Pretreated with Mycobacterial Fractions

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1 NFECTON AND MMUNTY, Sept. 1971, p Copyright 1971 Amerian Soiety for Mirobiology Vol. 4, No. 3 Printed in U.S.A. mmune Response to Sheep Red Blood Cells in Mie Pretreated ith Myobaterial Frations A. BEKERKUNST, E. YARKON,. FLECHNER, S. MORECK, E. VLKAS, AND E. LEDERER Department of Medial Bateriology, Hebre Universily-Hadassah Medial Shool, Jerusalem, srael, anld asit/ut de Chimie des Substanes Naturelles, Gif-sur-Yvette, Frane Reeived for publiation 4 May 1971 Ten mirograms of trehalose-6,6'-dimyolate (ord fator) injeted into the footpads of mie inreased the antibody response to sheep red blood ells (SRBC) subsequently injeted into the same sites. There is a relationship beteen the antibody response and the ellular reation indued loally and in the draining lymph nodes by ord fator, as judged by a muh eaker response hen antigen is injeted into the ontralateral footpads. The time intervals beteen injetion of ord fator and antigen ere from 5 to 2 days. A similar inreased antibody response to SRBC as evident after preliminary administration of Freund's omplete adjuvant or living BCG bailli into the footpads. There as no inreased antibody response in mie pretreated ith living BCG or Freund's adjuvant to SRBC injeted into the ontralateral footpads. Administration of ax D from human strains Peurois and Test as ithout any effet. Administration of SRBC emulsified in inomplete Freund's adjuvant ontaining 5,ug of ord fator indued a very strong antibody response in the mie as ompared to that after injetion of the same amount of antigen in inomplete Freund's adjuvant ontaining ax D or myobateria. The results are disussed. Ten mirograms of trehalose-6,6'-dimyolate (ord fator), a glyolipid derived from myobateria, indued, after injetion into the footpads of mie, histologial hanges similar to those folloing injetion of living BCG. Both materials indued in the draining lymph nodes formation of granulomas omposed of epitheloid ells, marophages, and lymphoytes as ell as marked hyperplasia of the lymphoid tissue in the paraortial one of the nodes and aumulation of marophages. No ellular reation as seen in the internal organs suh as liver, spleen, and lungs after 28 or 3 days of observation. Experimental infetion ith BCG enhanes the ativity of retiuloendothelial system and inreases formation of antibody against unrelated antigen (5, 12, 13). Relevant to these ativities are a number of observations revieed in the preeding paper (4). t has been assumed by the authors that the above ativities are losely onneted ith the ellular reation on the part of the host at the sites of lodgment of the bailli and their multipliation. t is oneivable that ord fator, hih auses a similar ellular response, ill indue a similar hange in the immune response of the host. MATERALS AND METHODS Animals. Albino mie of a loal strain ere used throughout all of the experiments. Preparation of myobaterial frations, of suspensions of living BCG bailli, and of emulsions as as desribed previously (3, 4). Sheep red blood ells. Sheep blood as olleted eekly in Alsever's solution and stored at 4 C. Before use, the erythroytes ere ashed three times ith about 15 volumes of phosphate-buffered saline (PBS), ph 7.2. After the final ash, the sheep red blood ells (SRBC) ere suspended in PBS as a 2% suspension (based on paked ell volume) for the serologial tests and as a 1% suspension for immuniation of the mie. A 1-ml amount of the 2% suspension ontained approximately 5 X 18 red blood ells. Hemagglutination test. ndividual mouse sera ere diluted in PBS, starting from a dilution of 1:2. To.25 ml of the serial tofold dilutions,.5 ml of the 2% RBC suspension as added. After inubation for 1 hr at 37 C, the tubes ere entrifuged for 1 min in a Clay-Adams Sero-Fuge, and the degree of agglutination as determined marosopially by gently shaking off the sedimented SRBC pellet. Titers ere reorded as the reiproal of the highest dilution in hih at least 1+ agglutination as observed. The titer ero as arbitrarily assigned to sera produing no agglutination at the dilution 1:

