HYPERSENSITIVITY REACTIONS OF. GUINEA PIGS INFECTED WITH I . I ' . I _ . Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY RONALD C.. GOREWIT 1971 Thth~ ; LIBRARY amnens ,; llllll !.!-r.'mc.'s.'! 4 WI ‘5 " ‘ -~-___4_~ H HYPERSENSITIVITY REACTIONS OF GUINEA PIGS INFECTED WITH Leishmania donovani By. .<% Ronald CE Gorewit A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1971 To My Beloved Sister Melissa Jo Gorewit ii ACKNOWLEDGEMENTS The author wishes to express his appreciation to Dr. D.W. Twohy for the use of his experimental resources, his criticism and his guidance during this investigation. I wish to express my gratitude to Drs. R.M. Corwin, W.D. Collings and D.E. Schoenhard for their helpful suggestions and evaluation of this thesis. I especially want to acknowledge my sincere gratitude to my parents, Mr. and Mrs. H. Gorewit, for their under- standing, encouragement and guidance throughout my academic work. iii TABLE OF CONTENTS DEDICATION . . . . . . . . . . . . ACKNOWLEDGEMENTS . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . INTRODUCTION . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . In Vivo Manifestations of Delayed-type Hypersensitivity . In Vitro Reactions Correlated with Delayed- Eype Hypersensitivity . . . . ; Antimicrobial Cellular Immunity and Delayed- type Hypersensitivity . . Delayed- type Hypersensitivity to Leishmanial Antigens . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . Experimental Hosts and Parasites . . Immunization and Sensitization of Guinea Pigs . . . . Skin Testing of the Guinea Pigs , , Collection of Serum . . . . . . Serological Tests . . Passive Cutaneous Anaphylaxis (PCA) . Macrophage Migration-inhibition Tests Statistical Considerations . . . . O O O O . O I RESULTS . . . . . . . . . . . . . Intradermal Tests for Hypersensitivity . Studies on the Induction Period, Intensity, and Duration of Skin Test Hypersensitivity in Guinea Pigs Infected with L. donovani Serological Tests . . . . . . . . iv Page ii iii vi vii 13 15 24 2L!- 25 26 27 28 29 30 32 33 33 39 43 Page Passive Cutaneous Anaphylaxis . . . . . #3 Tests for the Inhibition of MacrOphage Migration . . . . . . . . . . . 48 DISCUSSION . . . . . . . . . . . . . 52 SUMMARY 0 O O O O O O O C O O O C C O 62 BIBLIOGRAPHY . . . . . . . . . . . . . 65 Table 1. LIST OF TABLES Titers for precipitin tests 48 hours after addition of antisera . . . . Results of passive cutaneous anaphylaxis reactions 8 hours after intracardiac injection of antigens . . . . . Results of passive cutaneous anaphylaxis reactions 8 hours after intracardiac injection of antigens (HKMLD's used in place of HKHLD's) . . . . . . Percent inhibition of migration for peritoneal exudate cells in the presence and absence of test antigens vi Page 1+4 46 47 50 LIST OF FIGURES Figure Page 1. The diameter of intradermal skin reactions to antigens in guinea pigs infected with L. donovani . . . . . . . . . 35 2. A comparison of intradermal skin test reactions of guinea pigs super infected with L. donovani with animals receiving a single infecEion . . . . . . . 38 3. The development of skin sensitivity in guinea pigs infected with L. donovani. #2 vii INTRODUCTION Leishmania donovani is a parasitic protozoan which causes visceral leishmaniasis or kala-azar, a disease that is usually fatal in untreated human cases. The disease is endemic in certain areas of Africa, Asia, the Middle East and South America. The vector which transmits Leishmania donovani in- fection is the sandfly (Phlebotomus). When feeding on an infected vertebrate, the fly ingests aflagellated, intra- cellular forms called Leishman-Donovan (LD) bodies or ama- stigotes. The LD bodies deve10p into small elongate fla- gellated forms in the gut of the sandfly, which are known as leptomonads or promastigotes. After a period of time the leptomonads migrate anteriorly to the pharynx of the fly where they are in a position to be inoculated into the skin of the next host with the bite of the insect. In the vertebrate host, the leptomonads establish themselves as intracellular LD bodies after ingestion by macrophages. Eventually the parasites become distributed in monocytes and tissue histiocytes throughout the body. Humans and animals recovering from most forms of leishmaniasis have long-lasting resistance to reinfection. As in most infections specific antibodies are produced by the host to leishmanial parasites, but these antibodies are unable to protect the host from infection. Recent experi- ments carried out by Bray and Bryceson (1968) and Bryceson g: 3;. (1970 a,b) with L. enriettii and Miller and Twohy (1969) with L. donovani have suggested cellular immunity as the primary mechanism of resistance to the respective para- sites. Acquired cellular immunity is defined as that immunity which is mediated by cells rather than humoral factors. Under apprOpriate conditions such immunity can be trans- ferred with living cells, but not with serum. Delayed-type hypersensitivity, a slow developing inflammatory immunologi- cal response dependent upon sensitized cells rather than serum antibodies, is thought to be closely associated with cell mediated acquired resistance. The objective of this work was first to determine if guinea pigs infected with Leishmania donovani deVelop a hypersensitivity response to antigens from this parasite. After a hypersensitivity was demonstrated, an attempt was made to establish the time after infection when the maximum reaction occurred and to ascertain what portion of the reaction was a delayed-type reaction, mediated by cellular components. It is hoped that future work will show the _ relationship of delayed-type hypersensitivity to host im- munity to Leishmania donovani. LITERATURE REVIEW Zinsser (1921) is generally credited with the dis- covery that two fundamentally different types of intra- dermal reactions occur after sensitization with tubercle bacillus extracts: an immediate and a delayed reaction. Zinsser found that the immediate reaction was a transitory reaction which developed rapidly in animals sensitized against proteins such as horse serum etc. and was thought to be one of the manifestations of general protein hyper- sensitivity or anaphylaxis. The other type of intradermal reaction was the tuberculin type of skin reaction which developed more slowly, resulted in a more profound injury of tissues and was independent of the anaphylaxis response. Dienes and Mallory (1932) showed that the delayed- type hypersensitive response became apparent quite quickly after immunization and before humoral antibody could be detected in the host. They failed to passively transfer the reaction to normal hosts with serum from sensitized hosts. These authors found that delayed sensitivity could also be produced to simple proteins, such as ovalbumin, when these antigens were injected into‘tuberculous lymph nodes or testicles of infected animals. 3 4 Raffel (1948) and Freund (1956) found that if simple antigens were injected with the adjuvant-wax fraction of the tubercle bacillus (the esters of polysaccharide and higher alcohols with hydroxy fatty acids) or oil and water emulsions, animals developed delayed-type hypersensitive reactions to the former substances. Even though the exact mechanism of action of these adjuvants is still unknown they are still used to produce strong delayed-type hyper- sensitive responses. £2.1i12 Manifestations g£_Delayed-t e Hypersensitivity. The two types of hypersensitivity reactions can over- lap at a reaction site making it difficult to define a pure reaction based on the histoldgical appearance of lesions. Uhr 22.2l' (1957), Salvin (1958) and Cell and Benacerraf (1959) helped to differentiate between the hypersensitivity reactions by combining certain methods of immunization to elicit pure delayed reactions in guinea pigs. These methods included immunization with antigen-antibody complexes, using small amounts of antigen; employing modified antigens upon immunization; or testing the animals early in immuni- zation with microgram amounts of antigen. The reaction appeared between 4 to6 hours after antigen injection as a red indurated area which increased to maximum size and in- tensity by 18 to 24 hours. The site of reaction was flat when readily diffusable antigens were used or elevated and indurated when less diffusable antigens were employed such as, certain gamma globulins or dinitrophenylated poly-l-lysine. 5 Dienes and Mallory (1932) described the characteristic histological differences between immediate and delayed- type hypersensitivity reactions. During the first phase of the delayed-type hypersensitive reaction inflammatory edema occurred with an infiltration of polymorphonuclear IeuCocytes. The polymorphonuclear'leuCocytes were later replaced by large numbers of mononuclear'Cells. waksman (1960) described the invasive, destructive character of the delayed-type-hypersensitive lesion elicited by painting a hapten on the skin of sensitized animals. There was a destruction of the epidermal cells in contact with the in- vading mononuclear cells. Flax and Caulfied (1963) demon- strated that increased vascular permeability was associated with the delayed-type hypersensitivity reaction by showing the accumulation of intravenously injected colloidal carbon and trypan blue in the lesion. The exact type of mononuclear cells infiltrating delayed-type hypersensitivity lesions has been disputed. Studies on classical delayed hypersensitivity reactions, allograft rejection, allergic encephalomyelitis and aller- gic neuritis have indicated that the predominant cells are either lymphocytes or cells of the monocyte-macrophage series. Flax and Caulfield‘ (1963.), Wiener 52:0. 31. (1967) and Turk 23 3;. (1966 a,b) indicated that both cell types were clearly present. However, cell identification was complicated by transfbrmation of the lymphocytes. Pearmain 22.2l. (1963) established that specifically sensitized lymphocytes can transform into blast cells by exposure to antigen, in_zi££g, and Howard 22.2l0 (1966) believed that cells with the appearance of lymphocytes can develop into phagocytic cells. Volkman and Gowans (1965) and Van Furth and Cohn (1968) have shown that blood monocytes originate from rapidly dividing precursors in the bone marrow and that tissue macrophages (histiocytes) deve10p from blood monocytes. It appears that mononuclear cells with different morphological appearances can be present in delayed-type reactions and that the morphological appearance of these cells is not incontrovertible evidence of their function and origin (Howard 23.2lo 1966). Our knowledge of the cellular basis of delayed-type hypersensitivity has advanced rapidly since Landsteiner and Chase (19425 and Chase (19A5) first transferred tuber- culin delayed-type hypersensitivity to normal guinea pigs with peritoneal exudate cells, circulating leucocytes and lymphoid cells from sensitized animals. It was thought, until recently, that the transferred lymphocytes comprised most of the infiltrating cells in the inflammatory reactions. But McCluskey gt 2;, (1963) and Turk and Ort (1963) have shown, in transfer studies using H3 thymidine labeled donor and recipient guinea pig lymph node cells and spleen cells, that the great majority of the cells that make up the infiltrate in a delayed-type hypersensitivity reaction are not specifically donor cells by origin. Almost all of the cells in the lesions of delayed sensitivity were recently proliferated host cells. In most experiments, the specific accumulation of passively transferred sensi- tized cells could not be demonstrated at the reaction site, and in contrast 80 to 90 percent of the inflammatory exu- date cells were labeled if HB-thymidine was administered to the recipient animal instead of the donor animal. Lubaroff and waksman (1967) have shown that the macro- phage infiltrate is "hematogenous" and originates mostly from rapidly dividing bone marrow precursors of blood mono- cytes. Coe 23.2l0 (1966) have shown that these cells are necessary for the pathogenesis of the delayed-type hyper- sensitivity reaction because irradiation of the recipient animal previous to the passive transfer of sensitized cells renders it unable to exhibit the delayed-type hypersensiti- vity reaction. Presumably irradiation destroys the bone marrow precursor of the blood monocytes which are believed to be the effector cells of the delayed-type hypersensiti- vity reactions. There is very good evidence that actively sensitized lymphocytes carry the.immunological specificity and that their reaction with antigen in some way initiates or eli- cits the inflammatory reaction characteristic of delayed- type hypersensitive reactions in which the macrophages participate (waksman 22.2l' 1961; WOodruff and Anderson, 1964; Coe gt'gl. 1966; and Brent and Medawar, 1967). In litrgReactions Correlated flith_Delayedetypg,Hypg_- sensitivity. Various in_1i£rg manifestations are believed to be related to the delayed-type hypersensitivity response. Sensitized lymphocytes in response to antigen 1) undergo multiplication and blast transformation, 2) inhibit macro- phage migration and 3) destroy target cells. Pearmain gt al. (1963) has shown that lymphocytes from the peripheral blood, peritoneal exudate or lymph nodes of sensitized animals react to antigens, in'zitrg, by trans- formation to blast cells. Dutton and Eady (196A) and Paul gt‘al. (1968) have shown increased 32.13239 DNA synthesis in lymphocytes from host animals after antigen stimulation by incorporation studies with H3-thymidine. These experi- ments suggest that transformation and multiplication in vitrg by sensitized lymphocytes corresponds to the initial steps in delayed-type hypersensitive reactions. Pearsall and Weiser (1970) believe that the reasons for concluding that blast transformation reflects delayed sensitivity are as follows: The specificity of lymphocyte blast trans- formation with antigen parallels other delayed sensitivity reactions. In the course of sensitization, lymphocyte blast transformation can be shown before a positive skin reaction occurs. There have been many demonstrations of cell associated immune reactions closely linked to delayed-type hypersensiti- vity which involve the activity of both lymphocytes and macrophages. Only the more significant work is reviewed here. Rich and Lewis (1932) first reported that the migra- tion of sensitized mononuclear cell populations from ex- plants of guinea pig spleen and lymph nodes in culture were inhibited in the presence of the specific sensitizing antigen. Gangarosa Eiréi‘ (1955) pointed out that addition of PPD to spleen, lymph node and tonsil cultures from humans with delayed sensitivity to tuberculin had an effect on migration of macrophages. A better in,zi££g test for the inhibition of macro- phage migration was devised by George and Vaughn (1962) who determined the extent of migration of macrophages from capillary tubes. Peritoneal exudate cells from guinea pigs sensitized to PPD failed to migrate in the presence of PPD. Spleen cells showed no consistent response to the specific antigen. The results of Aronson (1931), Moen gt al_.. (1936), George and Vaughn (1962), Carpenter (1963), Heil- man §t_al. (1960), and David 23.3l' (1964 a,b) have demon- strated that the inhibition of migration is very specific for the appropriate antigen. David gt El. (1964a) showed that highly purified proteins such as purified protein derivative (PPD), ovalbumin, and diphtheria toxoid inhibited cells from specifically sensitized animals in minute concentrations and did not affect cells from normal animals when greater concentrations were used. The migration of peritoneal exudate cells from animals with delayed-type hypersensitivity 10 to ovalbumin was inhibited by ovalbumin. The effect was specific, since cells from the same animal were not inhi- bited by diphtheria toxoid. Cells from animals with de- layed-type hypersensitivity to diphtheria toxoid were inhibited by toxoid, but the cells from these animals migrated normally in medium containing the unrelated anti- gen ovalbumin. David 23.2l' (1964b) have also shown that when peritoneal exudate cells from guinea pigs exhibiting delayed hypersensitivity to tuberculin and normal animals were mixed in varying concentrations, only a few cells in the mixed population needed to be specifically sensitive to influence the behavior of the entire population. Bennett and Bloom (1968) have shown that cultures of sensitized lymphocytes in the presence of antigen are capable of synthesizing a factor or factors which inhibit the migra- tion of normal macrophages1 Lymphocytes from guinea pigs with a delayed-type hypersensitivity to old tuberculin which were exposed to PPD, in 31333, produced a substance which inhibited the migration of normal peritoneal macro- phages. The migration-inhibition factor (MIF) seemed to be distinct from antibody or antigen. The activity was thought to reside in a protein fraction with a molecular weight of about 65,000. The factor was nondialyzable, tryp- sin-sensitive and stable to heating at 560C for 30 minutes. When this fraction was injected into the skin of normal guinea pigs, reactions characterized by induration, erythe- ma, and mononuclear cell infiltration were produced. It 11 was proposed that in migration inhibition reactions sensi- tized lymphocytes, on exposure to antigen, produce MIF which in turn inhibits the migration of macrophages. Amos'gt‘al. (1967) has challenged the conclusion that macrophage migration inhibition is a reliable indicator of delayed-type sensitivity. They have reported that cytophi- lic antibodies from serum could passively sensitize normal macrophages to PPD and cause an inhibition of migration of macrophages. These results have been confirmed by Heise and Weiser (1969). It has been suggested by Amos and co- workers that not all of the mechanisms found to be respon- sible for migration-inhibition are associated with delayed hypersensitivity and that the migration inhibition test is not a measure of the degree of delayed sensitivity. Two mechanisms may contribute to the inhibition of migration of macrOphages in the tuberculin system. One may be MIF and the other may be the reaction of antigen with cyto- philic antibody on the surface of sensitized macrophages. Weiser gt'al.(l969) have shown that when cytophilic anti- bodies on sensitive macrophages in purified preparations were removed by trypsin, the cells were no longer sensitive to the macrophage migration-inhibition test. Suscepta- bility to the test was restored by exposing the trypsinized macrOphages to immune serum or to cytophilic antibodies eluted from sensitized macrophages by heat treatment at 56°C for 30 minutes. 12 Thor and Dray (1968) reported that normal human lymph node cells could be sensitized to the.macrophage migration- inhibition test with an RNA extract of human lymph node cells from tuberculin sensitive donors. These authors found the active factor in the extract to be between 4s and 28s to be RNAase-sensitive, and to contain a small amount of protein. Jureziz‘gtlal. (1968) reproduced these experiments in guinea pigs by transferring sensitivity to PPD and coccidioidin with RNA extracts obtained from guinea pig lymph nodes and spleen. To this date the cause(s) of migration inhibition remains an open question. Sensitized lymphocytes have been shown to have cyto- toxic effects in culture on normal and neoplastic types of target cells by Rosenau and Moon (1961), Wilson (1965), Brunner 23 EL (1968) and Govaerts (1960). Specific target cells in culture were killed by sensitized lymphocytes whose specific cytotoxic effect seemed to depend upon an immunoglobin receptor which caused them to adhere to tar- get cells. This reaction did not require complement. Wilson (1965) and Brunner gt 21. (1968) have shown that the cytotoxic effect of the immune lymphoid cells could be inhibited by treatment of the target cells with isoanti- serum from skin graft recipients. Brunner ethal. (1968) also showed that the cytotoxic effect could be inhibited by the isoantiserum 19s and 73 fractions and could be partially inhibited by actinomycin-D and by cyclohexamide at concentrations which effectively blocked DNA-dependent 13 RNA and protein synthesis. The isoantiserum is thought to cover or bind to the antigenic sites on the target cells and does not allow the cytotoxic reaction to occur. The exact mechanism of the cytotoxic effect of sensitized lymphoid cells for target cells remains unknown. The cytotoxic effect of lymphocytes for target cells seems to be stimulated by their contact with antigen on the surface of the target cell. Holm and Perlman (1967) have shown that cytotoxicity of nonsensitized lymphocytes for cells in culture can result from stimulation by non- specific mitogenic agents. The only prerequisite for cyto- toxicity of lymphocytes seemed to be the initiation of blast transformation and the close contact with the target cell (Benacerraf and Green, 1969). Current ideas on lympho- cyte cytotoxicity are reviewed by Granger gi‘gl. (1969). It is not known whether the synthesis and release of factors which cause the inflammatory reactions and affect macrophage functions (MIF) are distinct from those which produce cytotoxic effects. Antimicrobial Cellular Immunity_anQDelayed¢typg Hypersensitivity. Cellular immunity can be defined as that immunity which results from the activities of cells rather than humoral antibodies and which can be transferred with cells, but not with serum (Pearsall and Weiser, 1970). Antimicro- bial cellular immunity to a variety of intracellular bacteria and protozoa is dependent upon sensitized 14 lymphocytes and activated macr0phages and is usually elicited only by infection and not by immunization with dead cells or dead cell components (Suter, 1953; Elberg, 1960; Suter and Ramseier, 196A; Mackaness and Blanden, 1967; and Pearsall and Weiser, 1970). Antimicrobial cellu- lar immunity has been described for many intracellular parasites, including L, donovani (Mackaness and Blanden, 1967; Turk, 1967; Dannenberg, 1968; Pearsall and Weiser, 1970; and Miller and Twohy, 1969). In most infections where antimicrobial cellular immunity predominates, delayed-type hypersensitivity has also been demonstrated. It is believed that the state of delayed-type hypersensitivity may be an essential part of the cellular immune response (Mackaness and Blanden, 1967; Dannenberg, 1968; and Pearsall and Weiser, 1970). Although it seems clear that antimicrobial cellular immunity to infectious agents requires the prior sensiti- zation of the host, delayed-type hypersensitivity alone does not produce antimicrobial resistance. 'Antimicrobial resistance can occur only in the presence of sustained antigenic stimulation (Mackaness and Blanden, 1967). A brief review of two intracellular infections will be given to illustrate the points listed above. Mackaness (1962 and 1968) reported that within A days after infection, Listeria monocytogenes could incite an immunity in mice capable of protecting them against 10h 15 lethal doses of Listeria. This immunity was passively transferred with lymphoid cells, but not with serum (Miki and Mackaness, 1964) and was accompanied by a state of delayed—type hypersensitivity to the organisms (Mackaness, 1962). Mackaness (1964a)has found that delayed-type hyper- sensitivity appeared by the fourth day in mice infected with Brucella abortus, however the bacterial population continued to increase for 8 days post infection. After this 8 day period, resiStant macrophages appeared in the peritoneal cavities of the mouse. After the bacterial population had been reduced, host resistance and the number of resistant macrophages decreased, although the level of delayed-type hypersensitivity increased. The fact that specific immunity can be transferred with purified preparations of immune lymphocytes free of phagocytic cells given intravenously (Mackaness, 1968) indicates that lymphocytes play a role in cellular immunity and contribute to the formation of macrophages which destroy parasites. Since the phenomena of delayed-type hypersensitivity and cellular immunity are tested under completely different conditions, their interrelationship remains largely specu- lative. Delayed-t e Hypersensitivity £2 Leishmanial Antigens. Early experimental work on the sensitization and latter establishment of delayed-type cutaneous reactions to leish- manial antigens has been reviewed by Di Cristina and Caronia 16 (1914), Laveran (1916) and Rotberg (1952). Tests for delayed-type reactions that resembled the cutaneous tuberculin reaction were first carried out by Wagener (1923). Rabbits immunized with Leishmania tropica and Leishmania infantum developed delayed-type hypersensi- tivity reactions to skin test extracts of the corresponding leishmanial species. The reaction was characterized by an erythematous papule which reached its peak in 48 hours and persisted 72 hours to 5 days. The injection of a concentrated extract of either L. tr0pica or L, infantum produced necrosis and sloughing at the skin test site in animals previously immunized to either Leishmanial species. Rabbits immunized to one species of Leishmania were sensi- tive to the protein of both species. However, L. tr0pica produced the most pronounced reaction even in animals immunized to L. infantum. Wagener and Koch (1926) demon- strated delayed-type cutaneous reactions to L. tro ica, L. donovani, L. brasiliensis and L. infantum in rabbits and guinea pigs immunized with the respective leishmanial species. Jessner and Amster (1925) found that dogs infected with Leishmania tropica gave delayed-type hypersensitive reactions upon intradermal vaccination with cultural forms of L. tropica. Montenegro (1926) produced delayed-type cutaneous reactions (Montenegro reaction) that were similar to that of the tuberculin reaction in patients with mucocutaneous leishmaniasis which were skin tested with culture extracts 17 of L. brasiliensis and L. tropica. Marzinowsky (1928) observed an inflammatory response upon injection of live L. tropica in patients recovering from oriental sore. The erythema and edema persisted for 5 days. Buss (1929) demonstrated delayed skin reactions with phenolized sus- pensions of L. brasiliensis and L.tropica in patients with South American cutaneous leishmaniasis. The delayed skin reaction was characterized by a papule which appeared 48 hours and lasted from 4 to 5 days. Dostrovsky (193A and 1935), Dostrovsky and Sagher (19A6), Mayer and Malamos (1936) and Malamos(1937) used the delayed skin reaction (Leishman reaction) to diagnose human cutaneous leishman- iasis. Phenolized flagellates of L. tropica were used as the antigen. Senekji (l9Al) produced allergic reactions after skin test in patients with oriental sore. The reac- tions peaked 24 hours after the intradermal injection of anIL. tropica polysaccaride antigen. Senekji and Beattie (19A1) isolated both a polysaccharide and a protein frac- tion from L. tropica. Reactions against the polysaccharide fraction were less intense than reactions against the pro- tein fraction. Adler (1965) obtained delayed-type skin reactions in sensitive human volunteers from leishmanial exoantigens which were prepared from the filtered supernatants of L. tropica and L. mexicana cultures, from a saline extract of a hamster spleen infected with L. mexicana and from sera of hamsters infected with L. mexicana. Adler and l8 Gunders (1964) observed delayed cutaneous reactions at skin test sites injected with phenolized preparations of L. mexicana and L. tropica in a human volunteer that was previously infected with L. mexicana. Experiments by Glazunova (1966) showed evidence of local allergic reaction in the form of cellular infiltra- tion, induration and edema in guinea pigs after repeated injections of L. enriettii in the skin at various inter- vals after recovery from infection with this parasite. Adler and Halff (1955) were unable to produce delayed reactions to live parasites in guinea pigs infected with L. enriettii when skin tested with promastigotes of this parasite. Bray and Bryceson (1968) and Bryceson 23.3L. (1970a,b) have reported a delayed-type hypersensitive response occurring in guinea pigs infected with L. enriet- LLL'when skin tested with whole parasites or to antigenic extracts of the parasite. Bosyia (1967) obtained typical delayed-type hypersensitive reactions to various extracts of leishmanial species in SensitiZed guinea pigs. Inoculation of man with the relatively non-pathogenic organism called L. adleri led to the development of a typical leishmanin reaction upon skin testing with antigens from homologous and some heterologous species of Leishmania (Manson-Bahr and Southgate, 1964; Southgate, 1967). Adler and Nelken (1965) injected leucocytes and whole blood, obtained from a leishmanin positive donor which had recovered from oriental sore into normal pe0ple and could 19 not elicit a delayed-type reaction upon skin tests with phenolized promastigotes of L. troEica. ‘When Bray and Lainson (1965) injected leucocytes from humans and monkeys recovered from infections with L. mexicana and L. brasili- ensis into men, monkeys, rabbits, and guinea pigs, they failed to produce delayed cutaneous reactions with skin tests. Boysia (1967) was able to transfer a delayed hy- persensitive response to leishmanial antigens from sensi- tized to normal guinea pigs with lymph node cells. The number of cells injected, the route of injection and the degree of sensitivity in the donor animals seemed to in- fluence the delayed hypersensitive response in recipient animals. Some workers have shown that immediate-type hyper- sensitivity reactions may occur in animals and humans sen- sitized with leishmanial antigens. Berberian (1939) found an immediate-type allergic response in humans recovered from oriental sore after the intradermal injection of viable promastigotes of L. tropica. The immediate reaction lasted from 6 to 12 hours and was followed by a delayed- type reaction. Senekji and Beattle (1941) skin tested people recovering from oriental sore with suspensions of live promastigotes of L. tropica and observed an acute inflammatory response which lasted for 48 hours. Ansari and Mofidi (1950) observed a typical Arthus reaction in two humans recovered from oriental sore when they were skin tested with viable L. tropica promastigotes. The 20 reaction was visible at 4 hours post skin test. Adler and Gunders (1964) described an Arthus-type reaction in a volunteer that had recovered from.oriental sore when skin tested with viable amastigotes of L, mexi- gana. Erythema appeared at 12 hours post skin test. At 24 hours post skin test a "pale halo" surrounded the area of erythema, and at 48 hours the reaction had reached a maximum. Southgate (1967) and Southgate and Manson-Bahr (1967) described an Arthus reaction in humans sensitized with Leishmanial antigens when skin tested with viable L. donovani and L. adleri promastigotes. Dostrovsky and Sagher (1946) passively transferred an allergic reaction using serum from two cases of'oriental sore to four human volunteers. Passive cutaneous anaphylaxis at the site of injection of serum, from leishmanin positive subjects was found by Bray and Lainson (1965). Bray and Bryceson (1968) infected monolayers of guinea pig peritoneal cells with Leishmania enriettii and compared the uptake and growth of the organisms in macrophages from convalescent and from non-infected animals. They found that the macrophages from convalescent animals took up over twice as many organisms as did normal macrophages and that the growth of the parasites was not impared. Lympho- cytes from convalescent animals destroyed all infected macr0phages within 24-48 hours. The lymphocytes from normal animals caused less sloughing of infected macro- phages than sensitized lymphocytes. Bryceson gg‘aL. (1970a) 21 has reviewed the cell-mediated immunity in leishmanial in- fections. Bryceson states that the lymphocyte is the prime mediator of resistance to infection with Leishmania and that the macrOphage simply plays a supportive role for the parasite, providing food and shelter. Bryceson 22.2L. (1970b) studied the development of immunity in guinea pigs after intradermal injection of Leishmania enriettii and the use of 22.1112 and_Lp_y§L£2.techniques to characterize the immunological response to infection and artificial immunization. These authors found that artificial immuni- zation with soluble and insoluble antigenic extracts of .E- enriettii in Freund's complete adjuvant partially pro- tected animals against infection. Extracts of other leish- manial species failed to offer any protection against in- fection. Infection and artificial immunization were accom- panied by delayed hypersensitivity which was passively transferred with lymphoid cells. Cell-mediated immunity was examined $2.2l322 by the ability of soluble leishmanial antigens to cause blast cell transformation, inhibit macro- phage migration and to induce the production of lymphokine factors from lymphocytes from sensitive animals. Target cell destruction was shown where sensitized lymphocytes destroyed monolayers of parasitized macrophages. Cross reactivity of leishmanial antigens with mycobacterial antigens was shown in skin tests and in target cell destruc- tion. . Bryceson 23 filo (1970b) further showed that 22 phagocytic activity of macrophages from recovered animals was increased for homologous species of Leishmania, but not for heterologous species of leishmania. The growth of phagocytized Leishmania was not reduced. Passive cu- taneous anaphylaxis tests showed that circulating antibody to antigen was not present. Agglutination of antigen coated sheep erythrocytes in sera from infected or convales- cent animals showed no presence of antibody. However, some convalescent animals showed active cutaneous anaphylaxis. Antibodies were demonstrated by both of these techniques in immunized animals which showed anaphylactic and Arthus hypersensitivity, when skin tested with soluble antigens. Tremonti and Walton (1970) studied Lnfinggg reactions of cells in humans and guinea pigs infected with Leishmania brasiliensis. Peripheral lymphocytes from patients with V American cutaneous leishmaniasis showed a 2% or greater tranSformation to blast cells in the presence of leishmanin, but no significant transformation occurred in noninfected controls. Inhibition of macrophage migration in the pre- sence of leishmanin was slight in Leishmania-infected guinea pigs and was correlated with a slight skin test reaction to Leishmania brasiliensis antigen. Miller and Twohy (1969) showed that macrophages from superinfected mice displayed a resistance to intracellular forms of L. donovani in culture. The parasites in macrophages from normal mice multiplied for the first 72 hrs, but the parasites from superinfected mice either failed to multiply 23 within the macr0phages or decreased in numbers. When serum from superinfected mice was added to the culture it had no effect on the rate of parasite multiplication either in macrophages from normal mice or on the rate of destruction of parasites in cells from infected mice. The resistance shown by the superinfected macrOphages was thought to be associated with the source of cells. Kelly (1970) was unable to demonstrate the pronounced development of delayed-type hypersenSitivity to L. donovani in the mouse but was able to show destruction of macro- phages from normal mice, $2.2i2229 by spleen and peritoneal lymphocytes collected from mice 4 weeks after infection with L. donovani. MATERIALS AND METHODS EXperimental EQ§E§.§EQ Parasite Female guinea pigs (92132 porcellus) of the Hartley strain were used in most of the experiments in this investi- gation. Animals of similar ages were segregated randomly into experimental and control groups at the start of an experiment. The guineapigs were fed Wayne Guinea Pig Diet. The 5s (Sudan-3) strain of Leishmania donovani that was utilized in this investigation, was obtained from Dr. Leslie Stauber, Department of Zoology, Rutgers University and was prepagated in male Golden Hamsters (Mesocricetus auratus). Hamsters were infected with an intracardial in- jection of 30x106 LD bodies. One to 2 months later the spleen was removed, aseptically, minced up into small pieces with scissors and partially ground in a 15 ml teflon pestle homogenizer with 5 ml of NCTC 155 medium. The sus- pended homogenate was removed and the remaining spleen particles homogenized with succeeding 5 m1 aliquots of medium. All homogenate suspensions were pooled and centri- fuged 5 minutes at 132xg to remove large cell particles. The supernatant containing most of the parasites was 24 25 withdrawn and saved. The button was then resuspended with 10 ml of NCTC 135 medium and the above centrifugation repeated. The two supernatant fractions were pooled and centrifuged at 392xg for 10 minutes. The sediment con- taining the parasites was finally resuspended in 10 to 15 ml of NCTC 135 medium and the parasites counted in a Pet- roff-Hauser counting chamber using phase microscopy. The suspension was adjusted to the desired concentration by diluting the suspension with NCTC 135 medium. Immunization 22$ Sensitization gg'Guinea gigs Guinea pigs were infected by an intraperitoneal in- jection of 30x106 LD bodies of L. donovani prepared from hamsters as described above and suspended in 0.2 ml of medium. A spleen cell homogenate was prepared from normal hamsters by the same technique described for separating LD bodies from spleens of infected animals. A 0.2 m1 volume of the homogenate was injected into the peritoneum of designated controls to immunize against this tissue. Non- immunized controls were given an intraperitoneal injection of 0.2 m1 of NCTC 135 medium. Superinfected guinea pigs were infected with two intra- peritoneal injections of 30x106 LD bodies of L. donovani 8 days apart. Controls for the superinfected animals were sensitized with two intraperitoneal injections of 0.2 m1 of spleen cell homogenate or two intraperitoneal injections of 0.2 m1 of NCTC 135 medium 8 days apart. Other animals were given a single 0.2 m1 injection of LD bodies, hamster 26 spleen homogenate, or NCTC 135 medium. A 2 ml volume of an LD body suspension from hamsters containing 150x106 parasites/ml was heated in a water bath at 560 for 30 minutes and designated as heat-killed para- site antigen (HKHLD's). This antigen was stored in the freezer for future use. (HKMLD's) were also prepared from the spleens of infected C57B1/6J mice. The mice were in- fected with an intravenous injection of 50x106 LD bodies of L. donovani. Four weeks later the spleens were removed and a parasite suspension was prepared by the same technie ques employed for obtaining LD bodies from hamsters. Ex- perimental and control animals were lightly anesthetized with ether for immunization. §EEE Testing 2; 3L2 Guinealglgg All animals to be skin tested were restrained on a small animal platform. The hair was clipped from the ab— dominal area by an electric small animal clipper (Oster Model A2 with clipper size 40). The abdominal skin was first waShed with warm tap water, then with 70% ethyl alco- hol and allowed to dry. A.0.21mlvolume of each antigen was injected with a one-half inch 27 gauge needle at spaced intervals over the shaved abdomen. All skin test sites were observed and measured at 4-,7-,16-,24-,36-,48-, and 60 hours following injection. Since the diameter of the erythema generally corresponded to the diameter of indura- tion only the former was measured in inches with a circle template (Pickett, No 1200, Pickett and Eckel Inc., Chicago, 27 I11. ) . Guinea-pigs infected with L, donovani, immunized with hamster Spleen homogenate or injected with NCTC 135 medium were skin-tested 8 days post infection or immunization. The antigens employed on each animal were heat-killed LD bodies from hamster spleen (HKHLD's), live LD bodies from hamster spleen (LHLD's) and normal hamster spleen homoge- nate (NHSH). ‘Animals were also skin tested with NCTC 135 medium. Superinfected guinea pigs and their controls were skin tested 16 days post infection or immunization. The skin test antigens were the same as those used in the previous experiment. Five guinea pigs were used in each experimental and control group. In later skin-test experiments, guinea pigs infected with|L. donovani, immunized with hamster spleen homogenate and injected with NCTC 135 medium were tested'3-,7-,l4e, 21-,28-,35-,56-,77-, and 98 days post infection or immuni- zation. Two animals were used per immunization procedure at each time period listed. The three different antigens employed on each animal were heatqkilled hamster LD bodies (HKHLD's), heat-killed mouse LD bodies (KHMLD's) and normal hamster spleen homogenate (NHSH). Animals were also akin tested with NCTC 135 medium. i Collection pl Leg-gm Blood was collected 60 hours after each group of guinea pigs was skin tested. Thus, blood was collected at 28 9 intervals between 3-to 98 days post infection. Approxi- mately'20nfl of blood was aseptically withdrawn from the heart of each animal. The blood collected from each pair of guinea pigs in a group was pooled in a 50 ml sterile centrifuge tube and allowed to clot for 1-2 hours at room temperature. The blood was then stored in the refrigerator overnight. After centrifugation at 392xg for 10 minutes the serum was removed with a Pasteur pipette and stored at ~20°C for later use. Serological-nggg The Ouchterlony gel-diffusibn method (Crowle, 1961) was used to test for the presence of antibody to L, d222- Xflfll‘ The central antisera wells were filled with 0.21m1 of serum. A 0.2nfl.volume of HKHLD's and KHMLD's were each added to an antigen well. A 0.2nfl.portion of NHSH was added to the third antigen'well. Diffusion was allowed to preceed at room temperature for three days.} ' I Antibody—antigen precipitin tests were also carried out to determine the presence of humoral antibody to experi- mental antigens. Guinea pig' antiserum was clarified by centrifugation at 2,440xg for 30 minutes before it was used in the precipitin experiments. Serial two-fold dilutions of the antigens employed in the Ouchterlony gel-diffusion tests were made in 18x150 mm test tubes with distilled water. Undiluted antigens were in the concentrations employed for Skin tests. Aliquots of 0.5Iilof undiluted serum were added to 2.01m10f each of the antigen dilutions. 29 The test tubes were stoppered and placed in a water bath at 37°C for 30 minutes. The tubes were then refrigerated for 24 hours. After refrigeration the precipitates were resuspended by shaking the tubes and returned to the re- frigerator. After 48 hours the tubes were examined for the presence of precipitate. Passive Cutaneous Anapgylaxigigfigfl) Five groups of 4 guinea pigs each were used in the PCA experiments. Four animals were used to test each of the 5 periodic serum collections (sera obtained from infected and control guinea pigs which were skin tested 3-,7-,21-, 35-, and 77-days post-infection or immunization). Each animal was given an intradermal injection of 0.1 ml of each of the following sera: (1) serum from animals infected with L. donovani, (2) serum from animals sensitized with normal hamster spleen homogenate and (3) serum from normal guinea pig controls injected with NCTC 135 medium. The techniques for the preparation of the animals and injection of serum were similar to those employed in skin tests for hypersensitivity. Eight hours after the intradermal in- jection of serum, each animal was injected, intracardially, with .5cc of 1% Evan's blue dye in physiological saline mixed with 1.5 ml of one of the 2 following antigens and NCTC 135 medium: (1) HKHLD's, (2) NHSH. Intracardial in- jections were carried out using a one inch 20 gauge needle fitted to a 20c syringe. Observations were made at the serum sites at 10-,30-, and 60-minutes and 3-,8-,l6-, and 30 24-hours after the intracardiac injections. If bluing was observed at the test site, its extent was measured by the template used to measure hypersensitivity by skin tests. The above experiment was repeated a second time using heat-killed parasites collected from mice in place of the heat-killed parasites from hamsters. Two animals were tested at each time interval, one receiving HKMLD's and the second NHSH by intracardiac injection. Macrophage Migration-InhibitionLigggs. Peritoneal exudate cells were obtained from infected guinea pigs, guinea pigs immunized by an intraperitoneal injection.of'l.01fl.of Freund's complete adjuvant (5mg/ml killed dried Mycobacterium butyricum, Difco, Detroit, Michigan) and normal guinea pigs injected by the peritoneal route with NCTC 135 medium. The cells were collected 8 and 21 days postsinfection or immunization for the first series of experiments. In a later eXperiment an additional 31 day (post-infection) collection was made on one group of guinea pigs. To collect cells each animal was injected via the intraperitoneal route with 20 m1 of NCTC 135 medium cone taining1% heparin and 4pg of penicillin and streptomycin/ ml. The abdomen of each animal was kneaded gently and the fluid collected aseptically with a syringe and needle. The peritoneal exudates were diSpensed into sterile 40 ml centrifuge tubes and stored in an ice bath until the collections were completed. The exudates were centrifuged for 10 minutes at 392xg and the supernatants discarded. 31 The cells were then suspended in complete culture medium consisting of 50% NCTC 135 medium, 40% inactivated horse serum, 10% beef embryo extract and 4 ug of penicillin and streptomycin/ml and recentrifuged for 10 minutes at 392xg. The final sediment of cells was mixed with culture medium to yield approximately 10-20% cells by volume and asepe tically drawn into 1.4x75 mm capillary tubes. The tubes were sealed at one end with a flame. After centrifugation of the tubes at 392xg for 10 minutes, they were cut at the cell-medium interface. The broken ends of the capillary tubes containing the exudate cells were secured on sykes; Moore chamber cover slips with a dr0p of silicon grease before the chambers were assembled and filled with complete NCTC 135 culture medium containing one of the following antigens: 30x106 HKMLD's/ml, 30x106LHLD‘s/m1, 50% v/v NHSH, 50% v/v normal mouse spleen homogenate (NMSH), and 50 ug of PPD/ml (ParkeeDavis, Detroit, Michigan). The chambers were then placed in an incubator at 37°C for 1 hour. After one hour fresh medium and antigens were added to all chambers. After 24 hours of incubation the tubes were placed under a light microsc0pe and photographed. Thirtyeone days after the start of these macr0phage migration-inhibition tests, peritoneal cells were harvested to determine the effects of different concentrations of LHLD's as antigens on capillary tube macr0phage migration. Cells from both infected and normal guinea pigs were allowed to migrate under the influence of from 1- to 32 100x106 live LD bodies of L. donovani as antigen. Others wise the techniques used were those of the previous eXperi- ment. The extent of linear migration was measured at 3 radii from the capillary tubes and averaged. The data is presented as the average percent linear migration of exue date cells in the presence of antigen by considering the migration of cells in antigenefree chambers as 100% migra; tion. Statistical Considerations Statistical significance was determined using the Students "T" test. RESULTS Intradermal Lgsgs LEE Hypersensitivity Normal guinea pigs injected with NCTC 135 medium, guinea pigs infected with L. donovani and guinea pigs immunized with NHSH were used in the first skin test experiment. Each animal received intradermal injections of live parasites, heatskilled parasites, NHSH and NCTC 135 medium 8 days after infection or immunization. A cutaneous reaction deve10ped in response to both viable and heatekilled LD bodies in guinea pigs infected with L. donovani 16 hours after skin testing (Figure l). The ex- tent of erythema was similar for both antigens through the 48 hour peak of the reaction. In animals infected with L. donovani, injections of NHSH also produced an area of ery- thema and induration which became apparent as early as 16 hours after skin testing, but which was smaller than that observed for live and heatekilled LD bodies of L. donovani. This reaction toward NHSH subsided gradually after 16 hours, whereas the erythema from parasite antigens persisted for 60 hours. Guinea pigs immunized with NHSH and injected with NCTC 135 medium showed no measurable cutaneous response to the skin test antigens. 33 Figure 1. 3A The diameter of intradermal skin reactions to~ antigens in guinea pigs infected with L. dono- vani. Guinea pigs were given an intraperitoneal injection of 30x106 LD bodies and were skin tested 8 days after infection with heat—killed ' and live LD bodies 0f L, donovani from hamsters, NHSH and NCTC 135 medium. Guinea pigs immunized With NHSH and'injected with NCTC 135 medium Showed no measurable cutaneous response to the skin test antigens. Each point on the figure is the mean of the area of reaction for 5 animals. DIAMETER OF EIYTHIMA (mm) 16 I4 TO TO 35 *4 HKHLD's 0 E ——o LHLD's X‘ 4 NHSH - 30 4 . so 50' HOURS POST SKIN TEST '36 Since a cutaneous skin test reaction was observed as early as 8 days after infection against both live and heat; killed hamster LD bodies (LHLD's and HKHLD's) and normal hamster spleen homogenate (NHSH) it was decided to compare skin test reactions in superinfected guinea pigs with those in animals receiving a single infection. Guinea pigs were given two intraperitoneal injections of L. donovani, NHSH or NCTC 135 medium, 8 days apart and skin tested 8 days after the last injection. Other animals were given only the latter of the above.3 injections. All experimental and control animals were skin tested with separately spaced intradermal injections of HKHLD's, NHSH and NCTC 135 medium. A cutaneous reaction characters ized by erythema and induration became observable for all skin test antigens as early as 7 hours after intradermal injections, except for animals immunized once with NHSH and NCTC 135 medium which showed no observable skin reace tivity throughout the experiment (Figure 2). In superinfected animals the reaction against HKHLD's reached a peak by 24 hours but in singly infected guinea pigs it reached a peak 48 hours after intradermal injection. The cutaneous reactions against NHSH peaked by 16 hours after injection in both superinfected and singly infected animals. The results of this experiment indicate that _superinfection of guinea pigs with L. donovani enhances the degree of skin reactivity these animals have toward HKHLD's. The skin teSt response of the parasite is more Figure»2. 37 A comparison of intradermal skin test reactions of guinea pigs superinfected with L. donovani with animals receiving a single infection;" Guinea pigs were injected with 2 intraperitoneal injections of LHLD's, NHSH or NCTC 135 medium, given 8 days apart and skin tested 8 days after the last injection. Other animals were given a single injection of the inoculums listed above 16 days'prior to skin testing. All animals were skin tested intradermally with HKHLD's, NHSH and NCTC 135 medium.' Five guinea pigs were used in each experimental and control group. 38 (mun) vwaulma so ualawvuo I 5 _Q A no we 1093 BM... «.22 4mg A0 vameZ—"mnqmo $22.. r E- «mama £21” £15.. Pow 79 7. W At. 212.. MCvmaLIZCZfim—u $22.. ZIMI - 4mm._.m0 $241” *IxIPUw AZIMI .zmmnqmo <<=.: r9353. .330 (24.1” OIXI..U.« 621m: 39 intense than for NHSH in superinfected animals, the latter showing a more immediate reaction and reaching an earlier peak of reactivity. Superimmunization of guinea pigs with NHSH is necessary to elicit a skin test reaction toward the homologous antigen. _Studieseiss 619.9991:th .____..Per:i.oe. leteeeit¥&.n.i_m_1?eration_ o_f _S_k_i_r_1_ _T_s_s_’_t_ Hypersensitivity Ln Guinea Pigs Infected WLLL .E' donovani. The following experiment was designed to answer three questions raised by previous skin test experiments:1) When in the course of infection do guinea pigs first respond to skin test antigens?, 2) When in the course of infection . does the maximum skin test response occur?, and 3) What is the duration of the skin test response over the course of infection? Guinea pigs infected with L, SQQSVSQi’ animals immu- nized with NHSH, and controls injected with NCTC 135 medium were skin tested 3-,7-,14-,21-,28-,35-,56-,77-, and 98 days fellowing infection or immunization with separate Spaced intradermal injections of HKHLD's, HKMLD's and NHSH. Two animals were tested for each group at each time interval after infection. Since heatékilled parasites gave a simi- lar skin reaction to that of live organisms, only the former were used in this experiment. Heat—killed mouse LD bodies (HKMLD's) were used to determine if the skin test response was attributable to the parasites or to contaminating ham- ster spleen antigens. Skin test responses at 7, 24 and 48 40 hours after intradermal injection are given in Figure 3 a-c. Those animals immunized with NHSH and controls injected with NCTC 135 medium displayed no observable skin test re- ponse to the test antigens. Erythema and induration became apparent to HKMLD's and HKHLD's as early as 3 days post infection and 24 hours after skin injection (Figure 3b). A peak in cutaneous reactivity was reached for both parasite antigens by 48 hours after skin injection and 7 days post infection (Figure 3c). Both mouse and hamster sources of parasites gave similar cutaneous reactions with guinea pigs infected with L. donovani. The skin test response against L. dogg- zaLL'antigens could still be elicited up to 98 days post infection and the reactions lasted as long as 60 hours with final sloughing of cells and necrosis of tissue. A cutaneous reaction could be observed 7 days after infection and 16 hours after intradermal injection of NHSH. A maximum extent of cutaneous reactivity for NHSH occurred by 24 hours in animals skin tested 14 days post infection. NHSH gave less erythema than parasite antigens in animals infected with L. donovani, but on a temporal basis the reaction could not be considered an immediate-type of hyper- sensitivity. The skin test response against NHSH remained positive up to 14 days post infection, 60 hours post skin test. The pronounced erythema encountered before 24 hours upon the skin testing of guinea pigs infected with L. Figure 3. 41 The development of skin sensitivity in guinea pigs infected with L. donovani. The diameter of erythema is shown a5 7 Hours (Fig. 3a), 24 hours (Fig. 3b) and 48 hours (Fig. 30) after intradermal injections over 98 days of infection. Each point is the mean of measurements from 2 animals.— The antigens used for skin tests were HKMLD's (....), HKHLD's (0--0), and NHSH (x--x). NCTC 135 medium was injected as a non-antigenic control. Animals immunized with NHSH and con- trols injected with NCTC 135 medium displayed no observable skin test response to the test antigens. A II 7HOIIIS POST SIIN TIST MAHSTII OI IITTHIMA InlII 4 DAYS POST INPICTION OI IMMUNIZATION "I 24 noun '0" sum nu J K OATS POST IMICTI“ OI IWHTION DIMITII OT IIYTIIIIA InuI C no 48 noun '0" ms nu ammu- ov Immu In.) I ”'8 POST IUICTION OI INMUNIZATION L13 donovani suggested that part of the cutaneous reaction could be an immediate-type hypersensitive response. There- fore, an attempt was made to detect serum antibodies in animals infected with L. donovani and immunized with NHSH. Antisera from guinea pigs infected with L. donovani, immunized with NHSH or injected with NCTC 135 medium! were used in the Ouchterlony gel-diffusion tests. None of these antisera produced precipitin bands when allowed to react with antigen used for skin tests. Antibody-antigen precipitin tests were carried out with the antigens and sera used for the Ouchterlony gel- diffusion tests. Serial two-fold dilutions of the skin test antigens were made with distilled water used to produce soluble antigens. Aliquots of undiluted serum were added to each of the antigen dilutions. Sera ob- tained from animals immunized with NCTC 135 medium did not form precipitates to any dilution of the antigens. The titers for the precipitin tests are given in Table 1. Titers for all reactions were low with little signi- ficant differences between antigens at any one time period. A slight increase in titers for HKMLD's and HKHLD's is apparent from days 3-77 post infection with L. donovani. There is also a tendency for the reaction with NHSH to decline in sera collected from infected guinea pigs and from animals immunized with NHSH after 21 days of infection. Antisera obtained from guinea pigs infected with L. donovani, immunized with NHSH and injected with NCTC 135 44 TABLE 1. Titers for precipitin tests 48 hours after addition of antisera. ~ Serum collected Test Titer for Titer for (Days + 60 hours Antigen infected guinea guinea pigs Post immunization) Pigs immunized - e . with NHSH ~ HKHLD's NEG NEG 3 Days HKMLD's NEG NEG NHSH 1:1 1:1 NCTC NEG NEG - HKHLD's ‘IfiZ- lkl 7 Days HKMLD's 1:1 NEG NHSH 1:2 1:4 NCTC NEG NEG * HKHLD's ‘le 1:1 14 Days HKMLD's NEG 1:1 NHSH 1:8 1:8 NCTC ANEG NEG ‘HKHLDTs ‘154. 1152 21 Days. HKMLD's 1:2 1:1 NHSH 1:8 1:8 NGTC-. , NEG . NEG ‘ HKHLD's ‘1:4 ‘1:1 28 Days HKMLD's 1:4 1:1 NHSH 1:2 1:4 NCTG‘. NEG NEG HKHLDTs ‘1:4 1:1 35 Days HKMLD's 1:8 1:1 NHSH 1:2 1:2 NCTC “y NEG NEG - HKHLD's ‘1:4 NEG 56 Days HKMLD's 1:8 NEG NHSH 1:1 1:2 NGTC;._ ,NEG NEG HKHLDY s 1 :8 N EG 77 Days HKMLD's 1:16 NEG NHSH 1:2 1:2 NCTCJ, ,NEG NE ' HKHLDTs ‘1:4 NEG 98 Days HKMLD's 1:2 NEG NHSH 1:1 1:1 NCTC NEG NEG 45 medium were used in tests for passive cutaneous anaphylaxis reactions. Each animal was given an intradermal injection of each of the sera and injected intracardially with Evan's blue dye mixed with one of the two antigens tested and NCTC 135 medium. Positive reactions toward LD bodies from hamsters and NHSH became apparent 30 minutes after intracardial challenge in guinea pigs receiving serum from animals infected with .L- donovani and immunized with NHSH. The cutaneous response showed a maximum extent of bluing for each serum in animals injected with HKHLD's and NHSH 8 hours after intracardial challenge. Therefore, only the 8 hour measurements are re- ported (Table 2). Serum from infected guinea pigs and ani- mals immunized with NHSH gave similar reactions to NHSH and HKHLD's. But the reactions were less pronounced for the serum from animals infected with L. donovagi. Serum from animals injected with NCTC 135 medium gave no PCA response to the challenge antigens. Also no reactions were observed at any of the sera sites when NCTC 135 medium was injected. In a second experiment HKHLD's were replaced by HKMLD's in order to determine whether or not the PCA response was attributable, in part, to parasite antigens. Again positive reactions toward HKMLD's and NHSH became observable 30 minutes after intracardial challenge and a maximum extent of bluing occurred by 8 hours (Table 3). Normal animals receiving serum from animals immunized with NHSH gave no cutaneous reaction upon intracardiac 46 TABLE 2. Results of passive cutaneous anaphylaxis reactions 8 hours after intracardiac injection of antigens. Days after Serum Intracardiac Diameter of infection or from Test bluing in immunization animals Antigen millimeters Infected with NHSH 5.60 L. donovani HKHLD's 7.60 3 Immunized with NHSH 16.00 NHSH HKHLD's 9.65 Infected with NHSH 7.00 L. donovani HKHLD's 8.65 7 Immunized with NHSH ‘ 16.00 NHSH HKHLD's 10.4 Infected with NHSH 8.65 L. donovani ' HKHLD's 9.65 21 Immunized with NHSH 17.5 NHSH HKHLD?s 17.5 Infected with NHSH 8.65 L. donovani HKHLD's 9.65 35 Immunized with NHSH 17.5 NHSH HKHLD's 15.3 Infected with NHSH 4,84 IL. donovani HKHLD's 6.35 77 Immunized with NHSH 17.5 NHSH HKHLD's 15.3 1+7 TABLE 3. Results of passive cutaneous anaphylaxis reactions 8 hours after intracardiac injection of antigens (HKMLD's used in place of HKHLD's) Days after Serum ' Intracardiac Diameter of infection or from test bluing in immunization animals antigen millimeters Infected with NHSH 5.10 L. donovani HKMLD's 7. 60 3 Immunized NHSH 8. 40 I?“ with NHSH HKMLD's 0 Infected with NHSH 15.0 , .E- donovani HKMLD's 7. 89 f 7 Immunized with NHSH 17.30 , NHSH HKMLD's 0 - ~ .1 Infected with NHSH 11.2 L. donovani HKMLD's 10.4 2 l Immunized NHSH 16.8 with NHSH HKMLD's O Infected with NHSHTO 0.4 L. donovani HKMLD's 11. 2 35 Immunized NHSH 16.0 with NHSH HKMLD?s 0 Infected wiEE NHSHI 4.30 L. donovani HKMLD's 7.10 77 Immunized NHSH 9.65 with NHSH HKMLDts 0 48 injection with HKMLD's, but serum from infected animals showed comparable reactions to parasites from mice and to NHSH. This seems to indicate a positive reaction to the parasite proper. Serum from animals injected with NCTC 135 medium gave no PCA response toward the challenge anti- gens or NCTC 135 medium. _T_gr_s_1;s_ £95 £112 Inhibition_2_f_ Macrophage Migration Since a positive PCA response to L. donovani and NHSH antigens suggested the presence of an immediate-type hypersensitivity, it was necessary to determine if a dis- tinct delayed-type response could be demonstrated. Inhibi- tion of macrophage migration tests were attempted to de- lineate the latter response. Peritoneal exudate cells were collected from infected guinea pigs, guinea pigs immunized with Freundis complete adjuvant and normal animals injected with NCTC 135 medium. The cells were collected 8 and 21 days post infection or immunization in the first experiment. In a later experi- ment an additional collection was made at 31 days after infection or immunization. Inhibition of macrophage migration tests were carried out by observing the migra- tion of macrophages from capillary tubes in Sykes-Moore chambers to which antigen was added with the medium. Controls to determine the extent of normal migration lacked antigen in the medium. Peritoneal exudate cells collected from infected guinea pigs 8 days after infection showed a statistically 19 significant inhibition of macrophage migration when exposed to LHLD's (p40.01) but not to HKMLD's, NMSH or NHSH (Table 4). Macrophages from control guinea pigs injected with NCTC 135 medium and tested 8 days later showed no inhibition of migration to the test antigens including the presence of live parasites. Cells collected from animals immunized with Freund's complete adjuvant 8 days after immunization were not inhibited in their migration by PPD. Peritoneal cells harvested from animals 21 days post infection with L. donovani showed a statistically signifi- cant inhibition of their migration when exposed to either LHLD's or HKMLD's (p40.01), but not to NMSH or NHSH. Macro- phages from control guinea pigs injected with NCTC 135 medium and collected 21 days after injection did not respond to any of the test antigens. Cells harvested 21 days after immunization with Freund's complete adjuvant displayed a statistically significant degree of inhibition of migration when exposed to PPD (p10.01). Thirty-one days after the start of these tests, peri- toneal cells were harvested from infected animals to de- termine the effects of different concentrations of LHLD's as antigens on macrophage migration from capillary tubes. A statistically significant degree of inhibition was ob- served in infected animals when peritoneal cells were 6 LHLD's (Table 4). Although cells exposed to 1- 100x10 from control animals injected with NCTC 135 medium dis- played a statistically significant degree of inhibition TABLE 4. 50 Percent inhibition of migration for peritoneal exudate cells in the presence and absence of test antigens. Percentage of inhibition of migration is equal to mean migration with anti— - gen/mean migration without antigen x 100. Days post *SOurce oII‘ Antigen %:mi ration (mean infection or cells- from added .1 SD (% migration immunization animals Lg_vitro inhibition no anti- gen = 109) Antigen IfiTecfeT HKNEED‘ 3 75-3, 38 8 with L. NMSH 89 1 35 donova‘ni NHSH .86 1 35 , "__"-—' LHLD's 55 :_ 3** .. Injected - HKMLD‘s 102 1 2'5- - 8 with NCTC NMSH 114 _+_ 16 135 NHSH 98 _+_ 31 medium LHLD's . 931: 8' Immunized 8 with Freund's PPD 86 1 13 complete ad'uvant ' In ected HKMLD‘s. 64 1 18” 2l with L. NMSH 96 1 5 ' donovani NHSH 93 1.24 "'""'"" LHLD's ,38-1 3** Injected? HKMLDTs 100’:' 4.- 21 with NCTC NMSH 97 1 8 A 135 NHSH 92 1 23 1, medium 11g LHLD's .97 :_ 6 Immunized 'F 21 with Freund's PPD 39 1 4 complete ad'uvant . In ectedi 1x10 8 ‘_ 31 with 10xlo§ " 80 1 6** , L. 20x10 " 44': A** donovani 30x106 " 36,: A** —""' 50x106 " 38 1 ‘4** 75x106 " 60 1 4* 100x10§ " 84 1 5*- Ihjectecf "le00 1001 3- 31 with 10x106 98 1 2 NCTC 20x106 100 1 6 135 30x106 89 1 5* medium 50x106 93 1 6 75x106 100 1 9 100x10 98 1 6 ~*P<0.0§ **P<0.0l 51 of migration when peritoneal cells were exposed to 30x106 LHLD's (p40.05), the cells did not respond to lower and higher concentrations of parasites. DISCUSSION Skin test experiments carried out on guinea pigs infected with L. donovani in the absence of adjuvants suggested that a delayed-type hypersensitivity reaction to live and heat-killed parasites was present, as well as what appeared to be a more immediate-type cutaneous reac- tion to normal hamster spleen homogenate. Guinea pigs were infected with live parasites in order to study the phenomenon of hypersensitivity under conditions of infec- tion. At first an attempt was made to compare the reac- tivity of heat-killed and live parasites as skin test antigens. Similar reactions to both heat-killed and live parasites first occurred in tests given 8 days after in- fection or immunization and 16 hours after skin testing. The extent of erythema was similar for both antigens up to the 48 hour peak of reaction. Animals infected with L. donovani showed cutaneous reactions toward normal hamster spleen homogenate, indicating that the reaction observed toward the parasites might be due to contamina- ting antigens from hamster spleen. The reaction toward normal hamster spleen homogenate in infected animals be- came apparent as early as 16 hours after skin testing, but the erythema was less than that for the parasite and 52 53 subsided gradually after 16 hours. When attempts were made to further characterize cu- taneous reactions described above, it was found that erythema and induration deve10ped in response to both heat- killed mouse and hamster LD bodies as early as 3 days post infection. The maximum cutaneous reactivity for both para- site antigens was attained in animals tested 7 days after infection. Reactions peaked between 24 and 48 hours after skin injection. Animals responded with positive skin reac- tions to both L. donovani antigens up to 98 days after in- fection. These results suggested that animals infected with L. donovani show a strong delayed-type hypersensiti- vity reaction upon skin test with the parasite proper. The cutaneous reaction in infected animals toward normal hamster spleen homogenate showed some delayed-type charac- teristics. This was not surprising because delayed-type hypersensitivity reactions have been reported for many simple protein antigens, such as, ovalbumin, etc. (Dienes and Mallory, 1932; Benacerraf and Gell, 1959). The tran- sitory character of the response, as shown to normal ham- ster spleen homogenate, is usually not associated with classical delayed-type hypersensitivity reactions (Zinsser, 1921; Turk, 1967). The reactions described above were not indicative of anaphylactic-type-immediate hypersensitivity where a wheal and flare are observed directly after skin test. The epithelial necrosis which was observed after the reaction 54 subsided was most likely indicative of a severe cutaneous reaction and reflected upon the extreme sensitivity of the guinea pig (Dienes and Mallory, 1932; Waksman, 1960; and Uhr, 1966). The timing of the induction of erythema and induration corresponded to the so-called delayed-type hypersensitivity reactions observed by Boysia (1967). The duration of sensitivity after infection (up to 98 days) also corresponded to that described by Boysia (1967). Boysia (1967) observed cutaneous reactions using various leishmanial antigens in skin tests of guinea pigs sensitized with either heat-killed promastigotes of L. mexicana or alkaline extracts of L. donovani promastigotes in Freund's complete adjuvant. The reactions appeared 3 to 5 days post sensitization and 4 to 9 hours after skin testing. The reactions peaked in intensity between 16 to 24 hours. The subtle differences in the induction periods and peaks of the cutaneous reactions between Boy- sia's observations and this investigation could have been caused by several factors. One factor isthe immunologi- cal enhancing effects of Freund's complete adjuvant which Boysia used in immunization. Freund's complete adjuvant may have decreased the induction periOds for cutaneous reactions and the time after injection to the peak of cutaneous reactivity. Salvin (1958) reported that the induction period and the duration of delayed-skin test sensitivity were dependent upon the amount of sensitizing antigen injected and the type of antigen preparation used 55 to sensitize in either Freund's complete or incomplete adjuvant. The fact that infected animals were used in this investigation and not guinea pigs sensitized with extracts or heat-killed parasites may be influencing the reactivity of the skin tests. Boysia (1967) found that when a large dose of sensitizing antigen was used, either the induction period or duration of skin reactions was shortened and the opposite trend occurred when smaller amounts of antigen were used for sensitization. Wagener (1923) skin tested rabbits sensitized with a phenolized 6 suspension of 3X10 promastigotes of L. tropica or L. infantum and observed a delayed cutaneous reaction to the corresponding leishmanial species which peaked at 48 hours. Wagener and Koch (1926) skin tested guinea pigs 8 to 15 days after sensitization with an intraperitoneal injection of phenolized promastigotes of L. tropica. Peak reactions appeared 48 hours after intradermal injection of the homo- logous antigen. Bryceson 23'3L. (l970a,b) reported cu- taneous reactions peaking at 24 hours in guinea pigs infect- ed with 106 organisms of L. enriettii when they were tested with soluble and insoluble L. enriettii antigens. Peak tuberculin skin reactivities in guinea pigs have been re- ported to occur at 24 hours post infection (Koch, 1891; and Dienes, l930a,b). The route of sensitization or in- fection may also play a role in the induction period and durations of the skin test responses. Boysia (1967) sensitized guinea pigs via the foot pad route, but in this 56 study animals were infected by the intraperitoneal route. In this investigation some skin tests of infected guinea pigs produced a pronounced erythemabefore 24 hours. This suggested that part of the cutaneous response could be an immediate-type hypersensitive response. Since anti- bodies are closely associated with immediate hypersensiti- vity reactions, an attempt was made to detect serum anti- bodies in guinea pigs infected with L. donovani and con- trols immunized with normal hamster spleen homogenate. Evidence for the existence of serum antibodies to L. £222? X221 and normal hamster spleen homogenates have been found by qualitative antigen-antibody precipitin tests and passive cutaneous anaphylaxis reactions. In qualitative antigen- antibody precipitin tests, titers for all reactions were low with little significant difference between antigens at any one time period. It is possible that the distilled water used to dilute and solubilize antigens could have produced a precipitate which did not involve antigen-anti- body complexes, but such precipitates should have also occurred in normal serum controls and in reactions with heat-killed hamster LD bodies that were negative. Passive cutaneous anaphylaxis (PCA) reactions indi- cated that serum from guinea pigs infected with L. donovani and animals infected with normal hamster spleen homogenate possessed antibodies which reacted with heat-killed hamster LD bodies and normal hamster spleen homogenate antigens. At first these were thought to be anti-spleen homogenate 57 antibodies. But when PCA tests were carried out using heat-killed mouse LD bodies in place of heat-killed ham- ster LD bodies, serum from infected guinea pigs gave po- sitive PCA reactions toward both normal hamster spleen homogenate and heat-killed mouse LD bodies, confirming the presence of antibody to each antigen. The possibility of homologous antigenic determinants on mouse LD bodies and spleen homogenate is considered unlikely. Animals receiving serum from normal hamster spleen homogenate immunized animals showed reactivity only to normal hamster spleen homogenate. Bray and Lainson (1965) have reported positive PCA tests in guinea pigs and monkeys using sera from leishmanin sensitive patients and animals. Boysia (1967) failed to show the presence of humoral antibody in guinea pigs in- fected with either L. mexicana or L. donovani extracts mixed in Freund's complete adjuvant. Bryceson 23.21. (1970 a,b) failed to demonstrate the presence of circu- lating antibodies to L. enriettii by PCA tests or by agglutination of antigen coated sheep erythrocytes in sera of infected or convalescent animals, although some con- valescent guinea pigs showed an immediate-type of active cutaneous anaphylaxis. These workers did demonstrate the presence of antibody by PCA and hemagglutination tests in animals immunized with antigen extracts of L. enriettii in Freund's adjuvant. These animals also showed anaphy- lactic or Arthus-type hypersensitivity when skin tested 58 with soluble antigen. It can not be determined from this investigation whether or not the antibodies to L. donovani and normal hamster spleen homogenate take part in skin test reactivity. It also has not been determined whether the antibody or antibodies to L. donovani found in the serum are protective. Results of studies of serum antibody effects in hamsters infected with L. donovani suggest that they are not pro- tective against infection (Ada and Fulton, 1948; Gellhorn_ gL‘aL., 1946; Rossan, 1960; Stauber, 1954; Van Peenan and Miale, 1962; Rahman, 1966; and Miller and Twohy, 1969). Since PCA reactions to L. donovani and normal hamster spleen homogenate antigens showed the presence of anti- bodies to these antigens and antibodies are associated with immediate-type hypersensitivity responses and skin test experiments indicated that a pronounced cutaneous erythema was present before 24 hours after skin test, it was necessary to determine if a distinct delayed-type hypersensitivity response could be demonstrated. The inhibition of the migration of peritoneal macro- phages obtained from sensitized animals by specific anti- gen is an important test for the presence of delayed-type hypersensitivity. Bryceson 23‘31. (1970 a,b) showed the inhibition of migration of peritoneal macr0phages har- vested from guinea pigs infected with L. enriettii and from animals immunized with insoluble and soluble L. enriettii antigens emulSified in Freund's incomplete 59 adjuvant. Tremonti and Walton (1970) demonstrated inhibi- tion of migration of peritoneal exudate cells obtained from guinea pigs infected with L. brasiliensis and tested with leishmanin. In this investigation peritOneali cells were specifi- cally inhibited in their migration by LD bodies from mice and hamsters and not by spleen homogenates. Thus, a delayed-type hypersensitivity could be attributed to the parasite itself. Peritoneal exudate cells collected from guinea pigs as early as 8 days after infection showed a statistically significant degree of inhibition of migration, when exposed to live parasites. This inhibition occurred earlier than that seen to PPD in macrophages from guinea pigs immunized with Freund's complete adjuvant which usual- ly occurs in about 3 weeks after.immunization.(Bloom and Bennett, 1968). At 21 days post-infection or immunization, a statistically significant degree of inhibition of migra- tion was detected with cells from infected animals exposed to either heat-killed or live parasites. At this time peri- toneal cells collected from guinea pigs immunized with Freund's adjuvant showed a sensitivity to PPD. The results of the inhibition of macrophage migration tests found in this investigation bring out two questions that need serious consideration: 1) Why are live parasites a more effective antigen in inhibiting the migration of peritoneal macrophages from infected animals than heat- killed parasites? They produce a response in cells 6O harvested earlier in the course of infection and a higher percent inhibition when compared to heat-killed hamster LD bodies. And 2) Why do the live and heat-killed para- sites produce similar degrees of response in skin tests and a distinct quantitative dissimilarity of results in 12.11332 tests? These questions might be answered through an overview of the mechanisms thought to be responsible for the inhibition of macrOphage migration and a considera- tion of the possibility that antibodies may be involved in skin test reactions. It is conceivable that when sensitized lymphocytes come in contact with live parasites, the live parasites may be in some way more active in inducing the production of migration inhibition factor (MIF) or have antigenic determinants that have a higher affinity for macrophage- cell bound immunoglobins, thus promoting a more active inhibition of migration than that observed for heatehilled parasites. It may be that the activity of live parasites depends upon labile antigenic components that are altered in the heat-killed preparation. It is possible that the antibodies found in precipitin and PCA tests are involved in cutaneous reactions. A similar reactivity in infected animals toward heat-killed and live parasites might be explained by the equal specificity the antibodies have toward a common heat stable antigenic determinant. In the La 11222 tests where antibody is not involved different antigens may be responsible for the macrophage migration. 61 Live parasites may be able to induce the production of MIF in sensitive lymphocytes or react with macrophage-cell bound antibody earlier in infection because of the presence of an antigen that is produced only by living cells and is denatured in the heating process. Evidence presented in this investigation suggests that a non-classical form of delayed-type hypersensitivity is present in guinea pigs infected with L. donovani. The response is difficult to quantify based on the hetero- geneity of antigens acting within the system and the possibility of the involvement of both cell and serum mediated components. SUMMARY Guinea pigs infected with L. donovani showed delayed- type cutaneous reactions upon skin test with heat—killed and live parasites, as well as with normal hamster spleen homogenates. The skin test reactions for both live and heat-killed parasites were characterized by induration and erythema which first appeared 16 to 24 hours after intra- dermal injection and 3 and 8 days post infection. The reactions peaked between 16 and 48 hours after intradermal injection. In animals infected with L. donovani, skin tests with normal hamster spleen homogenate produced a smaller area of erythema and induration than parasite antigens. The reaction subsided gradually after 16 hours whereas the reaction from parasite antigens persisted for 60 hours. Guinea pigs superinfected with L. donovani showed cutaneous reactions as early as 7 hours after intradermal injections of leishmanial and spleen homogenate antigens. Superinfection of guinea pigs with L. donovani enhances the degree of skin reactivity these animals have toward heat-killed hamster LD bodies. The skin test response of the parasite was more intense than for normal hamster 62 63 spleen homogenate in superinfected animals, the latter showing more immediate character and reaching an earlier peak of reactivity. Superimmunizing guinea pigs with normal hamster spleen homogenate was necessary to elicit a skin test reaction toward the homologous antigen. Qualitative antigen-antibody precipitin tests and passive cutaneous anaphylaxis tests carried out on guinea pigs infected with L. donovani indicated the presence of antibody to both parasite and hamster spleen antigens. Lp'yiggg'tests showed a statistically significant inhibition of macrophage migration when peritoneal exudate cells were exposed to live parasites collected from in- fected guinea pigs as early as 8 days post infection. 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