Lepr. Rev. ( 1970) 41, 207-222

. Cell-Mediated Immunolog ica l Processes In Lep rosy*

J. L. TURK PTOjessor oj Pathology, Royal Oollege oj Surgeons oj England, Lincoln's Inn Fields, London, lV.O.2

A large number of orgsnisms such as viruses, protozoa, helminths, fungi and bacteria, especially mycobacteria, need cell-mediated immunological processes for their elimination. As well as being involved in protection, cell-mediated immunological processes are also involved in a number of allergic reactions to products derived from mycobacteria. Cell-mediated immunological processes can be demonstrated by a number of in vitro reactions. Leprosy can present with a wide range of different clinical patterns. The clinical spectrum of leprosy can be shown to depend on the degree of the cell·mediated immune response of the host against Mycobacterium leprae. Thus in tuberculoid leprosy therc is a high degree of cell-mediated immune response whereas in lepromatous leprosy such a response is virtually absent. There appears to be a constitutional prf'disposition to lepromatous leprosy. In addition to a specific loss of cell· mediated in,mune response against Myco. leprae, there is also a non-specific drop in the abiiity of patients with lepromatous leprosy to show other aspects of cell-mediated immune response, e.g. contact sensitivity and skin homograft rejection. There is also a relative impairment of the ability of lymphocytes to react in vitro. Lymph nodes from patients with lepromatous leprosy show a deficiency in those areas associated with the development of cell-mediated immune responses. The article includes a discussion on the possible causes of deficienciesin cell·mediated immune responses in lepromatous leprosy.

THE NATURE OF THE IMMUNOLOGICAL the subsequent train of events, which each leads RESPONSE to, is different. Thus, humoral antibodies may The body can respond to an antigenic stimulus in react with antigen on cell surfaces to produce 2 fundamentally different ways. The end result anaphylactic phenomena (asthma, hay-fever, of each is the production of a factor which will urticaria, etc.) or react with complement and be interact specifically with the antigen, setting off deposited in the walls of blood vessles, causing a train of events leading either to the elimination vasculitis, glomerulonephritis, arthritis, uveitis, of the antigen from the body or the induction of etc. The interaction between humoral antibodies an inflammatory reaction. In one case, the and the antigens of certain bacteria, such as specific antigen-reacting factor is a serum pneumococci or Salmonella, may increase the protein, an immunoglobulin molecule, which phagocytosis and elimination of these organisms we can refer to as a "humoral antibody". In the from the body. However, a large number of other, there is the production of lymphocytes micro-organisms cannot be eliminated by the which have a similar property of reacting action of humoral antibodies alone. These specifically with the antigen. The production organisms require the interaction of sensitized of these specifically sensitized lymphocytes is lymphocytes and macrophages for their elimina­ known as the "cell-mediated immune response" tion. It is becoming more apparent that cell­ Although both humoral antibody and sensitized mediated immune processes are necessary for the lymphocytes react specifically with the antigen, protection of the body from viruses, protozoa, helminths, fungi and many bacteria, including *Reprinted from Bull. Wld Hlth Org., 1969,41,779-792, by kind permission of Chief, Technical Puhlications, organisms as different as the treponemata, WHO. Brucella and the mycobacteria. 207 208 J. L. Turk

The need of cell-mediated immune processes what is often refened to as the paracortical for the elimination of mycobacteria has been area of lymph nodes 01 in the area of the white known for many years. Much of the early work pulp of the spleen around the central arceIiole. on this subject was performed on Mycobacterium The paracortical area of the lymph node is found tuberculosis, and started with the observation of situated between the true cortex and the Koch (1891), who demonstrated that if virulent medulla and between the lymph follicles and tubercle bacilli were injected subcutaneously germinal centres (Oort and Turk, 1965). into a guinea-pig which was already infected, the Both this area and the areas of lymphocytes animal reacted against the organisms differently in the white pulp of the spleen round the central from an uninfected animal. The rejection of the arteriole have been found to be dependent on organisms was associated with a massive in­ thymus integrity in late foetal and neonatal flammation which developed at the site of life (Parrott, de Sousa and East, 1966). The injection. A similar type of inflammation could differentiation of lymphocytes, probably sen­ be induced if the guinea-pig, or a similarly sitized by contact with antigen in the periphery infected human subject, was inoculated intra­ (lVledawar, 1958), into large pyroninophilic cells dermally with a culture filtrate of the micro­ (immunobJasts), which can be ;;hown to divide organisms (the tuberculin reaction). Subse­ into other small lymphocytes takes place in the quently, it has been shown that both the paracortical area of lymph nodes as part of a elimination of the organisms from the body and cell-mediated immune response (Oort and Turk, the inflammatory reaction ploduced by a 1965). Removal of the thymus in neonatal life sensitized person against the organisms or depletes these areas of lymphocytes and is culture fluid are caused by the reaction with associated with an inability to manifest such a sensitized lymphocytes and macrophages, cell­ response (Miller, 1961). mediated immunological reactions. Babies born with congenital thymic hypo­

Both the cell mediated immune response and plasia are found to be unable to produce a the humoral-antibody response are functions of cell-mediated immunological response and the the central lymphoid tissues of the body. An paracortical areas of their lymph nodes and immunological response may be considered to the area of the white pulp of the spleen round have a number of phases. There is first contact the central arteriole are deficient in lymphocytes with antigen and this is followed by a pha8e of and replaced by reticulo-histiocytes. A similar cellular proliferation, lymphocytes in the case of appearance is found in the lymphoid tissue of cell-mediated immune responses, and plasma animals treated with anti-lymphocyte serum cells for humoral-antibody production. Cellular (Turk and Willoughby, 1967) which also pre­ proliferation takes place mainly in the lymph vents the development of cell-mediated immune nodes, spleen or the lymphoid tissue of the gut. responses. Neonatally thymectomized animals, As a result of this, either sensitized lymphocytes babies with congenital thymic aplasia and or humoral antibody are present in a sufficient animals treated with anti-lymphocyte sera are concentration to react with the foreign sub­ able to produce humoral antibodies, have stances (antigens) on subsequent contact. normal levels of immunoglobulins in their Production of humoral antibody takes place serum and have normal numbers of plasma cells in the plasma cells which are found at the in their lymphoid tissue. cortico-medullary junction or in medullary As well as being involved in the protection of cords of lymph nodes or in the red pulp of the the body against a wide range of micro­ spleen, and is usually associated with the organisms, cell-mediated immune responses are formation of germinal centres in the cortex of the involved in a number of allergic responses to lymph nodes or the splenic white pulp. The substances produced by a number of micro­ proliferation of lymphocytes associated with organisms. These reactions, like the tuberculin cell-mediated immune reponses takes place in reaction mentioned above, take 24 to 48 hr to Cell-mediated Immu,nological Processes in Leprosy 209