2 VOL. 4, 1971 MMUNE RESPONSE TO SHEEP RED BLOOD CELLS 257 Antiglobulin test. After determination of the agglutination titers, the erythroytes ere ashed tie in PBS and reuspended in.2 ml of rabbit antimouse gamma globulin serum diluted 1:1 in PBS. The tubes ere entrifuged as above, and the Coombs titers ere determined in the same ay as in the hemagglutination test. The rabbit antimouse gamma globulin as a gift from D. Suliteanu. The dilution 1:1 as found to bring about the agglutination of SRBC after exposure to a ide range of subagglutinating antibody onentrations. Myobaterial ompounds. These ompounds ere prepared as desribed in referene 3. RESULTS Antibody response to SRBC after pretreatment ith ord fator, Freund's adjuvant, and emulsion. Five days after injetion of 1,ug of ord fator into the footpads of mie, in addition to a marked hyperplasia of the paraortial one, aumulations of marophages and ell-irumsribed granulomas ere present in the draining popliteal nodes. At that time, the ellular reation in the nodes as muh eaker after injetion of Freund's omplete adjuvant (4). t has been assumed that there is a relationship beteen the above ellular reation and the antibody response to an unrelated antigen. To test this assumption, the folloing experiment as arried out. Three groups of five mie ere injeted into the left hind footpad ith ord fator, Freund's omplete adjuvant, and emulsion, respetively. A fourth group served as the ontrol. After 5 days, the animals ere injeted ith a suspension of sheep erythroytes, and, at the intervals indiated in Fig. 1, blood as dran from the retroorbital plexus and the hemagglutination and antiglobulin titers of the sera ere established (Fig. 1). The strongest response as seen in the ord fatorpretreated group; this response as stronger than in the groups treated ith omplete Freund's adjuvant and emulsion. The differene as partiularly great 5 days after injetion of the antigen. The hemagglutination titer in the emulsion-treated group as less than 1:2, hereas in the ord fator group the average titer as 1:1. The differenes ere learly evident during the 26 days of observation both in the hemagglutination and antiglobulin titers. Sine ord fator injeted into the footpads of mie is apparently trapped in the draining lymph nodes and does not spread systemially (4), it as oneivable that the antibody response of mie to antigen administered into the ontralateral footpads ould be different from that injeted ith the antigen into the homolateral ones. To groups of five mie ere injeted ith ord fator into the left hind footpads. After 5 days, SRBC ere injeted in one of the group into the right ontralateral foodpads and in the other group into the homolateral ones. Starting from F 4 (' C71 (n s F.- -J m L 4 DAYS AFTER FRST NJECTON OF SR BC FG. 1. Antibody response to sheep red blood ells (SRBC) after pretreatment ith ord fator from Myobaterium kansasii, Freund's omplete adjuvant, and emulsion. The amount of ord fator administered as 1,ug in emulsion ontaining 2.5%Xo Bayol F. The amount of SRBC as 5 X 17 ells; all materials ere injeted in volumes of.2 ml.

3 258 BEKERKUNST ET AL. NFEC. MMUN. o 1 U) 8-4 Z=O ) u)8 6 -j _ /~~~~ C i,,~- -e - A.. -J CD 4 r A Cord-fator and SRBC1Left foot pad A Cord-fator left foot pad,srbc right foot pad (1 35 U D U 3 LU C:> X J D AYS AFT E R N J E C T O N OF S R B C FG. 2. Relationship beteen the antibody response and site of antigen injetion in mie pretreated ith ord fator. Other details as in legend to Fig. 1. L the 5th day after injetion of the antigen, the hemagglutination and antiglobulin titers ere determined. As an be seen from Fig. 2, the antibody response of the mie, although pretreated ith ord fator but injeted ith the antigen into the ontralateral footpad, as muh eaker than in the other group, hih reeived ord fator and antigen in the same footpad. Time relationship beteen injetion of ord fator and antigen and the antibody response. The ellular reation indued by ord fator in the draining lymph nodes of mie after injetion into the footpads persists for at least 3 days (4). t as reasonable to assume that if the above histologial hanges ere related to the antibody response, the latter ould also be inreased after intervals greater than 5 days. To test this assumption, groups of mie ere injeted ith 1,ug of ord fator into the left hind footpads and the sheep erythroytes ere injeted into the same site after intervals of 1, 15, and 2 days. After 5 days, the hemagglutination and antiglobulin titers in the sera of the animals ere determined (Table 1). t an be learly seen that the hemagglutination titers in the sera of ord fatortreated mie ere very great ompared to those of the emulsion-treated mie. After an interval of 2 days, the average hemagglutination titer in the former group as 24 versus less than 2 in the ontrol group. Similar large differenes ere also evident in the antiglobulin