states, “ There are strong grounds for suspecting that the condition is brought about by a specific serum haemolysin elaborated from the prolonged action of Plasmodium falciparum on the red blood corpuscles.” He suggests that, “ it is an altered chemical change which produces the so-called brassy corpuscles in malignant tertian. Corpuscles so altered act as foreign bodies and are thus capable of producing a specific haemolytic amboceptor which can act in the presence of complement on corpuscles so altered and not on normal red cells.” ROSS (1932), although stating that there is no doubt as to the reality of the appearance of the altered corpuscles that THOMSON describes, states that attempts to demonstrate the presence of such altered cells were very seldom successful during his investigation, and therefore considers the hypothesis regarding the role of such cells as open to criticism. FAIRLEY and BROMFIELD (1934) came to the conclusion that the intravascular haemolysis of blackwater fever was caused by a haemolytic agent arising in chronic subtertian malaria as a result of a metabolic breakdown precipitated by quinine or plasmoquine, or possibly by other factors such as chill or exhaustion. They considered their findings were against either the action of an immunological haemolysin or a direct drug effect on the corpuscles. Although the direct evidence is so unconvincing, there is considerable indirect evidence that an antibody acting on red cells is present in cases of blackwater fever. This will now be considered. It has repeatedly been observed that red cells from cases of blackwater fever show a tendency to autoagglutination. This in itself is suggestive of the presence of antibodies, but by itself is not conclusive, for autoagglutination of the red cells has been observed in many conditions that are not associated with acute intravascular haemolysis. For example, autoagglutination of the red cells is often observed in the blood of cases of human trypanosomiasis. However, even in this condition erythrophagocytosis has been noted, a phenomenon which suggests the presence of red cell opsonins.
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BLACKWATER FEVER.
Erythrophagocytosis has also been noted in cases of blackwater fever. (1924), in his observations on eight fatal cases of blackwater fever, noted that there was evidence of endothelial activity and the endothelial cells showed active phagocytosis of the red cells. YORKE, MURGATROYD and OWEN (1929) note that phagocytes containing red cells are frequently seen both in films of the peripheral blood and of the splenic pulp. Ross (1932) also reported that smears from the spleen of cases coming to autopsy showed the presence of phagocytosed red cells, and in several instances the phenomenon was also present in films of the peripheral blood. The engulfed red cells always appeared to retain their haemoglobin. For that reason he considered that the appearance indicated the presence of an increased erythrophagocytosis in the -disease. These authors all conclude that its importance in the haemolytic process is probably small, but that it may account for part of the reduction that occurs in the red cell count. However, its occurrence suggests the presence of red cell antibodies. Autoagglutination of the red cells was clearly seen in the two most recent cases of blackwater fever admitted to the Johannesburg Hospital. Both these cases in their early stages also showed a tendency for the red cells to be microcytic and hyperchromic, an observation in turn indicating that there was a tendency to spherocytosis. Spherocytosis, in cases of blackwater fever, was noted as long ago as I908 by CHRISTOPHERS and BENTLEY (1908). THOMSON (1924) confirmed this finding, noting that in stained preparations these small spherical erythrocytes assumed much darker red. than the other red cells when stained with Leishman.. More recently, in a case of blackwater fever admitted to the Children’s Hospital,. Johannesburg, FOY and KONDI (1943) studied this question in’detail and found that cells from a case of blackwater fever were more spherocytic than normal but less so than those of haemolytic jaundice. Spherocytosis of the red cells, of course, is one of the characteristic findings in familial haemolytic jaundice. DAMESHEK and SCHWARTZ (1938) suggest that this condition is caused by an autohaemolysin and has shown that one of the earliest demonstrable effects of a haemolysin’acting on a red cell is for the affected cell to become spherocytic. Accordingly, the occurrence of spherocytosis in blackwater fever favours the hypothesis that this condition is caused by a haemolysin. Two other important observations that suggest the presence of haemolysin similar in action to artificjally produced antibodies have been made by FOY and KONDI. FOY, KONDI and MOUMJIDIS (1941) showed that red cells transfused into a haemolysing case of blackwater fever from three different donors underwent haemolysis just as readily as did the blackwater fever patient’s own cells. This was interpreted as indicating that there is some circulating haemolysin that destroys all red cells that come into contact with it, irrespective of their origin. This finding is what would be anticipated if an immunological haemolysin is THOMSON
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GEAR.
