IMMUNOLOGICAL REACTIONS BETWEEN DEXTRAN POLYSACCHARIDE AND SOME BACTERIAL ANTISERA

IMMUNOLOGICAL REACTIONS BETWEEN DEXTRAN POLYSACCHARIDE AND SOME BACTERIAL ANTISERA BY JOSl~ ZOZAYA, M.D. (Frm the Mulfrd Bilgical Labratries, Sharp and Dhme, Glenlden, Pennsylvania) (Received fr publicatin December 21, 1931) The name dextran was given by Scheibler (1) t the mucus fermentatin prducts present in beet juices. He fund that this substance n hydrlysis yielded cnsiderable quantities f glucse and when xidized with nitric acid gave xalic acid. He was led t regard it as an anhydride f glucse. Many ther descriptins f this and similar substances are given in literature, but at present it is generally accepted that the prductin f the gum is due t the actin f bacteria n rich sugar media. Depending n the kind f sugar and the type f rganism used, different plysaccharides are btained. In the case f gum-prducing bacteria, large amunts f plysaccharides are prduced because the nutrient medium used is rich in sugar. Bas (2) recmmended a culture medium cntaining frm 5 t 1 per cent f carbhydrate. Accrding t the cmmnly accepted thery, it is assumed that mst, if nt all, carbhydratedecmpsing micrrganisms pssess synthesizing prperties which, during the early stages f their develpment give rise under favrable cnditins t the cndensatin f hexses and ther simple sugars t cmplex plysaccharides. In cases in which these synthetic plysaccharides readily imbibe water, a cllid, i.e., a mucus, is frmed. The mst imprtant f the bacteria capable f prducing dextran frm saccharse is Leucnstc mesenteriides. The methd f preparatin, purificatin and structure f the dextran prduced by this rganism will be fund described in frthcming papers (3). The material used in ur experiments was kindly supplied by Prf. Hibbert and shwn by analysis t be nitrgen- and ash-free. An antidextran serum has been prepared by the authr by adsrbing the dextran n clldin particles utilizing a methd reprted in a recent paper (). The relatin between the dextran plysaccharide 353

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js~ ZOZAYA 355 and varius bacterial antisera has nw been investigated and varius interesting crss-reactins have been btained while testing the specificity f this plysaccharide. The bacterial antisera utilized in the wrk were all prepared by injecting rabbits with whle frmalinized culture f the rganisms. The injectins were given intravenusly and treatment cntinued until tests n trial bleeding shwed a definite, specific plysaccharide precipitate. In a few cases antisera prduced in hrses by a similar technique were emplyed. TABLE II Titratin witk Dextran Plysaccharide against Several Antibacterial Sera (Cmpared wltk Hmlgus Serum R 12) Dilutin f plysaccharide... Antidextran R12... Antityphid... Anfimrgani... Anfisuipestifer... Antigaliinarum... Antiparatyphi A... Antiaertrycke... Anti- R pneumcccus... Antipneumcccus I... Antipneumcccus II... Antipneumcccus III... Antientercccus (1917)... Antistrep. ulcer, clitis (1916)... Antistrep. ulcer, clitis (1918)... Read after 5 hurs at 37 C. and vernight in ice bx. 1 Mre than thirty different antisera were investigated in rder t select the nes capable f reacting with the dextran at a dilutin f 1-5,. The results f these tests are shwn in Table I. The dextran was titrated with the reacting sera in varius dilutins, using fr cmparisn the specific antidextran sermn. The results f this test are shwn in Table II. It is f interest t ntice the apprximate cnstancy in the titratin f the majrity f the antisera, the value lying between 32, and 6,. The variatin can dubtless