test. The kinetis of the antibody response are shon in Fig. 3. t is evident from the table that the greatest differenes in the antibody response are present 5 days after in,etion of the antigen. Antibody response of mie to SRBC pretreated ith ax D. The ellular reation to administration of emulsions of ax D from human strains Peurois and Test into the footpads of mie as muh eaker loally and in the draining lymph nodes than that indued by ord fator (4). Aording to White and ollaborators, ax D from human strains injeted in inomplete Freund's adjuvant into the footpads of guinea pigs auses a granulomatous reation not only loally but in the draining lymph nodes and remote organs. This as paralleled by an inreased antibody response to ovalbumin inluded in the ater in oil emulsion. Cord fator, on the other hand, did not indue a granulomatous response and as inative in inreasing the antibody response (16). Therefore, it as of interest to ompare the above substanes under onditions used in this study. Groups of five mie ere injeted into the hind left footpads, respetively, ith ax D from the human strain Test, ord fator, ord fator plus ax D, and emulsion ithout an additive. After 5 days, the mie ere injeted ith SRBC. Starting from the 5th day after injetion of the antigen, blood as dran from the mie at different time intervals, and the hemagglutination and antiglobulin titers ere determined. As an be seen from the results presented in Fig. 4, a strong antibody response as evident only in the ord fator-pretreated groups. The group of mie injeted ith ax D alone reated in a similar ay to the emulsiontreated mie. A similar result as obtained in a separate experiment after administration of 1 Ag of ax D from the human strain Peurois. Comparison beteen the antibody response of mie pretreated ith lving BCG, ord fator, and omplete Freund's adjuvant. t has been already indiated that the ellular reation to ord fator in the draining lymph nodes is indistinguishable from that to living BCG bailli, but in some respets it is very similar to that indued by Freund's omplete adjuvant (4). Aording to

4 VOL. 4, 1971 MMUNE RESPONSE TO SHEEP RED BLOOD CELLS 259 TABLE 1. Time relationship beteen adminiistrationt of ord fator and sheep red blood ells (SRBC) anid the anitibody response nterval Hemagglutination titersb Antiglobulin titers beteen injetions Materialof ord ije fator and inee SRBCa 1C Mean Mean (days) 1 Cord fator , Emulsion Cord fator ,28 1,281 1,28 2,56 5,12 2,34 Emulsion Cord fator ,28 1,28 2,56 1,216 Emulsion a Amounts of ord fator and SRBC as in Fig. 1. b Titers ere determined 5 days after injetion of SRBC. o Sera no. NTERVALS BF.'W.FFN NJECTON OF CORD FACTOR AND ANTGEN m1 1 DAYS 15 DAYS 2 DAYS R D' S 1 W 8 / 6, 1'nU Uf _, - 8 ax x e 6 _ 'x ),r-- -X x x 4 o Cord-fator i 2 - x x EmuLsion _ O D AY S AFT ER NJECTON OF SR B C FG. 3. Time relationship beteen administration of ord fator atid sheep red blood ells (SRBC) and the atitibody response. Other details as in legend to Fig. 1. Freund, the adjuvant ation of inomplete and omplete Freund's adjuvant is due to the persistene of antigen at the site of injetion, to the slo systemi dissemination of the antigen, and to the ellular reation evolved. n addition, the antigen should be inluded into the emulsion, otherise antigen and adjuvant injeted into separate sites are no more effetive than the injetion of antigen alone (7). t has been assumed in this study that the inreased antibody response indued by Freund's omplete adiuvant is due mainly to the ellular reation aused apparently by ord fator of the myobateria. To test this assumption, the folloing experiment as arried out. SRBC ere injeted into the footpads of groups of mie 18 days after injetion of living BCG, ord fator, or omplete Freund's adjuvant into the same sites. Five days afterards, the hemagglutination titers ere determined in the sera of individual mie. The results are presented in Table 2. t an be seen that the titers in the groups treated ith BCG, ord fator, and Freund's adjuvant are muh higher than those in the emulsion-treated