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concerned, thus donors’ cells on transference to the recipient would become sensitized by the haemolysin present in the recipient’s circulation. The ‘cells are thus rendered susceptible to the action of complement and so to haemolysis. Later, FOY et al. (1945) showed that the red cells from a case of blackwater fever, when transfused into a normal individual, were haemolyzed as rapidly as those remaining in the patient. This observation is also what would be anticipated if an immunological ,haemolysin were concerned, for the patient’s red cells are already sensitized by the autohaemolysin, so that, even if transferred to a healthy person, they remain susceptible to the action of complement and are haemolyzed. That an antigen-antibody reaction is probably concerned in the pathogenesis of blackwater fever is also suggested by a consideration of other conditions characterized by intravascular haemolysis. Two of the best known of these are paroxysmal haemoglobinuria and incompatible blood transfusion. Both are known to be due to red cell antibodies which can be demonstrated in vitro. A condition analogous to blackwater fever can be brought about experimentally by the injection of an artificially prepared haemolytic serum into experimental animals. CHRISTOPHERS and BENTLEY (1908) and DUDGEON (1920), who investigated the effects of haemolytic sera on animals, showed that the pathological appearances were the same’ as produced in blackwater fever. THOMSON (1924) remarks “ there is so marked a similarity between the action of a true specific serum haemolysin and an attack of haemoglobinuric fever that there can be little doubt that the mechanism is the same, and if we can produce such a condition by continual attacks of malaria the problem is solved.” It is known that blackwater fever rarely affects an individual who has been resident in a malarious area less than 6 months, but thereafter the chances of developing an attack increase with length of residence. The greatest number of cases occur in individuals who have lived for from 1 to 3 years in an area where malaria is hyperendemic, and who, during this time, have usually suffered from repeated attacks of malaria often inadequately treated with quinine. This time lag between the first infection with malaria and the development of blackwater fever in itself suggests a process of sensitization. As the time taken for sensitization exceeds 6 months, it is reasonable to conclude that the antigenic value of the hypothetical autoantigen is relatively low, so that repeated attacks of malaria are necessary before a state of sensitivity develops. It may well be that, in addition to malaria, treatment by antimalarial drugs is a necessary factor for the development of this state. The evidence discussed above may now be summarized :1. Autoagglutination and spherocytosis of the red cells in cases of blackwater fever have been observed.
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BLACKWATER FEVER.
2. Erythrophagocytosis is commonly seen in smears made from the spleen and occasionally in films of peripheral blood. 3. Red cells from healthy donors haemolyze as rapidly in a case of blackwater fever as the patient’s own cells. Red cells, when transfused into a normal person from a case of blackwater fever, haemolyze as rapidly as the red cells remaining in the patient. 4. Blackwater fever is closely simulated by paroxysmal haemoglobinuria and incompatible blood transfusion, conditions both caused by known and demonstrable anti-red cell antibodies ; and by the effects of artificially produced haemolytic sera in experimental animals. 5. There is usually a lag period between the first attack of malaria and the onset of blackwater fever. This time interval suggests .a process of sensitization. All these facts are readily explained on the assumption that an autohaemolysin is concerned in the pathogenesis of blackwater fever. Other hypotheses put forward cannot satisfactorily account for them. Indeed, it may be concluded that blackwater fever is almost certainly caused by an autohaemolysin. One of the most puzzling features of blackwater fever has always been its sudden and dramatic onset. Sir PATRICK MANSON put this puzzle in his question to Dr. THOMSON in 1922. “ Malaria is undoubtedly one of the factors but what pulls the trigger ? ” In Stitt’s Tropical Diseases (STRONG, 1942) it is stated that it is conceivable that a haemolysin may be free in the blood stream or Bound in certain cells, but it is not clear what suddenly frees it or precipitates its action. It is considered that the hypothesis put forward in this paper also accounts for the sudden onset of blackwater fever. In this connection it is relevant to discuss the site of formation of the haemolysin. There has been a considerable amount of experimental work on the site of formation of antibodies, and an extensive literature dealing with this subject has accumulated, but it is not intended to review it here. Suffice it for the present to note that much evidence has been presented favouring the view that antibodies are produced by the reticula-endothelial system of cells. The spleen is the largest depot of this system of cells in the body. Several observations have been made which show that the spleen is intimately connected with serum immune bodies. It is known that the removal of the spleen seriously undermines the resistance of rats to an infection with Bartonella murk. An infection which previously was easily tolerated, after removal of the spleen becomes rapidly fatal. In the field of human medicine, removal of the spleen is known to benefit the clinical condition of cases.of familial haemolytic anaemia, a condition which is possibly caused by autohaemolysins (DAMESHEK and SCHWARTZ, 1938). Splenectomy also benefits many cases of chronic thrombocytopenic purpura, and some cases of chronic leucopenia. Incidentally, it is considered very likely
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that these two conditions are also caused by autoantibodies, and will be considered from this point of view of this hypothesis in more detail at a later stage. It was noted, incidentally, in a paper on cases of Rhodesian sleeping sickness (GEAR and DE MEILLON, 1939), that smears made from the spleen of experimentally infected animals showed few intact trypanosomes, although degenerating forms and remnants of degenerated forms, were numerous. In contrast, smears made from all the other organs, including the lymph glands, It seemed from this observation that showed numerous intact trypanosomes. the spleen is the principal site of the formation of trypanosomicidal antibodies. It is reasonable to expect that it is also the principal site of formation of other circulating antibodies. It is likely, then, that haemolysins responsible for the acute intravascular haemolysis of blackwater fever would also be formed principally in the spleen. It would be expected, then, that when a person had become sensitized, any factors causing a sudden contraction of the spleen so expressing its contained haemolysins into the general circulation, would result in an attack of blackwater fever. On referring to the known precipitating causes of blackwater fever it is found that they are usually given in this order, quinine, chill, exertion and violent emotion. These, the chief exciting causes of this fever, have one effect in common, they cause contraction of the spleen. Thus it becomes clear why the onset of an attack of blackwater fever is so sudden and dramatic. It is only when a sudden contraction of the spleen occurs that sufficient haemolysin is liberated to cause intravascular haemolysis. At other times it seems probable that the haemolysin is mopped up as it is formed and the number of red cells so sensitized is not too large to be removed by the reticula-endothelial cells before intravascular haemolysis takes place. In that case one would expect that cases of subacute or chronic haemolytic anaemia, not associated with intravascular haemolysis, would occur. That such cases do occur has been made clear by FAIRLEY, BROMFIELD, FOY and KONDI (1938), who described cases of haemolytic anaemia associated with chronic malaria in Macedonia. It is very interesting to note that these cases occurred in Macedonia, a region where more cases of blackwater fever are seen than anywhere else in the world. It seems possible, then, that these cases are more or less benign manifestations of the process which in more violent form results in blackwater fever. Several writers on blackwater fever have considered that in some way or other the spleen is involved in the pathogenesis of blackwater fever. BLACKLOCK and MACDONALD (1928) believed that the sudden massive haemolysis is occasioned by the over-production of sarcolactic acid, a normal constituent of the blood and tissues. This is caused by anoxaemia particularly in the spleen, where the adherence of malaria infected red cells to each other and to the vascular endothelium interferes with the circulation. They point out that the agencies chill, exertion, quinine, which precipitate an attack, all cause
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FEVER.
contraction of the spleen, and this they consider accentuates the existing anoxaemia, so that lactic acid formation and accumulation increase to haemolytic concentrations and leakage of haemoglobin into the portal circulation results. This leakage becomes increasingly apparent as circulation through the spleen becomes re-established, leading to haemoglobinaemia and haemoglobinuria. ROSS (1932) studied cases from the point of view of this hypothesis and concluded that the theory on which it was founded was fallacious. FAIRLEY (1940) suggests that a perversion of the activity of the reticuloendothelial system occurs as a result of chronic malaria, which induces a considerable hypertrophy of this system in general and of the spleen in particular. He suggests that an escape into the blood stream from a pathological reticula-endothelial system of the intracellular ly& enzyme, normally responsible for the destruction of effete red cells, occurs and is responsible for the intravascular haemolysis. VINT (1941) puts forward a hypothesis of the causation of blackwater fever based on experiments of FAHRAEUS (1939), who showed that under certain conditions red cells undergo a change he called stabilization. VINT suggests that a similar change occurring in the splenic vein is responsible for the liability of red cells in cases of blackwater fever to haemolyze. Fov,and KONDI (1943), who found that the red cell fragility to lysolecithin was increased in cases of blackwater fever, state, “ The enlargement of the spleen in so many of these haemolytic conditions is regarded by many as of considerable significance. The production of lysolecithin as a result of the separation of the cells and plasma in this organ has led to the suggestion that this powerfully haemolytic substance may play a part in the haemolyses of certain of these conditions.” Although the hypotheses put forward by these authors differ, often very considerably, in their basis, they all bring forward evidence that in some way or other the spleen is intimately concerned in the pathogenesis of blackwater fever. It is considered that the role assigned to the spleen in the hypothesis here put forward accords with the observed facts more satisfactorily than the other hypotheses of the causation of the condition. Most of these hypotheses do not explain what might be called the specific nature of blackwater fever, a specificity that is clearly explained by the assumption that a biological haemolysin is concerned. It is now opportune to suggest the probable sequence of events leading up to an attack of blackwater fever. Red cells infected with malarial parasites, possibly only after treatment with an antimalarial drug or red cells altered by or combined with an antimalarial drug, become autoantigenic, and in response to this autoantigen an antibody or haemolysin is produced by the reticula-endothelial system, particularly by the spleen. The titre of this haemolysin is boosted by each repeat attack of malaria. When the circulation of the blood through the spleen is
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free, this haemolysin is mopped up by the red cells and these sensitized red cells are removed by the reticula-endothelial cells as they are sensitized and before a demonstrable intravascular haemolysis occurs. However, when the circulation through the spleen is impeded, and the spleen becomes congested, as it does in an attack of malaria, which also provides a secondary stimulus to boost the titre, this haemolysin accumulates, Factors which cause a sudden of quinine, chill, and contraction of the spleen, such as the administration exertion, now would sudden!y express into the general circulation sufficient haemolysin to sensitize a large number of red cells and their haemolysis intravascularly would result in haemoglobinaemia and haemoglobinuria, followed by the other signs and symptoms of blackwater fever. The acceptance of this hypothesis of the pathogenesis of blackwater fever may be of practical value. If the haemolysin is produced by the spleen and expressed from it by conditions which cause that organ to contract, it is obvious that it is desirable to prevent such contraction, especially sudden contraction. Measures to ensure this would be :Placing the patient at absolute rest in bed lying flat. Placing his mind at rest. Avoidance of drugs which would cause smooth muscle contraction. It would also be desirable, if such were available, to administer a drug which would prevent the union of antibody and antigen. The commonly accepted method of treatment of cases of blackwater fever aims at achieving these objects, thus to quote Stitt’s Tropical Diseases (STRONG, 1942), “ Absolute rest in bed, avoidance of chilling, and good nursing are prime considerations in treatment.” Recently BURKITT (1943) wrote that, when treating cases of blackwater fever in East Africa, he was struck by the great restlessness of mind and body characteristic of that disease, and to counteract this gave 8 to 10 grains of sodium luminal intramuscularly. In eight cases so treated in the evening, all traces of blackwater had disappeared by the following morning. He also reported that Dr. J. K. GREGORY, of Nairobi, has had a series of thirty cases all treated by intravenous injection of 15 grains of phenobarbitone and all, “ cured at once.” He remarks that he has used the unscientific expression “ cured at once ” because it gives the picture exactly, A reasonable explanation of the success of this sedative treatment has been given in this discussion. The advisability of ihe administration of quinine to cases of blackwater fever is still a vexed question. Arguing on the basis of the hypothesis here put forward, it would seem that, once the spleen had contracted and expressed its haemolysin, there would be little danger in treatment with quinine unless it was withheld for a sufficient time to once again allow a considerable amount of haemolysin to dam up in the spleen. However, as then it would be a real danger it would seem that treatment with quinine should not be given for the concomitant malaria unless mepacrine is not available. Although the
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BLACKWATER FEVER.