356 DEXTRAN POLYSACCHARIDE AND ANTISE1LA be accunted fr by variatin in the ptency f the sera when tested against their wn specific antibdy The high titer f the specific antidextran serum is cnspicuus. Having bserved the grupings f these antisera int species each grup was then studied separately. The first grup is the Salmnella and it included E. typhi. The results f the titratin f the sera, with the dextran at 1-5, dilutin, are shwn in Table III. It is f interest t nte that S. schttmuelleri (Para B) des nt give a reactin with dextran, but this exceptin can be explained by the fact that this serum des nt give specific carbhydrate precipitates. Perhaps the variatins with the sera are related t the differences in amunt f antidextran antibdies which may parallel thse f the specific anticarbhydrate antibdies. TABLE III Titratin f Antisera f the Salmnella Grup and E. typhi with Dextran Plysaccharide (1-5,) Antiserum.. Mrganl Suipe- Galli- Aer- [ Para A [ Para B I Typhid ] Cntrl I Cntrl I/2 [ [ [ l [ [ [ 1/13 113113[1311 1/8 I 1 I 13 [ ] 2 112 [ ] 1/16 [ [ 13 /1 2 I I x I 2 [ Read after 5 hurs at 37 C. and vernight in ice bx. T ascertain whether the antidextran antibdy was absrbed by the specific plysaccharide, absrptin tests were made with each sera. On ne sample the specific carbhydrate was absrbed and n the ther the dextran. Each sample was then titrated with the hmlgus plysaccharide and with the dextran. The results are fund in Table IV. N difference is t be bserved in any f the absrbed sera, demnstrating that the antidextran antibdy is nt assciated with the specific carbhydrate antibdy, but is a definite entity. The secnd grup studied was the pneumcccus. In Table V the results are given f the titratin f different antipneumcccic sera, type-specific and anti-"r" frm Types I and II. The type-specific sera gave weaker reactins than the anti-r sera. T ascertain whe-

js~ ZOZAYA 357 ther the precipitate btained in the type-specific sera was due t the "C" substance, absrptin tests were made with the specific and C carbhydrates. The absrbed sera were then precipitated with the de~tran, giving the same results as n the cntrl sera that were nt absrbed This eliminates the type-specific r the grup antibdies (anti-c) as the surce f the precipitate with dextran. These results TABLE IV Results f Precipitin Test with Hmlgus Plysaccharide (1-1,) and Dextran n Sera Absrbed with Same l'est... ~e rtlnl.., Titratin f sera with hm]- ] gus plysaccharide l-l, Titratin f sera after absrptin Titratin f sera with dextran with hmlgus SSS I-5, after being absrbed with its wn plysacchafide 1-1, with hmlgus plysacchar/de E 1/2 1/ 1/8 1/16 3 3 -- 3 2 2 3 1 2 3 3 3 ['est... Titratin f sera with dextran Titratin f sera with hmlgus Titratin f sera with dextran 1-5, plfsaccharide 1-1, after absrptin with dextran after absrptin with dextran 1/2 1/ 1/8 1/16 f 3 1 3 3 3 3 2 2 2 3 2 3 2 2 3 2 2 1 3 2 2 Read after 5 hurs at 37 C. and vernight in ice bx. are in accrd with thse bserved with the Salmnella grup as reprted abve. The third grup studied was the Streptcccus viridans (Bargen) f different types (Strains 1915, 1916, 1918 and 1838); a strain f Streptcccus faecalis (191), and anther f entercccus (1917). These rganisms are clsely related. Table VI shws the results f the precipitin test with dextran, It is f great interest t nte that