5 26 BEKERKUNST ET AL. NFEC. MMUN. ) u) llj a) l -4- a) D CD QD DAYS AFTER FRST NJECTON OF SRBC FG. 4. Antibody responise to sheep red blood ells (SRBC) of mie pretreated ith ax D and ord fator. Tlhe amount of ord fator as 5,ug; of ax D (Test), 5,ug; of SRBC, 5 X 17 ells. All malerials ere injeled in volumes of.2 ml. ontrol group and that they are quite lose and statistially not different from eah other. Loal effet of living BCG and Freund's omplete adjuvant on the antibody response in mie. Cord fator and living BCG injeted into the footpads of mie spread only to the draining lymph nodes and apparently are trapped there. No ellular reation as seen in the internal organs suh as liver, spleen, and lungs after 28 or 3 days of observation (4). A similar lak of ellular response as also observed by others in the organs of mie after injetion of Freund's adjuvant into the footpads (6, 7). The similarity in the antibody response seen in Table 2 seemed to be due to the similarity in the histologial hanges indued by ord fator, BCG, and the myobateria in Freund's ater in oil emulsion. f the adjuvant effet of Freund's adjuvant and of a BCG infetion is due to the ellular reation evolved in the tissues of the host, there should not be an inreased antibody response after injetion of the antigen into a site at hih no ellular reation is antiipated. To test this, the folloing experiment as arried out. To groups of five mie ere injeted ith living BCG into the left hind footpads, and to other groups ere injeted ith Freund's omplete adjuvant into the same sites. After 15 days, the groups of mie ere injeted ith SRBC, as indiated in Table 3. The hemagglutination titers ere determined 5 days after injetion of the antigen. The results indiate learly a diret relationship beteen the ellular reation indued by the multiplying BCG bailli and the myobateria in Freund's adjuvant and the inreased antibody response. The hemagglutination titers in the sera of animals injeted ith SRBC into the ontralateral footpads ere at TABLE 2. Antibody response of mie to sheep red blood ells (SRBC) after previous treatmenit ith living BCG, ord fator, and omplete Freund's adjuvan t Material injeted Hemagglutination titers in individual miea Avg Emulsion BCG Cord fator from M. kansasii (1,ug) Freund's adjuvant a SRBC ere injeted into the footpads of the mie 18 days after injetions of emulsions, BCG, ord fator, or Freund's adjuvant. The hemagglutination titers ere determined 5 days after injetion of the antigen. Emulsion ontained 1.25% of Bayol F, BCG as a suspension prepared from an 8-day ulture in Dubos broth medium ith Teen 8, and optial density of the suspension as 6 Klett units. Volumes of injeted material ere.2 ml.

6 VOL. 4, 1971 MMUNE RESPONSE TO SHEEP RED BLOOD CELLS 261 TABLE 3. Loal effet of living BCG and Freund's omplete adjuvant on the antibody response of mie to sheep red blood ells (SRBC) Hemagglutination titers in Antiglobulin titers in individual Material Mateial injeted njeed Site of injetion individual mie mie of SRBC' Avg Avg BCG Homolateral , BCG Contralateral Freund's Homolateral adj uvant Freund's Contralateral adj uvant a SRBC (5 X 1') ere injeted into the footpads of the mie 15 days after injetions of BCG and Freund's adjuvant. The hemagglutination titers ere determined 5 days after injetion of the antigen. BCG as a suspension prepared from a 12-day ulture in Dubos broth medium ith Teen 8; optial density of the suspension as 9 Klett units. Volumes of injeted material ere.2 ml. TABLE 4. Anitibody response of mie to sheep red blood ells (SRBC) in inomplete Freund's adjuvant (FA) in the presene of ord fator, ax D, or myobateriaa Medium Hemagglutination titers in individual mie Antiglobulin titers in individual mie Avg Avg Saline FA , CFAb , FA plus ax D FA plus ord fatord 1, ,56 2,56 2,56 1, ,92 a b SRBC (5 X 17) ere injeted in saline or FA in the presene of ax D, ord fator, or myobateria. Complete Freund's adjuvant. Amount of ax D from strain Peurois, 1,g. d Amount of ord fator from M. kansasii, 5,g. levels seen in animals that ere not treated at all ith BCG or Freund's adjuvant. Antibody response to SRBC in inomplete Freund's adjuvant in the presene of ord fator, ax D, or myobateria. The above results indiate that ord fator is apparently responsible for the adjuvant ativity of the myobateria hih are present in the mineral oil of the emulsion as ell as for the ation of multiplying BCG bailli. t as of interest to determine ho ord fator ould affet the antibody response after administration of antigen, emulsified in inomplete Freund's adjuvant ontaining a small amount of ord fator, and to ompare this ith other adjuvants. A suspension of SRBC in saline as emulsified in equal volumes of inomplete Freund's adjuvant ith and ithout addition of ord fator and ax D. A similar emulsion as also prepared in omplete Freund's adjuvant. A.2-ml amount of the preparations as injeted into the footpads of groups of mie. After 11 days, blood as dran from