evidence is not altogether convincing, it is generally accepted that the regular taking of prophylactic quinine decreases the liability to blackwater fever. It is known that the adequate treatment of malaria by quinine likewise diminishes the chances of blackwater fever. Yet the irregular taking of prophylactic quinine, and the inadequate treatment of malaria by quinine, are recognized as most important factors in rendering a patient prone to blackwater fever. A reason for even his paradoxical state of affairs emerges from this discussion. It has already been noted that the antigenic value of the autoantigen is probably low, and that it usually requires a prolonged exposure to its action before a state of sensitivity develops. Such a prolonged action would be ensured by irregular prophylactic quinine, and by inadequate treatment, both of which would allow a latent infection to persist and to progress, but which would also prevent such an infection from frankly asserting itself, thereby lessening the body’s natural development of immunity. Also because of the inadequate treatment relapses are frequent and provide the secondary stimuli which result in boosting the titre of an autohaemolysin. Accordingly, from the point of view of prophylaxis, it is important that, if a person is taking prophylactic quinine, he should do so regularly, but more important still should he contract malaria it is essential that his treatment should be thorough. It seems that thorough treatment, by eliminating the potential autoantigen, guards against the development of the autohaemolysin. In cases of chronic malaria that have already suffered from blackwater fever, it is essential that they should be thoroughly treated, because such thorough treatment eliminates the autoantigen and it appears that once this is eliminated the titre of the autohaemolysin rapidly diminishes. This was clearly demonstrated in a case of recurrent blackwater fever induced by quinine described by FAIRLEY and MURGATROYD (1940). The capacity of quinine to produce blackwater fever appeared in this case to be directly or indirectly related to persisting malarial infection, which was associated with a demonstrably enlarged spleen. Following effective treatment by atebrin, and disappearance of the splenomegaly, the administration of quinine was no longer followed by haemoglobinuria. Regarding the prevention of the union of antigen and antibody, it is relevant to note that such properties have been ascribed to acetyl salicylic acid. This therapeutic effect no doubt accounts for its great value in relieving the symptoms of acute rheumatic fever, a condition which it seems very likely will also be found to be caused by an autoantigen-antibody reaction taking place in tissues rendered autoantigenic by union with haemolytic streptococcus toxin. As far as can be ascertained, aspirin has not been credited with any therapeutic effect in cases of blackwater fever. It would be interesting to know the results of a trial of this drug over a large series of cases, though no striking effect could be anticipated, because, once haemoglobinuria has occurred, the
JAMES
311
GEAR.
However, its administration may process has in most cases expended itself. be of value in the remittent type of case. The other lines of treatment such as blood transfusion for combating the effects of the intravascular haemolysis, although they are of the utmost importance and often life-saving, will not be considered in detail as they do not immediately concern our theme. It is, however, pertinent to discuss briefly other conditions characterized by intravascular haemolysis, noting whether any feature of them has any bearing on this hypothesis. Of these conditions attention has already been drawn to the effects of an incompatible blood transfusion and to paroxysmal haemoglobinuria, both of which are caused by demonstrable antibodies. Of other conditions, one of the most common is acute haemolytic anaemia following the administration of certain drugs. Some of these, such as phenylhydrazine, act directly on the corpuscles. In others the reaction appears to result from an undue sensitivity. In recent years, the drugs most commonly concerned in the latter group are those or the “ sulpha ” group. We have seen two cases of acute haemolytic anaemia followThese will not now be described ing the administration of sulphapyridine. in detail, suffice it to note that in each case, towards the end of a course of treatment, acute haemolytic anaemia suddenly developed, characterized by intravascular haemolysis as shown by haemoglobinaemia and haemoglobinuria. It is recognized that these accidents are rare and usually they are attributed to an idiosyncrasy of the patient. This idiosyncrasy is usually considered to be of an allergic nature. In the hypothesis here discussed a rational explanation for their development is found which can be formulated as follows :Red cell + sulpha compound or derivative Red cell + antibody (haemolysin) Sensitized red cell + complement
= Autoantigen. = Sensitized red cell. = Haemolysis.
In the cases noted above, one was fatal, dying of uraemia following anuria, the other rapidly recovered after withdrawing the drug. As the condition developed soon after exposure to the drug, it would appear that red cells linked to the “sulpha” drugs have a high antigenic value,which accounts for the relatively rapid development of haemolysin, but as this antibody, unlike the haemolysin of blackwater fever, apparently acted only on red cells that had come into contact with the drug, recovery was rapid and complete. We have also seen two cases of malaria in their primary attack, which towards the end of a course of treatment with atebrin, suddenly developed an acute haemolytic anaemia, characterized by haemoglobinaemia and haemoglobinuria. Although these cases being associated with malaria were considered to be cases of true blackwater fever, it is our opinion that they were more directly comparable with the type of haemolytic anaemia in the two cases noted above, resulting from an acquired sensitivity to the drug. It seems, too, that the cases of quinine haemoglobinuria of which many have been recorded also belong
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BLACKWATER
FEVER.
to this group. The acute haemolytic anaemia of favism likewise appears to belong to this group. Possibly similar in pathogenesis were two cases of aplastic anaemia, recently seen in the Johannesburg Hospital. One followed a full course of treatment with sulphapyridine for pneumonia, but it is of interest to note that the sikns of aplastic anaemia did not become evident until 3 months later. The other similarly followed a course of treatment with sulphapyridine and sulphanilamide for tonsillitis. It is also of interest to note that we have under observation one case of chronic agranulocytosis that first manifested symptoms 10 months after the second full course of treatment with sulphapyridine for gonorrhoea. The blood film of this case in which the leucocyte count was usually in the neighbourhood of 2,000, was characterized by a considerable proportion ( f 20 per cent.) of disintegrating neutrophil leucocytes. A similar blood picture can be artificially produced in experimental animals by the injection of an antileucocyte antiserum. Another apparently analigous case at present under observation is that of a patient who, following a full course of treatment for pneumonia with sulphapyridine, developed thrombocytopenia. This first manifested itself 1 month after recovery from pneumonia and now has persisted for 2 years. It is relevant to note that thrombocytopenia can be produced experimentally by the injection These interesting cases will be described and discussed of antiplatelet serum. They are mentioned now because it seems that a in detail in another paper. rational explanation of their pathogenesis is to be found in the hypothesis set out in this paper. The more fundamental problems of the constitution of antibodies and of complement have not been discussed. It seems probable that such a study may show that it is possible to correlate some of the, at present, apparently diverse theories of the pathogenesis of blackwater fever. SUMMARY
A hypothesis of the pathogenesis of blackwater fever, suggested by the observation that liver emulsion from a rhesus monkey dead of yellow fever, stimulates the formation of antiliver antibodies when injected into another rhesus monkey, is put forward and discussed. This hypothesis may be formulated thus :Red cell + malarial parasite or Red cell + antimalarial drug \ = Autoantigen or i Red cell -+-malarial parasite i + antimalarial drug I
JAMES
Autoantigen + R.E. cell system Red cell + haemolysin Sensitized red cell + complement
GEAR.
313
= Autoantibody haemolysin. = Sensitized red cell. = Haemolysis.
It is noted that, in cases of blackwater fever, the red cells show autoagglutination and spherocytosis and that erythrophagocytosis is commonly seen in smears of the spleen, and, occasionally, in films of peripheral blood: Further, it has been observed, red cells from healthy donors haemolyze as rapidly in a case of blackwater fever as the patient’s own cells and, on the other hand, red cells from .a case of blackwater fever, transfused into a healthy recipient, haemolyze as rapidly as the red cells remaining in the patient. There is usually a considerable interval between the first attack of malaria and the onset of blackwater fever, during which the patient suffers from repeated attacks of malaria, often inadequately treated with quinine. It is noted, too, that blackwater fever is closely simulated by the effects of an incompatible blood transfusion by paroxysmal haemoglobinuria and by the effects of the injection of an artificially produced haemolytic serum into experimental animals. All these observations are readily explained on the assumption that a biological autohaemolysin is concerned in the pathogenesis of blackwater fever. Other hypotheses put forward cannot satisfactorily account for them, especially for the specific association of blackwater fever with malaria. It is concluded, therefore, that an autohaemolysin is almost certainly concerned in the production of blackwater fever. The site of formation of this autohaemolysin is discussed, and it is considered that it is mostly produced in the spleen. The boosting of the titre of the autohaemolysin resulting from repeated attacks of malaria often inadequately treated, and factors producing a sudden contraction of a congested spleen, in which autohaemolysin has accumulated, are considered likely to precipitate an attack and to account for the sudden dramatic onset of blackwater fever. The treatment of the condition in the light of this hypothesis is discussed, and it is considered important to prevent such sudden contractions of the spleen. It is also important, in order to avoid the development of a state of sensitivity, to treat malarial infections adequately. Other conditions characterized by intravascular haemolysis are briefly discussed. It is noted that apart from incompatible blood transfusion, and paroxysmal haemoglobinuria which are known to be caused by biological antibodies, such conditions as the acute haemolytic anaemias following certain drugs and of favism can also be reasonably explained. Cases of aplastic anaemia, of agranulocytosis, and of thrombocytopenia, are briefly described, and it is noted that an explanation for their development is possibly to be found in the hypothesis here enunciated. D
314
BLACKWATER
FEVER.
REFERENCES. BLACKLOCK, D. B. & MACDONALD, G. (1928). The mechanism of blackwater fever and certain allied conditions. Bvit. med. J., ii, 145. BURKITT, R. W. (1943). Treatment of blackwater fever. Ibid., i; 737. CHRISTOPHERS, S. R. & BENTLEY, C. A. (1908). Blackwater fever. Sci. mem. med. s&t. Dep&India, No. 35, p. 51 (quoted from THOMSON (1924)). (1908). Note on the phagocytosis of red blood corpuscles in the spleen of a case of blackwater fever. Ind. med. Guz., p. 81 (quoted from Thomson (1924)). DAMESHEK, W. & SCHWARTZ, S. 0. (1938). H emolysins as the cause of clinical and experimental hemolytic anemias ; with particular reference to the nature of spherocytosis and increased fragility. Amer. J. med. Sci., 196, 769. DUDGEON, L. S. (1920). Blackwater fever. J. Hyg., 19, 208. FAHRAEUS, R. (1939). The erythrocytes-plasma interface and the consequences of its diminution. Lancet, ii, 630. FAIRLEY, N. H. (1940). Peculiar haemolytic hypochromic anaemia associated with postmalarial splenomegaly of Banti’s type. Trans. R. Sot. hop. &led. Hyg., 34, 173. & BROMFIELD, R. J. (1934). Laboratory studies in malaria and blackwater fever. Part II. Blackwater fever. Haemoglobinaemia. Ibid., 28, 141. -, ----, FOY, H. & KONDI, A. (1938). Nutritional macrocytic anaemia in Macedonia. A preliminary report. Ibid., 32, 132. & MURGATROYD, F. (1940). R ecurrent blackwater fever induced by quinine. Ibid., 34, 187. FoY,~:.~~&~ KONDI, A. (1938). Spl een puncture findings in blackwater fever. Ibid.,
f -, -> -,
.
-, a normal -
Ibi$.,
37,
& MOUMJIDIS, individual during (1943). Lyso-lecithin 1. REBELO, A., & fever circulation
A. (1941). Transfusion haemolytic crisis. Ibid., fragility in blackwater
of
black.water
fever
blood
into
35, 119. fever
and
haemolytic
jaundice.
-, SOEIRO, A. (1945). Survival of transfused red cells in blackwater and of blackwater red cells in normal circulation. Ibid., 38, 271. GEAR, J. H. S. & DE MEILLON, B. (1939). Laboratory investigations of two cases of trypanosomiasis contracted in Ngamiland, Bechuanaland. S. Afr. med J., 13, 233. Ross, G. R. (1932). Researches on blackwater fever in Southern Rhodesia. London School of Hygiene and Tropical Medicine, &fern. Ser., No. 6. SCHWENTKER, F. F. & COMPLOIER, F. C. (1939). The production of kidney antibodies by injection of homologous kidney plus bacterial toxins. J. exp. Med., 70, 223. & RIVERS, T. M. (1934). The antibody response of rabbits to injections of emulsions and extracts of homologous brain. Ibid., 60, 559. SMADEL, J. E. (1936). Experimental nephritis in rats induced by injection of anti-kidney serum ; preparation and immunological studies of nephrotoxin. Ibid., 64, 921. STRONG, R. P. (1942). Stitt’s Diagnosis, Prevention and Treatment of Tropical Diseases. 6th ed. London : H. K. Lewis. THOMSON, J. G. (1924). Researches on blackwater fever in Southern Rhodesia. London School of Tropical Medicine, Res. Mem. Ser., 6, 75. VINT, F. W. (1941). Some recent researches on the spleen and their possible relationship to blackwater fever. East Afr. med. J., 18, 162. YORKE, W., MURGATROYD, F. & OWEN, D. U. (1929). Observations on five cases of blackwater fever. Trans. a. Sot. trop. Med. Hyg., 23, 335.