358 DEXTRAN POLYSACCHARIDE AND ANTISERA Antisera 191 and 1916 shw a distinct difference in their reactin twards dextran, and as is knwn they are extremely difficult t differentiate by any ther methd (except absrptin f specific anticarbhydrate antibdies). All f these strains are immunlgically different, their plysaccharides are characteristic, yet while Strains 1916, 1917 and 1918 have TABLE V Titratin f Antipneumcccus Sera (S and R Types) with Dextran Plysaccharide (1-5,) Serum... Pncumcccus I 1/2 a/ 3 2 1/8 1 1/16 1 Pneum- Pneum- Pneum- Pneum-[ Cntrl [ Cntrl cccus II.. cccus III cccus R I cccus R II ) + g 12.... [ 3 3 [ 1 1 / 3 3 2 1 2 Read after 5 hurs at 37 C. and vernight in ice bx. TABLE VI Titratin f Sera frm Streptcccus viridans (Bargen) and Similar Organisms, witk Dextran Plysaccharide (1-5,) Strain N... 191 1915 1916 1917 " 1918 1838 1/2 1/ 1/8 1/16 1-- Read after 5 hurs at 37 C. and vernight in ice bx. antidextran antibdies, the ther three have nt. This wuld appear t suggest fundamental differences in mlecular cmpsitin f the plysacchar[des. T determine if the antidextran antibdy culd be absrbed by the specific carbhydrate, Sera 1916, 1917 and 1918 were absrbed with their hmlgus plysaccharide and then the dextran precipitin test repeated. There was n nticeable difference, this being in agreement with the results btained with the Salmnella and the pneumcccus grups.

~OS~ ZOZAYA 359 DISCUSSION Avery, Heidelberger and Gebel (5) have already described the crss-reactins between the Pneumcccus Type II antiserum and the B. friedlaenderi plysaccharide and vice versa. This crssing they attribute t a similarity in a prtin f the cmplex mlecule r a very similar cnfiguratin f atms. Immunlgical similarity between B. anthrax, meningccci, B. subtilis and B. mesentericus has als been shwn (6) and the same explanatin ffered as that by the abve authrs. These experiments suggest that sme f the bacterial plysaccharides may cntain several active antigenic grups in their mlecular structure. Certain f these may be mre active in stimulating antibdy frmatin r be acted upn by antibdies f a simpler nature. As immunizatin prgresses, increasing amunts f the cmplex specific antibdies wuld be frmed in respnse t the cmplex bacterial plysaccharide. In a study f the plysaccharides f the Salmnella grup their actin has been fund t be a specific ne in agreement with Furth and Landsteiner (7). At the same time it has been pssible t shw (unpublished wrk) that the plysaccharides frm S. aertrycke and S. suipestifer crss mutually with the respective antisera. The results in the absrptin tests with all the grups studied suggest that the dextran antibdy is a distinct entity, fr in all cases it remains after absrptin f the specific carbhydrate antibdies. It may be that this is a simpler antibdy, the primitive r immature stage f the mre cmplex ne which is cnstantly being frmed in prcess f immunizatin t the bacterial plysaccharides. This wrk suggests a pssible applicatin f the immunlgical methd t the study f the chemical structure f cmplex carbhydrates. SI.~MM.ARY Dextran, the synthetic plysaccharide prduced by Leucnstc mesenteriides frm saccharse, reacts immunlgically with antisera frm pneumccci, sme f the Salmnella and sme f the types f Streptcccus viridans (Bargen). This immunlgical relatinship is independent f the specific

36 DEXTRAN POLYSACCHARIDE AND ANTISERA antiplysaccharide antibdies f these sera, suggesting the existence f a distinct antibdy prduced by an active grup f the specific bacterial plysaccharide," which is similar r identical t the active grup f the dextran plysaccharide. BIBLIOGRAPHY 1. Scheibler, C., abstr. Wagner's Yakresb. ckem. Technl., 1875, 21,79. 2. Bas, F., Bickem. Z., 1917, 131, 8. 3. Tart, H. L. A., and tiibbert, H., persnal cmmunicatin. Hibbert, It., and Brauns, F., persnal cmmunicatin.. Zzaya, J., Science, 1931, 12, 27. 5. Avery, O. T., Heidelberger, M., and Gebel, W. F., Y. Exp. Med., 1925, 2, 79. 6. Zzaya, J., J. Exp. Med., 1931, 5, 725. 7. Furth, J., and Landsteiner, K., Y. Exp. Ivied., 1928, 7~ 171.