the mie, and hemagglutination and antiglobulin titers ere determined in the sera of the animals (Table 4). The strongest antibody response as evident in the group of mie injeted ith SRBC in inomplete Freund's adjuvant ontaining 5,ug of ord fator. DSCUSSON t is lear from the above results that trehalose- 6, 6'-dimyolate (ord fator) in "physiologial" amounts is able to hange the antibody response of mie to an unrelated antigen. There is also no doubt that this hange is due to the ellular reation indued in the draining lymph nodes by ord fator. There are three harateristi ellular omponents in the above reation: hyperplasia of the lymphoid tissue in the paraortial one, aumulation of marophages, and formation of granulomas omposed of marophages, epitheloid ells, and lymphoytes. The question of hih of the above omponents plays the most important role in the inreased antibody response is diffiult

7 262 BEKERKUNST ET AL. NFEC. MMUN. (N O X 2 kr x _ 1 CD Lr J D 6 m 4 -j CḎ 2, l A-Cord-fator x-emuls ion -Normal mie E n -1- p r% r A n in ) n 2 n / n a n U U U U JU 4U ;3U DAYS AFTER NJECTON FG. 5. Antibody response to sheep red blood ells ofmie injeted ith ordfalor and antigen al ihe same lime. Mie ere injeted ith 1 tug ofordfator in emulsion ontaining 1.25% Bayol F. to anser at present. t is oneivable that all three are important in vie of hat is knon about antibody formation. There is relatively little and no onlusive information in the literature about the ontribution of loal granulomas to antibody formation. Aording to Fishel et al. (1), the degree of granulomatous reation has an intimate relationship to the development of antibody, a vie hih is at variane ith that onluded by others (15). Askonas and White demonstrated that the loal granuloma in guinea pigs produed by myobaterial ax dissolved in inomplete Freund's adjuvant as relatively inative in the in vitro synthesis of antibody (2). This is in ontrast to results obtained in the rabbit and horse in hih the granuloma itself made a major ontribution to the antibody prodution (1). Whatever its role in antibody formation, there seems to be little doubt that the granulomatous reation plays a protetive role in infetion ith tuberle bailli (3, 8, 17), not by virtue of irulating antibodies but rather beause of the large numbers of ativated marophages. Another fat hih emerged from this study is the loal harater of the immunologial response. Antigen injeted into the footpad as probably trapped in the draining lymph nodes here it ame into ontat ith immunoompetent ells, the proliferation of hih as enhaned by administration of ord fator. Were it different, the antibody response ould be the same, not ithstanding the site of antigen injetion. t is oneivable that the number of antibody-forming ells from the draining lymph nodes affeted by ord fator ould be relatively very large, ompared ith suh ells from the ontralateral nodes or spleen of the animal. t is also oneivable that by multiple injetions of ord fator at different sites the ellular reation ould evolve not only loally and in the draining lymph nodes but also in the internal organs, suh as liver, spleen, and lungs. A point of interest to onsider is the time relationship beteen injetion of ord fator and antigen and the antibody response. t seems that the latter is stronger as judged by the hemagglutination titers after a longer interval of 15 and 2 days than after 5 and 1 days. This is ompatible ith the intensity and persistene of the ellular reation and its loaliation. t is present not only in the popliteal and inguinal nodes but also in the more distant lumbar nodes. The latter may be even more involved after a longer interval than the inguinal nodes, hih are probably bypassed to some degree by ord fator (unpublished data). The antigen, hen it spreads through the lymphati system, apparently omes into ontat ith a larger number of immunoompetent ells, and hene the stronger antibody response after a longer interval from the time of ord fator administration. t is noteorthy that the above time relationship beteen injetion of the adjuvant and antigen and the immunologial response is ompletely different from that observed ith endotoxin. The latter has to be injeted either together ith the antigen or ithin a relatively short time before or after antigen (14). The same is true ith the Bordetella pertussis adjuvant (9). n this study, to get an inreased antibody response, ord fator had to be injeted before the antigen. Administra-