develop and include the "reaction of immunity" this is injected intradermally, it will produce an to vaccinia virus, the mumps skin test, skin inflammatory reaction which has many of the reactions to streptococcal antigens such as those characteristics of a tuberculin reaction (Bennett found in mixtures of streptokinase and strepto­ and Bloom, 1968; Pick et al ., 1969). dornase, to fungal antigens such as coccidio­ The effect of the interaction between specifi­ mycin and histoplasmin, and to protozoal cally sensitized lymphocytes and antigen on the antigens such as the Montenegro test for ability of macrophages to eliminate myco­ leishmaniasis. The reaction of rejection of a bacteria has also not been completely elucidated. skin homograft is another example of a cell­ However, Berthrong and Hamilton (1959) have mediated immune response, so also is contact observed the intracellular growth of these sensitivity to simple chemical agents such as organisms in macrophages. Normally, tubercle 2,4-dinitrochlorobenzene (DNCB) which attach bacilli multiply intracellularly and form cords readily to skin proteins. which engorge the cells so that they eventually Following contact between sensitized lympho­ burst. Macrophages from immune animals do not cytes and antigen in the periphery, a soluble allow the bacilli to grow intracellularly in this substance is released which has a direct effecton manner, and they survive longer than non­ macrophages. The nature of the substances on or immw:;te macrophages although they may have within the lymphocytes, with which the antigen still ingested the organisms. At the same time reacts specifically, is not known. However, many it has been shown that macrophages fr om people feel that, because the reaction resembles immune animals have a high rate of dehydro­ humoral antibody reactions so closely, the genase enzyme activity (Allison, Zapposodi and reacting molecule must be related in some way Lurie, 1962), and an increase in lysosomal to an immunoglobulin molecule. Thus it has enzymes (acid phosphatase, b-glucuronidase and been suggested that it might be a polypeptide cathepsin). Lysosomes of macrophages from chain such as the Fd fragment of the immuno­ animals immune to mycobacteria are also globulin molecule, or some similar peptide probably more fragile than normal, as indicated chain carrying an amino-acid sequence specific by the release of considerable amounts of to the particular antigen for which it is coded lysosomal enzymes, especially b-glucuronidase, to react. into the plasma following the intravenous The soluble substances released by the injection of an endotoxin (Saito and Suter, incubation of specifically sensitized lymphocytes 1965a, b). The little work that has been pub­ with specificantigen are in the molecular-weight lished on the phagocytosis of Myco . leprae by range of approximately 70,000. These sub­ macrophages suggests, as will be discussed later, stances can be shown to have a direct effect on that in tuberculoid leprosy, where there is a the cell surface of macrophages, decreasing the high degree of cell-mediated immunity, macro­ surface electrostatic charge, increasing their phages can readily lyse phagocytosed Myco. adhesibility both to each other and to similarly leprae, whereas in lepromatous leprosy, where charged surfaces, and decreasing their per­ cell-mediated immunity is deficient, the organ­ meability to certain organic chemicals with isms are still phagocytosed but not lysed. molecular weights from 600 to 900, such as Moreover, the lysis of Myco. leprae by macro­ diphosphopyridine nucleotide and triphospho­ phages from patients with lepromatous leprosy is pyridine nucleotide. The relationship of these immunologically specific (Beiguelman, 1967 ). changes to the development of an inflammation A few in vitro reactions have been studied in the skin with erythema, induration and a extensively over the last few years in relation to mononuclear cell infiltrate is not yet completely cell-mediated immunity. One of these is related clear. However, the soluble mediator can be to the increased adhesiveness of macrophages isolated after culture of specifically sensitized following the release of a soluble substance after lymphocytes with specific antigen and, when the interaction of specifically sensitized lympho- 210 J. L. Turk

cytes with specific antigen. The macrophages are duction is highly controversial, there is a direct grown in tissue culture in small capillary tubes. relationship between the ability of lymphocytes Under normal conditions, they migrate from to be transformed by PHA and the ability these tubes in a fan-like manner. However, if of individuals to show cell-mediated immune they are in close contact with antigen and phenomena. Thus an inability both to be specifically sensitized lymphocytes, or incubated sensitized to show contact sensitivity to DNCB together with the soluble substances released and to have their lymphocytes transformed by from specifically sensitized lymphocytes in­ PHA occurs in patients with congenital thymic cubated with antigen, they fail to migrate from aplasia, Hodgkin's disease and sarcoidosis. the tubes. For this reason, the soluble substance released by incubation of specifically sensitized THE SPECTRUM OF CLINICAL LEPROSY cells with antigen is referred to as the "migration inhibition factor" and the reaction is referred It has been recognized for a considerable time to as "inhibition of macrophage migration that leprosy may present clinically in a wide in vitro". variety of forms. The pleomorphism of the The other reaction performed in tissue culture presentation of the disease was first rationalized is more related to the changes which occur to by the separation of those forms of the disease lymphocytes in lymph nodes following first where the presentation was one of peripheral contact with antigen, during the development of nerve damage fr om the form where the lesions a cell-mediated immunological response. It will mainly took the form of nodular deposits in the be remembered that after first contact with skin. The association of anaesthetic patches in antigen, probably in the periphery, lymphocytes the skin with a surrounding raised erythematous differentiate into large cells in the paracortical edge or with a large erythematous plaque led to area of lymph nodes, prior to division into other the des0ription of these lesions as "tuberculoid" lymphocytes. These large cells which are owing to their occasionally resembling the lesions synthesizing DNA, and which can thus incor­ of lupus vulgaris. The nodular lesions are porate radioactively labelled thymidine, are generally referred to as "lepromatous" as they are referred to as "immunoblasts". Similar trans­ typical of this form of disease. Thus leprosy has formation of small lymphocytes into "blast" become divided into 2 polar forms, "tuberculoid" cells can be produced in tissue culture in vitro. leprosy and "lepromatous" leprosy. However, However, this occurs most frequently if the more recently many descriptions have been person or animal has had a previous exposure made of patients who may :;how,at one and the to the antigen. Under these conditions, no same time, manifestations of both polar forms more than 10 % of the small lymphocytes are of the disease. This form of leprosy has been traniSformed. There are a number of substances referred to as "dimorphous" or "borderline" which will regularly transform 80 to 90% of the leprosy. Occasionally, patients may present small lymphocytes, without any known, previous with just one or more hypopigmented macules. exposure of the individual to the substance. The These cases may be difficult to classify into one substances, or non-specific mitogens, include or other of the more florid forms of the disease, phytohaemagglutinin (PHA), a similar mitogen and are referred to as "indeterminate" leprosy. from pokeweed (Phytolacca americana), strepto­ However, the disease rarely stays in this form lysin S and the filtrate of Staphylococcus aureU8 and may develop with the production of more cultures. Similar transformation can be obtained typical tuberculoid or lepromatous lesions. It is by treating the lymphocytes with an anti­ thought that the indeterminate state is probably immunoglobulin allotype serum. Although the the earliest form of the disease and that many relationship between lymphocyte transforma­ patients pass through it before they develop the tion with specific antigen and cell-mediated typical manifestions of tuberculoid or leprom­ immune response or humoral antibody pro- atous leprosy. Cell-mediated Immunological Processes in Leprosy 211

Thus, clinically, leprosy forms a spectrum there will be an increased infiltration with small between the 2 polar forms-tuberculoid and lymphocytes as the bacterial load gradually lepromatous leprosy-and patients may present decreases. with varying lesions depending on the degree In an attempt to provide a scheme whereby to which tuberculoid or the lepromatous the patient can be assigned at any time to the elements contribute to the disease. Apart from correct place on the spectrum, Ridley and those with the 2 polar forms, patients may vary Jopling (1966) attempted to characterize a from time to time in the degree to which the number of points on the leprosy spectrum, understanding, however, that the disease in a 2 elements contribute to the disease. number of patients was mobile and that the Thus the disease may oftenbe considered to be patients could only be assigned to one place on "mobile". A patient with leprosy at the tuber­ the scale at a particular point in time. By closely culoid end of the spectrum may move to a more correlating the clinical state of the patient with borderline condition, or a patient with borderline the histological appearance of the lesions, Ridley leprosy may move to the lepromatous end of the and Jopling were able to define 5 points on the spectrum as a result of an exacerbation of the spectrum. The 2 polar forms of the disease at the disease. Conversely, on sulfone therapy a patient tuberculoid and lepromatous ends of the may move from the lepromatous end of the spectrum are referred to as TT and LL, re­ leprosy scale towards a more borderline position, spectively, while the central position, dimor­ or a patient with borderline leprosy may move phous or borderline leprosy, is referred to as BB. towards a tuberculoid position. They added 2 other points, BT between TT and The lesions in the 2 polar forms ofleprosy show BB, and BL between BB and LL. It was marked histological differences. In tuberculoid recognized that patients with single-lesion TT leprosy, the granuloma is invariably infiltrated rarely, if ever, swing forward across the spec­ with a dense collection of small lymphocytes and, trum, and patients with true polar LL also despite well-developed collections of histiocytic rarely swing back across the spectrum. However, cells (macrophages), Myco. leprae are not found within the range ET-BL there could be consider­ on standard methods of examination. This is in able variation from year to year. In south-east marked contrast to the appearance in leprom­ Asia, lepromatous leprosy appears to be more atous leprosy where small lymphocytes are labile than it is in other parts of the world and absent or scanty and the macrophages are patients have a form of lepromatous leprosy packed with leprosy bacilli which may form which is far less stable than that found in LL globi. The tissues throughout the body may be patients in other parts of the world, presenting infiltrated with macrophages containing pro­ histologically with a slightly different picture liferating bacilli which, it appears, cannot be in which the granuloma consists of undifferen­ eliminated. As with the clinical state, the tiated histocytes, often with a fibrocytic appear­ histological appearance shows a broad spectrum ance. These patients, intermediate between between the 2 polar forms, the main variable BL and LL with a potentiality to revert to BL being the proportion of small lymphocytes on treatment, are sometimes referred to as LI present in the lesion, and the intensity of (Ridley and Waters, personal communica­ parasitism of the macrophages by mycobacteria. tion). It must, however, be emphasized that the Also, in parallel with the trend towards the points TT, BT, BB, BL, LI and LL are arbitary lepromatous end of the scale as the disease points on what is a spectrum of disease. Patients worsens, lesions may show a decrease in the can be at any particular time at points inter­ number of small lymphocytes and an increase in mediate between the 5 .marker points, i.e. the number of bacilli present. Similarly with TT jBT, BTjBB, BBjBL, BLjLI or LIjLL, treatment, as the clinical condition swings back depending on the state of their disease at that towards the tuberculoid end of the spectrum time. 212 J. L. Turk

THE IMPORTANCE OF THE SPECTRUM Tile lepromin test is not in itself an indication CONCEPT IN ASSESSING THE STATE OF of infection with Myco. leprae, but indicates that RESISTANCE OF THE PATIENT TO HIS the patient has had previous contact and has DISEASE been immunized with these antigens which are probably common to a number of mycobacteria. The correlation between the lepromin test and Normal subjects can be immunized to produce the state of clinical leprosy has been accepted for positive lepromin tests by repeated intradermal many years. Thus patients with tuberculoid injections of the killed antigen. However, it leprosy are well known to be strongly lepromin­ does indicate that the patients are capable of positive, while those at the lepromatous end of mounting a strong cell-mediated immune re­ the scale are lepromin-negative. The presence of action against Myco. leprae associated with a positive lepromin test in a patient with in­ marked lymphocytic infiltration. determinate leprosy would indicate that he is A positive lepromin test and the extensive more likely to develop tuberculoid leprosy, lymphocytic infiltration in the lesions of patients whereas a negative lepromin test would with tuberculoid leprosy of the TT type indicate indicate a considerable risk of lepromatous that these patients have a high degree of cell­ leprosy developing. mediated immunity directed against Myco. At this stage it is profitable to digress slightly leprae . This means that they have in their and discuss the nature and importance of the circulation sensitized lymphocytes capable of lepromin test. The antigen used is an extract of activating macrophages and interacting with lepromatous tissue containing particulate mat­ Myco. leprae antigens. This would account for erial, especially particulate mycobacteria, as their lesions having the appearance of typical well as antigens derived fr om the human tissue cell-mediated allergic reactions (i.e. prolonged from which the extract is derived. The reaction erythema, induration and infiltration with small appears in 2 phases: a typical, delayed hyper­ lymphocytes). It would also account for the fact sensitivity reaction-the Fernandez reaction­ that mycobacteria are absent from the lesions. maximal 48 to 72 hr after intradermal inocu­ Thus they also have a high degree of cell­ lation, and a papular granulomatous reaction­ mediated immunity and are capable of the Mitsuda reaction-which develops 3 weeks eliminating Myco. leprae from the body, al­ after inoculation. It is the Mitsuda reaction though they over-react with an intense allergic which is used by most leprologists to indicate reaction to the antigens at the same time. whether a patient with leprosy is at the tuber­ In tuberculoid leprosy of the BT type culoid end of the scale. Although this reaction lymphocytic infiltration is of the same degree takes place as long as 3 weeks after inoculation, as that in TT leprosy and the extent of the and is not a typical delayed hypersensitivity allergic reaction in the skin is as intense. reaction, it is probably evidence of a high However, more than one lesion is present and degree of cell-mediated immunity directed mycobacteria can be found in the lesion, against the mycobacterial antigens. As the indicating that the degree of cell-mediated inoculated material is particulate, it is retained immunity is less, so that not all organisms are in the tissues for a long period and does not immediately eliminated. The lepromin test is diffuse always as rapidly as does tuberculin. also not so intensely positive as in the polar However, it may be that in the form in which form (TT). Throughout the BB and BL forms, it is injected the antigen is not readily available cell-mediated immunity appears to be in­ to react with the sensitized lymphocytes of a creasingly less as indicated by the negative cell-mediated immune process and possibly the lepromin test, decreasing lymphocytic infitration 3-week latent period is the time taken for the in the lesions and increasing numbers of myco­ mycobacterial antigens to be exposed and made bacteria that can be found. available, perhaps by tissue enzymes. This loss of cell-mediated immunity increases Cell-mediated Immunological Processes in Leprosy 213 so that in the LI or polar LL forms of leprosy Evidence that leprosy in its various forms there may be no lymphocytic infiltration and occurred more frequently in certain families in the mycobacteria proliferate throughout the endemic areas has been cited by a number of body as though the body possessed no defence workers since it was first demonstrated by mechanisms towards the organisms at all. Danielsen and Boeck in 1848. Jamison and Mycobacteria are found growing in colonies and Vollum (1968) have recently cited evidence forming globi within the macrophages in an which suggests that leprosy occurs in families apparently undisturbed manner. in which there is a naturally occurring weakness Thus the spectrum of clinical leprosy fr om in their ability to develop a cell-mediated TT to LL appears to reflect a massive cell­ immune response. When tuberculin-negative mediated immune response at the one extreme children with a family history of leprosy were to a complete absence of cell-mediated immunity vaccinated with an avirulent strain of myco­ at the other. The main problems to be discussed bacterium containing antigens which cross­ in understanding the various manifestations of reacted with Myco. tuberculosis (vole tuber­ leprosy are why in some patients there is this culosis vaccine) only 18% were converted to high degree of cell-mediated immunity and in tuberculin positivity, whereas 90% of children others cell-mediated immunity is absent, why who came from families where there was no some patients starting with lesions indicating a history of leprosy were converted to tuberculin high degree of immunity can develop lesions positivity by the vaccine. This would indicate indicating a progressive loss of immunity, and that children from families in which leprosy why some patients with little or no immunity occurs are less able to develop a strong cell­ can, on chemotherapeutic treatment, develop mediated immune response, than children from lesions of a different type, indicating that they families in which leprosy does not occur. are regaining a certain degree of immunity A similar relation between weakness of an against the organisms. This latter state may be ability to show a cell-mediated immune response particularly distressing since, at the same time and the development of leprosy can be shown in as they begin to eliminate organisms from their experimental animals. Under normal conditions body, they will start to develop allergic re­ it is only possible for Myco. leprae to proliferate actions, affecting not only the skin but also the locally and for a limited number of generations nerves, of a cell-mediated immune type directed when inoculated into mice and other rodents, against residual antigen. The resulting disease and after a period of 8 months, the organisms are may not only be temporarily more severe than rejected by the body. However, if adolescent their original lepromatous state, but also may mice are thymectomized and irradiated with result in permanent scarring or nerve damage. 900 R, a process known to suppress the ability of the body to develop a cell-mediated immune response, the mycobacteria are able to spread A CONSTIT UTIONAL PREDISPOSITION TO throughout the body and produce a disease LEPROMATO US LEPROSY resembling leprosy in man (Rees et al., 1967). Lepromatous leprosy seems not to occur in a The signs include nodular swellings of the skin fixed proportion of leprosy infections. In a similar to those seen in patients with leprom­ population in which the rate of leprosy infection atous leprosy. If the mice received lymphoid is decreasing there is not a parallel fall in the tissue from animals with a normal state of cell­ incidence oflepromatous leprosy. It has therefore mediated immunity, the infection was far less been suggested by Newell (1966) that the severe than in those animals which had been development of lepromatous leprosy in an thymectomized and irradiated but had not infected person is a host-determined characteris­ received lymphoid cell replacement. If animals tic that is present in a fixed proportion of all were allowed to develop an infection like people everywhere. lepromatous leprosy and were then given an 214 J. L. Turk inoculation of lymphocytes the nodular swellings no record is given of what proportion of patients became inflamed and subsided, after which the reacted to the 100-[lg dose only and what lesions developed a condition resembling that proportion to both the 100- and 50-[lg doses, seen in borderline leprosy with nerve damage this report shows some unsatisfactory features. (Rees and Weddell, 1968). This would indicate The authors also found it difficult to explain that in experimental animals, a condition why a similar state of anergy or inability resembling lepromatous leprosy can be produced to be sensitized with DNCB did not occur by eliminating cell-mediated immunity, and that in patients in the same part of the lepromatous the lepromatous state can swing towards the spectrum but who were having attacks of borderline state if cell-mediated immunity is erythema nodosum leprosum. Also, it is difficult restored partially by a transfusion of viable to interpret these results because the patients lymphocytes capable of mounting an immune were only classified broadly into lepromatous or reaction against M yeo. leprae. dimorphous leprosy without any assessment as This suggests also that the constitutional to whether they were moving in the clinical defects predisposing to the development of spectrum of leprosy. We do not know whether lepromatous leprosy might be a weakness in the patients classified as lepromatous had the thymus fu nction in the young person, or an polar LL form of the disease 01' a form between equivalent defect in the adult. LL and BB. In another report by Bullock (1968), the Ridley-Jopling scale (TT-LL) was used to place THE ABI LITY TO SHOW OTHER ASPECTS OF the patients more accurately according to the CELL-MEDIATED IMMUNITY IN clinical and histological state of their disease. LEPROMATOUS LEPROSY As well as pre-existing sensitivity to lepromin, A generalized depression of the ability to mount tuberculin, Candida and trichophytin, the a cell-mediated immune response has been ability of patients to become actively sensitized demonstrated recently, by a number of authors, was assessed by their response to picryl chloride. in patients with lepromatous leprosy (Waldorf Picryl chloride is a much weaker sensitizer than et al., 1966; Bullock, 1968). The general dis­ DNCB in experimental animals. It is known that ability to show delayed hypersensitivity re­ DNCB will sensitize some 90% of normal actions was demonstrated by an inability of individuals, and Bullock (1968) was able to patients with lepromatous leprosy to become sensitize 28 out of 30 normal people with this sensitized with DNCB (Waldorf et al., 1966). agent. Although patients in this series were Altogether, 75% of patients with lepromatous originally classified according to the Ridley­ leprosy without erythema nodosum leprosum Jopling scale, patients with TT and BT leprosy failed to be sensitized to DNCB whereas only are referred to as tuberculoid and patients with l out of 5 patients with dimorphous (border­ BL and LL as lepromatous. In the patients with line) leprosy failed to be sensitized in this way; lepromatous leprosy who had been under treat­ the technique used to assess whether sensitivity ment for less than 18 months, 70% could not be had developed was to skin-test the patients with sensitized with picryl chloride; however a 100 and 50 [lg of DNCB in acetone. The result discrepancy again exists because only 50 % of is curious since it has been found that the these patients were tuberculin-negative. More­ application of 100 [lg of DNCB in acetone can over, 1 0% of the patients were lepromin­ produce a non-specific inflammatory reaction in positive, suggesting that they were closer to the unsensitized individuals (Turk and Waters, borderline or tuberculoid end of the spectrum 1969). Moreover, 6 out of the 17 patients who than the other patients in this group. could not be sensitized with DNCB were shown In a second group of lepromatous patients at the same time to be able to mount a cell­ who had been treated for longer than 18 months, mediated immune reaction to tuberculin. As only 47 % could not be sensitized with picryl Cell-mediated Immunological Processes in Leprosy 215

chloride. In this group, 40 % were lepromin than DNCB which acts by modifying the body's positive. A further problem in interpreting this own proteins. The inability to respond to result is that as many as 55% of the patients DNCB does not therefore indicate a complete with tuberculoid leprosy failed to be sensJtized anergy in respect to cell-mediated immunity, but with the picryl chloride. There appears to be no suggests that this property is only somewhat logical explanation why patients with tubercu­ weakened. loid leprosy fail to be sensitized to picryl chloride when 90 % of controls can be sensitized IN VITRO REACTIVITY OF LYMPHOCYTES easily. One possible thought is that the 2 FROM LEP ROMATOUS PATIENTS populations are not identical and that the controls come from a population which is more The failure of lymphocytes fr om patients with readily sensitized to picryl chloride than the impaired cell-mediated immunity, by non­ group from which the patients with tuberculoid specific mitogens such as PHA, has been des­ leprosy are derived. One explanation that cribed in a number of diseases. These include Bullock (1968) himself has suggested is that in primary thymic dysplasia, Hodgkin's disease, tuberculoid leprosy cell-mediated immunity is sarcoidosis, Sjogren's disease and primary deviated so strongly to produce the reaction biliary cirrhosis. It is possible that in these against Myco. leprae that no reserves are avail­ conditions the lymphocytes which are capable able to react with other antigens. The report of of being stimulated are those that are under the increased immunological activity following control of the thymus in neonatal life and are therapy in patients with lepromatous leprosy is thus the same class of lymphocytes that can be interesting and has been observed in other stimulated to proliferate into "sensitized groups of patients (Waters, personal communi­ lymphocytes" in a cell-mediated immune re­ cation). However, difficulty is obtained in sponse. As has been discussed above, consider­ interpreting these results as BL and LL able evidence is accumulating that there is an patients are grouped together and it would impaired ability of patients with lepromatous be important to know whether increased leprosy to manifest a wide range of cell­ ability to be sensitized to picryl chloride was mediated immune reactions. These include associated with clinical evidence of an increase contact sensitivity to simple chemical sensitizers in the immune state, such as a movement and delayed hypersensitivity to antigens from across the immunological spectrum of the Candida and trichophytin. Moreover, experi­ disease from LL to BL. ments by Job and Karat (personal communi­ The finding of patients who were tuberculin­ cation, cited by Hart and Rees, 1967) in 4 sensitive but unable to be sensitized to DNCB, patients with lepromatous leprosy indicate poses an interesting problem. These patients t,hat these patients show prolonged survival could have a degree of cell-mediated immunity of allogeneic skin grafts. In normal cultures of high enough to respond to antigens to which lymphocytes, approximately 80% of the cells they were exposed before they developed can be transformed into lymphoblastoid type leprosy, but insufficient to respond while they cells by the addition of PHA. Dierks and had their leprosy. Another possibility is that Shepard (1968) found that the cells from 8 active contact-sensitizing agents provide a weaker lepromatous patients had a low response to antigenic stimulus than bacterial antigens. In PHA (only 10% of the cells being transformed recent experiments (Turk and Waters, 1969) it by this mitogen). Of these patients, 4 had has been found that patients with leprosy who erythema nodosum leprosum and this did not could not be sensitized to DNCB could, however, affect the response of the patients. In another be sensitized to develop delayed hypersensitivity series, 3 patients with active lepromatous to keyhole-limpet haemocyanin (KLH). KLH leprosy were found to have a markedly diminish­ provides a more powerful antigenic stimulus ed response. One surprising result in this series 216 .f. L. '['urk was that 3 patients with tuberculoid leprosy autologous plasma. When the cells were sus­ also had a diminished response to PHA. The pended in homologous plasma from normal reason why patients with lepromatous leprosy subjects no depression of activity was found. fail to be stimulated with PHA and show a When the cells of normal subjects were sus­ diminished ability to manifest delayed hyper­ pended in the plasma of some lepromatous sensitivity reactions, could be explained as subjects depression in the ability of these cells being due to a general failure to manifest to be transformed by SLO was observed. How­ cell-mediated immune reactions. This might be ever, plasma from lepromatous patients did not genetic as suggested by Jamison and V ollum suppress the response of lymphocytes fr om (1968). However, other causes should be normal subjects to PHA. Thus the plasma from considered, such as the changes which are some patients with lepromatous leprosy contains seen in the lymphoid organs of such patients a factor which can inhibit the response of normal which will be discussed in another section. lymphocytes to SLO. Much might be learned Whether these changes themselves are also about the depression of cell-mediated immunity hereditary and due to thymus dysfunction in leprosy if the nature of this fa ctor could be will also be discussed. discovered. More difficult to understand is the diminished A similar phenomenon has been described by responsiveness of the lymphocytes of patients Melli et al. (1968) in patients treated with with tuberculoid leprosy to respond to PHA. heterologous anti-lymphocyte serum. Mter 14 to This may be associated with the diminished 21 days of treatment, there was a severe de­ ability of these patients to develop delayed pression of the response of the patients' lympho­ hypersensitivity to picryl chloride. One sug­ cytes to PHA. However, this only occurred if the gestion that has been put forward (Bullock, cells were cultured in autologous plasma. In the personal communication) is that the lymphoid presence of homologous plasma the response of tissue of such patients is responding so strongly the cells was normal. Correspondingly, the . to antigens from Myco. leprae that there is no plasma of patients treated in this way would residual activity to respond to other antigens. inhibit the ability of PHA to transform lympho­ This would explain an inability to respond to cytes from normal people. Melli et al. (1968) an antigen, but it is difficult to see how this considered, reasonably, that the factor present effectcould cause an inability of the lymphocytes in the plasma of patients treated with anti­ to respond to non-specific mitogenic stimulation lymphocytic serum, which inhibited the trans­ with PHA. formation of lymphocytes by PHA, was in fact Extremely interesting experiments have been an anti-lymphocyte antibody. It would thus described by Bullock and Fasal (1968). In these appear that a factor is present in the plasma of experiments, a depression of DNA synthesis, both patients with lepromatous leprosy and indicative of the transformation of lymphocytes those treated with anti-lymphocytic serum into blast cells was found in lymphocytes from which could inhibit the response of normal patients with lepromatous leprosy when exposed lymphocytes by non-specific mitogens. In both to PHA and streptolysin 0 (SLO) which also these cases it should be easy to find out whether acts as a mitogen probably through its antigenic the factor involved is an immunoglobulin, and properties. A similar depression in the trans­ thus an antibody. If an anti-lymphocytic anti­ formation of lymphocytes of patients with body is present in the circulation of patients with tuberculoid leprosy was found, as was described lepromatous leprosy, this could have consider­ by Dierks and Shepard (1968). However, one able implications. It is now well known that of the most fascinating aspects of this in­ anti-lymphocytic antibodies act specifically by vestigation was that the depression of response suppressing the circulating pool of long-lived to SLO was found only when the lymphocytes small lymphocytes, influenced by the thymus from lepromatous patients were suspended in in neonatal life, which are necessary for the Cell-mediated Immunological Processes in Leprosy 217

normal manifestation of a cell-mediated im­ IN VITRO EXAMINATION OF MACROPHAGE munological reaction. Treatment of animals with ACTIVITY IN LEPROSY anti-lymphocytic antibody has an effect very Although much work has been done on the similar to thymectomy in neonatal life, in behaviour of macrophages in experimental depleting the paracortical area of lymph nodes animals with tuberculosis, little study has been and the corresponding area of the spleen of these made of macrophage activity in patients with small lymphocytes, without affecting the lym­ leprosy. Beiguelman (1967) has developed a phocytes in the lymph follicles, thought to be of technique for testing the lysogenic ability of direct bone-marrow origin, the germinal centres macrophages for Myco. leprae. He observed the and the plasma cells in the medulla, involved in behaviour of macrophages from leprosy patients humoral antibodies production. Thus the pre­ maintained in a tissue-culture medium after the sence of an anti-lymphocytic antibody in the addition of the dead leprosy bacilli. The macro­ circulation selectively inhibits cell-mediated phages from both lepromatous and tuberculoid immune processes without affecting the pro­ patients actively phagocytosed the dead leprosy duction of most types of circulating antibody. If bacilli; the macrophages from the tuberculoid an anti-lymphocytic antibody was present in the patients completely lysed the ingested bacilli, circulation, this could account for the anergy in so that they became free of lipids. However, the respect of cell-mediated immunity in certain macrophages from the lepromatous patients were patients with lepromatous leprosy. How could unable to lyse the organisms and became such an antibody develop? Such an antibody transformed into typical lepra cells. Their would be an "autoantibody". Autoantibodies of cytoplasm was found to contain numerous a wide variety have been found in patients with bacilli and droplets of lipid material which lepromatous leprosy; they include the rheuma­ stained readily with Sudan Black. A similar toid factor, thyroglobulin autoantibodies, anti­ study was reported by Hanks (1947 , pp. 21, 31, nuclear factor and the Wassermann antibody 38) who cultured phagocytic cells morpho­ which is an autoantibody directed against heart logically resembling fibroblasts from patients muscle (Bonomo and Dammacco, 1968). The with leprosy. Those from patients with tuber­ presence of these different autoantibodies may culoid leprosy were able to lyse the organisms, be due to the exposure of hidden antigenic whereas the cells from patients with lepromatous groups as a result of tissue damage caused by the leprosy degenerated and released the organisms presence of such large amounts of micro­ free into the medium. Although unable to lyse organisms throughout the body. A similar wide Myco. leprae, cultured macrophages from spectrum of autoantibodies is found in a number patients with lepromatous leprosy have been of other chronic infective diseases such as reported to be able to lyse Myco. lepraemurium tuberculosis and syphilis. In experimental and Myco. tuberculosis (Beiguelman, 1967). animals the production of autoantibodies can The inhibition of migration of macrophages be stimulated by the injection of an emulsion of in vitro in the presence of sensitized lymphocytes homologous tissues in oil containing myco­ and antigen is now a technique regularly used bacteria, thus reproducing the antigenic stimu­ in the study of cell-mediated immunity. This lus which the body receives from its own tissues technique, developed originally in experimental in certain chronic infective diseases. animals by Rich and Lewis in 1932 using tissue Thus an important step forward in under­ explants, has been adapted to free-cell sus­ standing the mechanism of the loss of cell­ pensions by George and Vaughan (1962). More mediated immunity in patients with lepromatous recently, methods have been described where leprosy will be the characterization of the factor this technique can be adapted for use with which inhibits the transformation of lympho­ human tissues. In one of these methods (Thor. cytes by SLO in the plasma of some of these 1967; Thor and Dray, 1967) human lymph-node patients. cells are cultured as a free suspension for 72 hI' 218 J. L. 'Turk prior to being placed in the capillary tubes. difficult and involves cultivation of the lympho­ The lymph-node cells then grow out in a fan­ cytes prior to the release of the factor; being a like manner, similar to that seen in cultures of 2-stage cultivation procedure, it is not an easy guinea-pig macrophages. In the presence of technique but could be adapted to leprosy specific antigen they are inhibited from mi­ research under carefully controlled experimental grating. conditions. Another modification is that described by Extensive research on cell-mediated immunity Soberg and Bendixen (1967). In this modifi­ in vitro is thus recommended along 2 lines. The cation, peripheral blood leucocytes are used in first, using the leucocyte-inhibition technique of the capillary tubes and inhibition of the Soberg and Bendixen (1967) or Thor et al. (1968), migration by specific antigen. Both adaptations should give information on the presence of of the leucocyte migration technique appear to sensitized lymphocytes capable of secreting a correlate well with the presence of delayed migration inhibition factor after reaction with hypersensitivity. The first technique has the Myco. leprae or soluble antigens derived from disadvantage that it uses lymph-node cells and the organisms. This substance can be presumed needs biopsy, whereas the second technique uses to act on leucocyte cell surfaces causing in­ peripheral leucocytes. Moreover, the second creased stickiness of these cells. The second technique has been performed with particulate approach should be an extension of the work of organisms, especially Brucella abortus. The Beiguelman (1967) to investigate what it is that development of the cells' ability to be inhibited causes the macrophages of tuberculoid leprosy from migrating in the presence of specific patients to lyse Myco. leprae after phagocytosis, antigen correlates well with the known course in whereas the macrophages from lepromatous time of the development of cellular, rather than patients cannot lyse the organisms. It could humoral, immunity (Soberg, 1968). perhaps be shown that there was inhibition of The technique of Soberg and Bendixen (1967) leucocyte migration by Myco. leprae using the lends itself as an in vitro approach which could blood from tuberculoid leprosy patients and be used to investigate cellular immunity against that Myco. leprae fails to inhibit the migration of Myco. leprae in humans. The antigen could be leucocytes from lepromatous leprosy patients. killed Myco. leprae, similar to that used by Moreover, a soluble substance could be obtained Beiguelman (1967) to investigate macrophage from the culture supernatants of leucocytes fr om activity. The technique of Thor (1967) is more tuberculoid leprosy patients with Myco. leprae, difficult in that it uses lymph-node cells which which might be able to stimulate the macro­ are more difficult to obtain than peripheral phages from patients with lepromatous leprosy blood leucocytes and also entails a 72 hI' culture to lyse Myco. leprae. of the cells as a free-cell suspension, with the associated risk of any long-term tissue culture. THE HISTOLOG ICAL APPEARANCE OF In further experiments, Thor et al. (1968) LYMPHOID TISSUE FROM PAT IENTS WITH incubated peripheral blood lymphocytes with LEPROSY soluble antigen for 24 to 48 hr, then added the supernatant to a suspension of guinea-pig As has been mentioned above, the lymph nodes peritoneal macrophages in capillary tubes. The from experimental animals treated in various incubation of specifically sensitized human ways to suppress cell-mediated immunity show lymphocytes with the specific antigen causes a particular histological appearance. Neonatal the release of a migration inhibition factor thymectomy or treatment with antilymphocyte similar to that described by Bennett and Bloom serum causes a marked depletion of lymphocytes (1968) which inhibits the migration of the from the paracortical areas of the lymph nodes guinea-pig macrophages. This technique, al­ and these cells are replaced by pale-staining though also using peripheral blood cells, is reticulo-histiocytes. The germinal centres and Cell-mediated Immunological PTOcesses in LepTOsy 219 their marginal cuff of small lymphocytes are of cell-mediated immunity) there was evidence unaffected by this pl'ocess, as also are the of the beginning of repopulation of the para­ plasma cells at the cortico-medullary junction cortical areas with small lymphocytes. SOIDe of and in the medullary cords. A similar picture is the capillaries in the paracortical area were found in the lymphoid tissue of children with dilated and were surrounded by a cuff of small congenital thymic aplasia or with the Wiskott­ lymphocytes. At the tuberculoid end of the Aldrich syndrome which is also associated with spectrum a completely different picture is seen. a failure on the part of the patient to show The paracortical areas are well developed and cell-mediated immune reponses. However, packed full of small lymphocytes; an occasional lymph follicles, germinal centre formation and immunoblast can also be found. Germinal plasma cell proliferation in the medulla, which centres are not seen in the epitrochlear nodes and are associated with humoral antibody formation only an occasional plasma cell is seen. This is are unaffected by this process. consistent with the observation of Hanks (1961) In a recent study, a sirr�ilar histological that no significant levels of antibody are found pattern has been found in the lymph nodes of in those forms of the disease (tuberculoid) that patients with lepromatous leprosy (Turk and are characterized by very small numbers of Waters, 1968). The lymphocytes in the para­ bacilli, strong lepromin reactions and a frank cortical area of the lymph nodes were almost tendency to self-healing. On the other hand, completely replaced by reticulo-histiocytes. patients with lepromatous leprosy, who have no These cells were morphologically similar to the immunity to infection, possess large amounts of cells seen replacing the lymphocytes in the circulating antibody to mycobacterial antigens. paracortical area of lymph nodes from the A similar finding was made by Rees et al. (1965) guinea-pigs treated with anti-lymphocyte serum who found precipitating antibody in the sera (Turk and Willoughby, 1967). These cells also of all patients with lepromatous leprosy directed had striking phagocytic activity and in ad­ against a number of different mycobacterial vanced cases they were seen to have ingested antigens and crude homogenates of leprosy large numbers of Myco. leprae. However, this tissue. In contrast, antibodies directed against appears to be a secondary phenomenon as in mycobacterial antigens were only rarely found cases that were nearer the borderline part of the in the sera of patients at the tuberculoid end of spectrum, often there were no bacilli within the leprosy spectrum. these cells. The infiltration was confined ex­ clusively to the paracortical areas of the lymph ERYTHEMA NODOSU M LEPROSUM nodes and can be considered as due to drainage Although the antibodies against mycobacterial of these cells from areas of histiocytic infiltration antigens do not contribute to the elimination of in the periphery. The germinal centres were the mycobacteria, they are in a position to normal and their marginal zone of small 1ympho­ react with mycobacterial antigens in vivo. It is cytes was not decreased. The medullary cords of probable that the interactions between these the lymph nodes were unaffected and contained antibodies and mycobacterial antigens exposed large numbers of plasma cells . In bo rderline as a result of chemotherapy underlie the leprosy there was some infiltration of the condition known as erythema nodo sum lepro­ paracortical areas with these histiocytic cells sum. This condition takes the form of an acute but there were considerable numbers of small vasculitis in the skin associated with poly­ lymphocytes still present. In one case, where morphonuclear leucocytic infiltration. It may be the biopsy specimen had been taken after associated with iridocyclitis, arthritis and even treatment for 7 months (during which the glomerulonephritis. This symptom complex is patient had moved clinically from the leprom­ reminiscent of that found in serum sickness and atous end of the spectrum to borderline leprosy, is then associated with the circulation of indicating that he had regained a certain degree antigen-antibody complexes formed in slight 220 J. L. Turk

antigen excess which circulate and become fection in mice following a combination of deposited in small blood vessels, producing in adolescent thymectomy and deep X-irradiation. the skin lesions which resemble the Arthus However, it is more likely that this is due to phenomenon. There is no doubt that the skin drainage of histiocytes from peripheral lesions. lesions of erythema nodosum leprosum have Such cells passing down the afferent lymphatics many features in common with the Arthus will tend to accumulate in the paracortical reaction as seen in experimental animals and areas. Although the appearance of lymph nodes may even have a haemorrhagic centre. resembles those seen under conditions where Erythema no do sum leprosum always occurs there is thymus deficiency, there is no evidence in that part of the leprosy spectrum where that this plays any role in these appearances cell·mediated immune processes against the in leprosy. There is a suggestion from the mycobacteria are absent or minimal and under work of BuJlock and Fasal (1968) that an conditions where there are large amounts of anti-lymphocytic antibody could be present in circulating antibodies against mycobacterial the plasma of such patients. Moreover, Gaugas antigens, so high that they can be detected by (1968) has found that a combination of thy­ techniques as crude as precipitation in agar mectomy at 6 weeks of age and treatment with (Rees et al., 1965). antilymphocytic antibody will allow Myco. leprae to proliferate to the extent of reproducing many of the features of lepromatous leprosy. As POSSIBLE CAUSES OF DEFICIE NCES OF has been discussed above, such an antilympho­ CELL-MEDIATED IMMU NITY IN LEPROSY cytic antibody could be developed as an auto­ Depression of cell· mediated immunity in leprom­ immune phenomenon as a result of extensive atous leprosy is probably due to an interaction tissue damage during infection with Myco. of a number of different factors and cannot be leprae. attributed to a single mechanism. Another non-specific factor that could be In the first place, there is no doubt that in relevant to the depression of cell-mediated patients with lepromatous leprosy a funda­ immunity in leprosy is that the ability to mental constitutional defect contributes con­ develop delayed hypersensitivity is suppressed siderably to the eventual deficiency. This is in guinea-pigs pretreated with Freund's adjuvant indicated by the observation that lepromatous (Jankovic, 1962). The infiltration of tissues with disease is a host-determined characteristic that Freund's adjuvant containing mycobacteria, is possessed by a fixed proportion of all people might produce conditions similar to those found everywhere (Newell, 1966). This observation is in leprosy. Guinea-pigs treated in this way are supported by the finding that the children of not ouly not able to produce cell-med.iated parents with leprosy showed a marked re­ immune reactions when actively stimulated with duction in their ability to become converted to antigen but also have a reduced ability to tuberculin positivity when vaccinated with vole manifest passively transferred reactivity (Asher­ tuberculosis vaccine, as compared with normal son and Allwood, 1969). chilch·en (Jamison and Vollum, 1968). All these factors will contribute to a non­ The appearance of lymph nodes from patients specific depression of cell-mediated immunity. with lepromatous leprosy is very similar to that However, except in the polar form of leprom­ seen in children with congenital thymic aplasia atous leprosy, the inability to show cell­ (Turk and Waters, 1968) and suggests that the mediated immunity is specific to Myco. leprae defect might be due to an inherent deficiency in and a non-specific depression does not occur in thymus control of cell-mediated immunity. The other parts of the spectrum where bacillary suggestion is supported by the observation of growth may be little controlled. Thus a specific Rees et al. (1967) that a disease resembling immunological tolerance must play a certain lepromatous leprosy can be produced by in- role in the ability to respond to the organism. Cell-mediated Immunological Processes in Leprosy 221

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