8 VOL. 4, 1971 MMUNE RESPONSE TO SEEEP RED BLOOD CELLS 263 tion of both at the same time did not affet the antibody response (Fig. 5) unless the antigen as retained at the site of ation of ord fator by inorporating the former into the inomplete Freund's adjuvant as a ater in oil emulsion. Under the latter onditions, antigen and adjuvant do not separate; they spread together. At the time hen the ellular reation evolves in the draining lymph nodes, the antigen is at hand to exert its effet on the immunoompetent ells, the proliferation of hih is apparently part of the above reation. The similarity in the inreased antibody response after the preliminary treatment ith ord fator, living BCG, or Freund's omplete adjuvant seems to indiate a ommon mode of ation expressed in the similarity of the ellular reation to the above materials. The lak of inreased antibody response in mie injeted ith antigen in the presene of ax D seems to indiate that ax D has no adjuvant effet in the mouse as it has in the guinea pig (16). t is noteorthy that ax D is also inative in hiken (White, personal ommuniation). t is knon that omplete Freund's adjuvant indues, in addition to the inreased antibody response, a hypersensitivity state of the delayed type to unrelated antigens inluded in the emulsion. The remarkable inreased antibody response to SRBC in the presene of 5,ug of ord fator in inomplete Freund's adjuvant raised at one the question hether, under these onditions, a hypersensitivity state to the red ells has been reated. The above question and some further impliations are under investigation. ACKNOWLEDGMENTS This investigation as supported by Publi Health Servie grant from the National nstitutes of Health and Ligue Nationale Frangaise Contre Le Caner. LTERATURE CTED 1. Askonas, B. A., and J. H. Humphrey Formation of speifi antibodies and -y globulin in vitro. A study of the syntheti ability of various tissues from rabbits immunied by different methods. Biohem. J. 68: Askonas, B. A., and R. G. White Sites of antibody prodution in the guinea-pig. The relation beteen in vitro synthesis of anti-ovalbumin and -y globulin and distribution of antibody ontaining ells. Brit. J. Exp. Pathol. 37: Bekierkunst, A.,. S. Levij, E. Yarkoni, E. Vilkas, A. Adam, and E. Lederer Granuloma formation indued in mie by hemially defined miiyobaterial frations. J. Bateriol. 1: Bekierkunst, A.,. S. Levij, E. Yarkoni, E. Vilkas, and E. Lederer Cellular reation in the footpad and draining lymph nodes of mie indued by myobaterial frations and BCG bailli. nfe. mmun. 4: Bioi, C., C. Stiffel, B. N. Halpern, and D. Mouton Reherhes sur le meanisme de l'immunite nonspeifique produite par les myobateres. Rev. Fr. Etude Clin. Biol. 5: Carter, R. L., D. G. Jamison, and R. L. Vollum Histologial hanges evoked in mie by Freund's inomplete adjuvant. J. Pathol. Bateriol. 95: Carter, R. L., D. G. Jamison, and R. L. Vollum Histologial hanges evoked in mie by Freund's omplete adjuvant. J. Pathol. Bateriol. 97: Dannenberg, A. M., Jr Cellular hypersensitivity and ellular immunity in the pathogenesis of tuberulosis: speifiity, systemi and loal nature, and assoiated marophage enymes. Bateriol. Rev. 32: Finger, H., M. Bartoshek, and P. Emmerling Time relationships beteen injetion of antigen and adjuvant. 1. Adjuvany of Bordetella pertussis given at various times before primary antigeni stimulus. nfe. mmun. 2: Fishel, E. D., E. A. Kabat, H. C. Stoerk, M. Skolnik, and A. E. Beer Suppression by ortisone of granuloma formation and antibody in guinea-pigs reeiving egg albumin ith Freund's adjuvant. J. Allergy 25: Freund, J The mode of ation of immunologi adjuvants, p n Advanes in tuberulosis researh, vol. 7. S. Karger, Basel. 12. Halpern, B. C., C. Bioi, C. Stiffel, and D. Mouton Correlation entre lativite phagoytaire du systeme retiuloendothelial et la prodution d'antiorps antibateriens. C. R. So. Biol. 152: Leis, P., and D. Loomis Allergi irritability. The formation of anti-sheep hemolyti amboeptor in the normal and tuberulous guinea-pig. J. Exp. Med. 4: Neter, E Endotoxins and the immune response. Curr. Top. Mirobiol. mmunol. 47: Weigle, W., F. J. Dixon, and D. Deihmuller Effet of myobateria on antibody prodution by ater in oil emulsions of soluble protein antigens. Pro. So. Exp. Biol. Med. 15: White, R. G., L. Bernstok, R. G. S. Johns, and E. Lederer The influene of omponents of M. tuberulosis and and other myobateria upon antibody prodution to ovalbumin. mmunology 1: Youmans, G. P., and A. S. Youmans Reent studies on aquired immunity in tuberulosis. Curr. Top. Mirobiol. mmunol. V 48: