Purification and characterization of erythrogenic toxins IV. Communication: Mitogenic activity of erythrogenic toxin produced by Streptococcus pyogenes strain NY-5

Zbl. Bakt. Hyg., I. Abt. Orig. A 251, 15-26 (1981) 1 2

Central Institute of Microbiology and Experimental Therapy, lena, GDR Institute of Hygiene and Epidemiology, Prague, Czechoslovakia

Purification and Characterization of Erythrogenic Toxins IV. Communication: Mitogenic Activity of Erythrogenic Toxin Produced by Streptococcus pyogenes Strain NY-5 lsolierung und Charakterisierung von erythrogenen Toxinen IV. Mitteilung: Mitogene Aktivitat des vom Stamm NY-5 des Streptococcus pyogenes gebildeten erythrogenen Toxins

HEIDE KNOLL 1, VERA HRtBALOVA 2, DIETER GERLACH 1, and WERNER KOHLER 1 With 6 Figures· Received June 24, 1981

Summary Some in vitro reactions involved in the mitogenic activity of a purified erythrogenic toxin from the group A streptococcus strain NY-5 (ET NY-5) were studied. The optimal dose in the human blood lymphocyte transformation test was 1 to 10- 1 ,ug/ml lymphocyte culture, the maximum of 3H-thymidine incorporation was on day 3 or 4. The mitogenic activity showed the signs of nonspecificity, nevertheless, a specific mitogenic effect could not be ruled out either. When Con A and ET NY-5 were added simultaneously in high or low doses at the beginning of lymphocyte cultivation, antagonism or an additive effect was observed, respectively. Incubation of lymphocytes with ET NY-5 resulted in a decrease of mitogenic activity in the supernatants. Erythrocytes had no similar binding activity. ET NY-5 acts as a T-cell mitogen; 98 Ofo of ET NY-5-stimulated lymphoblasts formed E rosettes with sheep red blood cells. Thin-layer isoelectric focusing experiments revealed two mitogenic peaks corresponding to toxin types A and C in the ET preparation.

Zusammenfassung Das aus Kulturfiltrat von Streptococcus pyogenes, Stamm NY-5 durch Athanolausfallung mit nachfolgender Ionenaustausch- und Gelchromatografie isolierte erythrogene Abbreviations used in this paper: ET - erythrogenic toxin SPE - streptococcal pyrogenic exotoxin SR - stimulation ratio - lymphocyte transformation test LTT Con A - Concanavalin A

16

H.Kn611, Vi Hftbalova, D.Geriach, and W.K6hler

Toxin (ET NY-5 ) wurde auf einige Charakteristika seiner mitogenen Aktivirat gegeniiber Lyrnphozyten aus humanem peripheren Blur untersucht, Als optimale stimulierende Dosis wurde 1 bis 10-1 ,ug/ml Lymphozytenkultur ermittelt, die maximale 3H-Thymidininkorporation erfolgte am 3. oder 4. Tag. ET NY-5 stimuliert Lymphozyten unspezifisch, doch kann eine zusatzliche spezifische Stimulation nicht ausgeschlossen werden. Werden der Lymphozytenkultur gleichzeitig 2 Mitogene, Con A und ET NY-5, zugefiigt, rritt in hohen Dosen cine antagonistische, in niederen Dosen eine additive Wirkung ein. Lymphozyten, nicht aber Erythrozyren, besirzen ET NY-S bindende Eigenschaften, Inkubation von Lymphozyten mit ET NY-5 bewirkt eine Abnahme der rnitogenen Aktivitar in den entsprechenden Uberstanden, ET NY-S wirkt als T-Lymphozytenmitogen: autoradiographische Untersuchungen in Kombination mit der Bildung von Spontanrosetten zeigten, daR 98 Ufo der ET-stimulierten Lymphoblasten Rosetten mit Schafblurerythrozyren bilden. Isoelektrische Fokusierung der ET NY-5 und nachfolgende Testung der mitogenen Akrivitat der Eluate im Lymphozytentransformationstest zeigte, daR in der ET-Praparation die Toxine vom Typ A und C enthalten sind.

Introduction In the first paper of the present series (6), purification and characterization of an erythrogenic toxin preparation (ET NY -5) was described. The erythrogenic toxin was isolated from a culture filtrate of Streptococcus pyogenes strain NY-5 by differential precipitation with ethanol and purified by ion-exchange chromatography and gel chromatography. In Ouchterlony precipitation the toxin gave a precipitation line with antiserum to purified streptococcal pyrogenic exotoxin A and was designated, in accordance with accepted criteria, as erythrogenic toxin type A. A further paper (10) dealt with some in vivo activities of the purified toxin. The toxin induced erythema and swelling on intracutaneous injection into adult guinea pigs. The pyretic reaction to an intravenous injection into rabbits was characterized by late onset and protracted rise up to hour 5. The white blood cell count after intravenous injection displayed marked transient granulocytopenia and protracted lymphopenia. The method of pyrogenic cross-tolerance revealed the presence of the two toxin types A and C in ET NY-5. Erythrogenic toxins have been reported to stimulate lymphocytes in vitro (13, 22, 19, 7, 14,21, 1) and in vivo (8). This study was undertaken to investigate some in vitro interactions of erythrogenic toxin with lymphocytes. The nonspecific activation of T lymphocytes may have some importance in the pathogenesis of certain streptococcal diseases.

Material and Methods Mitogens. Erythrogenic toxin (ET NY -S) from Streptococcus pyogenes strain NY-5 was prepared and purified as described by Gerlach et al. (6). Concanavalin A (Con A) was a product of Pharmacia Fine Chemicals, Uppsala, Sweden; the stock solution contained ],OOO,ug dry matter per ml. Dilutions of both mitogens in base Eagle's minimal essential medium (MEM) were used. Lymphocyte transformation test. The test was performed as described previously (17). Briefly, lymphocytes were separated from heparinized peripheral blood of healthy adult

Mitogenic Activity of Erythrogenic Toxin

17

donors by gradient centrifugation in Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden). After a washing with Eagle's MEM (Staatl. Institut fiir Immunpraparate und Nahrrnedien, Berlin), the cells were suspended to a concentration of 1 X 106 cells/ml in Eagle's MEM containing 100 IU penicillin and 100 pg streptomycin per ml and supplemented with 20 Ofo inactivated calf serum. One millilitre aliquots were cultivated in the presence of varying concentrations of ET NY-5, Con A or without any additive at 37°C in round bottomed tubes (12 X 100 mm) tightly closed with rubber stoppers for the periods indicated below. Sixteen hours before harvesting, 1 pCi of 3H-thymidine (spec. activity 15.5 Ci/mM; Zentralinstitut fiir Kernforschung, Dresden-Rossendorf) was added to each tube. Termination of labelling and measurement of radioactivity were done, with slight modification, ad modum Waite and Hirschhorn (31): washed cells were lysed by adding 0.1 ml IN NaOH and heating at 56°C; then 0.1 ml of 0.25 Ofo solution of serum albumin per ml and 2.0 ml of cold 6 Ofo trichloroacetic acid were added. The tubes were kept at 4 °C for 60 min and the precipitation was repeated once more. The precipitate was then dissolved by adding 0.2 ml hydroxide of hyamine (10-X, Packard Instrument Co., Inc., Downers Grove, Illinois) for 10 min at 56°C. Ten millilitres of a toluene-based scintillation fluid containing 0.5 Ofo PPO and 0.D3 Ofo POPOP was added and the radioactivity was counted in a Packard Tri-Carb liquid scintillaton spectrometer. The mean uptake values were expressed either in counts per minute (cpm) or as stimulation ratio (SR): cpm in cultures with antigen cpm in cultures without antigen E-Rosettes. The method of ]ondal et al. (11) was used. Briefly, 1 X 106 human lymphocytes (either freshly prepared or precultivated for 72 hours with or without ET NY-5) in a volume of 0.2 ml Eagle's MEM were mixed with the same volume of 1 Ofo sheep red blood cells in saline. The suspension was centrifuged at 100 g for 3 min, incubated at 37°C for 5 min and then at 4 °C for 16 hours. The cells were gently resuspended and at least 100 lymphocytes were counted. Only lymphocytes binding more than 3 erythrocytes each were considered rosette-forming. Autoradiography. Lymphocytes cultivated with or without ET NY-5 and labelled with 3H-thymidine as in the transformation test were washed twice with Eagle's MEM and prepared to form E-rosettes as described above. One to 2 drops of resuspended cells were smeared on slides, dried and fixed by methanol for 15 min. The slides were covered with film (ORWO autoradiographic plates K 106, VEB Film Fabrik Wolfen) and placed in a light-tight box at 4 °C for 5 days. After developing with Orwo Developer Solution M-H 28, the films were fixed and Giemsa stained. The slides were examined for the numbers of transformed cells with incorporated label that formed or did not form rosettes. Polyacrylamide gel electrofocusing was carried out on Ampholine PAGE Plates (LKB Producter AB, Stockholm) according to the instruction of the producer. The samples (20 pi each) were applied to filter paper strips, which were placed on the surface of the gel. The samples were electrofocused for 2 h at 800 V on a water-cooled plate (5). On completion of the separation the pH gradient was measured with a microelectrode. Reference gels were fixed in 10 Ofo trichloroacetic acid and stained with Coomassie blue R250. The ET concentrations in the samples were 1,000 pg/ml for staining and 100 pg/m! for testing mitogenicity of eluates.

Results Lymphocyte transformation The mitogenic activity of ET NY-5 was tested in comparison with Con A activity. 2 Zbl. Bakt. Hyg., 1. Abt. Orig. A 251

18

H . Knoll, V. Hr ibalova, D. Gerlach, and W. Kohler

a) Dose dependence of response. Human lymphocytes were stimulated with 10 to 10- 4 f-lg ET NY-5 (100 J~ g had a depres sing effect) or 100 to 1O-~ f-l g Can A. Figure 1 shows a typical patt ern of lymphocyte stimulation after 72 h. There is a sharp peak in Can A-stimulated lymphocyte cultures for the value of 10 f-lg Con Aim! lymphocyte cult ure . The mitogenic respon se to ET NY-5 showed a broader peak for concentration s of 1 to 10-\ Ji g/mI.

SR

100 80

60

40

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Fig. 1. Dose dep endence of mitogenic activity of ET NY-5 A-A and Con A e-e. 10

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Fig. 2. Ti me dependen ce of mitogenic activity o ~ ET NY-5 and Con A. 0 -0 100 J~g, . -11 10 J~g, 0 - 0 1 ug, 6.- 6. 10- 1 .ug and • 10- 2 ,ug mitogen/ml lymphocyte culture, +- + control without stimulant.

Mitogenic Activity of Erythrogenic Toxin

19

b) Time dependence. Lymphocytes were stimulated with 100, 10, 1 or 10- 1 flg Con A or with 10, 1, 10- 1 or 10- 2 flg ET NY-SimI lymphocyte culture. The reaction was followed at 24-h intervals. As is seen in Fig. 2, the optimal Con A concentration gave maximum stimulation at 72 h; with lower concentratios, the maximum was shifted to later h. After stimulation with ET the maximum response occured after 72 to 96 h, and again there was a shift to later days with lower concentration of toxin. Effect of ET NY-S on Can A-induced lymphocyte stimulation Experiments were performed to study the simultaneous action of various doses of both of the nonspecific mitogens. The aim was to find whether the induction of DNA synthesis would be additive or whether antagonistic or synergistic interaction would be involved. Both mitogens were added simultaneously at the start of lymphocyte cultivation. The intensity of stimulation was tested after 72 h. SR 100

80

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Fig. 3. Effect of ET NY-S on lymphocyte stimulation induced by Can A. The crosses mark the stimulating effect of ET NY-S alone.

As may be seen in Fig. 3, a supraoptimal concentration of Con A caused a marked suppression of DNA synthesis; the addition of ET NY-S did not influence the degree of suppression. In the presence of the optimal Con A concentration, a supraoptimal concentration of ET NY-S (10 flg/ml) caused a pronounced suppression of DNA synthesis, while lower doses of toxin did not substantially influence the Con A-induced DNA synthesis. In the presence of suboptimal Con A concentrations, ET NY-S increased the DNA synthesis in relation

20

H , Knoll, V, Hf ibalova, D, Gerlach, and W, Kohler

to the dose used ; with the optimal concentration of to xin (1 Ilg per ml) t he degree of D N A synthesis was less than the sum of the individual responses and also less than the response to toxin alone; bu t with suboptimal doses of ET NY-5 (10- 1 to 10- 2 Ilg/ml) an additive effect was observed (Table 1). Table 1. Effect of ET NY-5 on DNA synthes is (expressed as stimulation rat io the presence of suboptimal concent rations of Con A Ca n A ,ug/ml: SR: ET NY-5 ,ug/ ml: 10 1 10-1 10- 0

SR: 20,8 52.2 49.4 28.1

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31.0 32.3 55.0 34.5

=

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Bindin g of ET NY-5 by lymphocytes and erythrocytes Lymphocyte s or er ythrocytes were incubated for 45 min with ET NY-5 (fo r test conditions see legend to Fig. 4 ) and th e supern atant s were tested for ET NY-5 SR 40

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Fig. 4. Residual mitogenic activity in supernatants of lympho cytes and erythrocytes preincubated with ET NY-5. 0.7 X 107 human blood lymphocytes suspended in 1 ml Eagle's base MEM were incubat ed with 10 ug ET NY-5 at 37 ° C for 45 min, The cells were separated by centrifugation and the supernatants were added in amounts of 10, 100 and 200 Iii to I-ml aliquots of a lymphoc yte suspension to test their stimulating activity in the transformation test @. Controls were ET NY-5 incubated in Eagle's MEM without lympho cytes ill and supernatants of lympho cytes @ or erythro cytes ® incubated without ET NY-5. The binding activity of erythrocytes was tested similarly, using a 20 010 suspension of human erythrocytes G).Transformation responses in 3-day lymphocyte cultures are given.

Mitogenic Activity of Erythrogenic Toxin

21

content in the lymphocyte transformation test. As demonstrated in Fig. 4, incubation resulted in a decrease of mitogenic activity in the respective supernatants. Erythrocytes had no similar binding activity. Repeated freezing and thawing of lymphocytes after the incubation did not cause disengagement of ET NY-5 into the supernatant. E-Rosette formation by ET NY-S-stimulated lymphocytes

To see whether ET NY-5 stimulated T or B lymphocytes, autoradiography combined with E-rosette formation was performed after cultivation of lymphocytes for 72 h. The results are summarized in Table 2. Cultivation of lymphocytes for 72 h in the absence of ET led to a low, nonsignificant rise in the number of rosette-forming cells. The presence of ET NY-5 did not significantly influence the proportion of rosette-forming cells. Autoradiographic studies revealed that 98 % of ET NY-5-stimulated lymphoblasts with incorporated 3H-thymidine formed E-rosettes with sheep red blood cells (Fig. 5). No labelled rosette-forming cells were found in control cultures without ET NY-5. Table 2. Influence of ET NY-5 on E-rosette formation "/0 E-rosettes

control Before cultivation ALter 72 h cultivation

lO,ug ET NY-5

n

m

20

51.9 67.6

56

3.1 2004

n

m

20

51.9 73.1

57

18.6 18.0

n = number of tests m = mean values s = standard deviation

Two peaks of mitogenic activity after polyacrylamide gel electro focusing of ET NY-5

Immediately on completion of isoelectric focusing, the polyacrylamide gel was cut into 5 mm slabs, each of which was eluted in 1 ml of Eagle's MEM at 4°C overnight. Aliquots a 100 ,Ill of the eluates were added to I-ml volumes of lymphocyte suspension and cultivated for 72 h. Mitogenic activity was obtained in two peaks: a lower one at pH 6.8 and a higher one at pH 5.5 (Fig. 6). The peaks of mitogenic activity corresponded with the stained bands in the gel.

Discussion Ability to induce lymphocyte transformation has been reported for several streptococcal products. Supernatant fluids from cultures of nearly all group A streptococcal strains induce nonspecific blast transformation (29, 16, 25). A lymphocyte-transforming activity has also been found in streptococcal cellular and

22

H.Knoll, V.HHbalova, Di Gerlach, and W.Kohler

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cytoplasmic extracts (4, 12, 20), M-Protein preparations from type I, 12 or 19 group A streptococci obtained by hot hydrochloric acid extraction or by phageassociated lysin and purified by ion-exchange chromatography contain a nonspecifically mitogenic protein that can be separated from the M protein by further gel chromatography or immunochromatography on immobilized type-specific anti-

Mitogenic Activity of Erythrogenic Toxin ,..=.;;;c-:--- .....-"II"'II---,I

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Fig. 6. Separation of mitogenic activity of ET NY-S by thin layer isoelectric focusing. • - . pH gradient __• lymphocyte stimulation with 100 ,ul aliquots of gel eluates.

bodies (17). In resolving whether the mitogenic activity of streptococcal products is specific or nonspecific, purity of the preparations is a crucial point. Erythrogenic toxin has been described to stimulate lymphocytes both in vitro (13,22, 19, 7, 14, 21) and in vivo (8). This mitogenic effect seems to be nonspecific. A high degree of 3H-thymidine incorporation was obtained in nonintentionally pres ensitized individuals of different animal species and in man - both adult and newborn (22, 19, 7, 8, 6, 15, 1). There are also some findings that have been interpreted as evidence of an immunologically specific mechanism of ET mitogenic activity (1, 23). In a previous paper (6), the purification and characterization of an extracellular protein of strain NY-5 of Streptococcus pyogenes was described; the protein was identified biochemically and serologically as erythrogenic toxin type A. In testing its in vivo activities (10), we found that ET NY-5 induced erythema and swelling after intracutaneous injection into adult guinea pigs. It was pyrogenic for rabbits. Intravenous administration led to changes in circulating leucocyte counts, with marked transient granulocytopenia and protracted lymphopenia. The method of cross tolerance revealed that ET NY-5 contains erythrogenic toxins type A and C. Quantitative parameters in the human-blood lymphocyte response to ET NY-5 correspond with the type of reaction to a nonspecific mitogen. In comparison with Con A, the dose-response curve is broader, without a sharp peak. A similar dose-response curve was obtained with staphylococcal enterotoxins (28, 32). With the optimal dose of 1 fig/ml, which is comparable with the optimum values obtained for other preparations of purified streptococcal toxins (19, 1), maximum stimulation was obtained on day 3 or 4. However, while Con A stimulation produces a time-response curve with a very sharp peak at day 3, the intensity of 3H-thymidine incorporation after ET NY-5 stimulation decreases more slowly with cultivation time. A similar kinetics of response was observed by Nauciel in human lymphocytes (19) and by ourselves in rabbit lymphocytes (30). A kinetic study on guinea pig lymphocyte transformation showed a sharp peak on day 4 after 1.0 fig of toxin; a later maximum was obtained after a higher dose (1). In the present study, when the kinetics were observed up to day 9, the lymphocyte cultures of some persons displayed a "second wave" of incorporation in the pres-

24

H.Knoll, V. Hfibalovd, D.Gerlach, and W.Kohler

ence of ET NY -5. This effect was not observed in Con A-stimulated lymphocyte cultures (unpublished data). The reason may be that ET NY-5 stimulates human lymphocytes both non specifically and specifically. The simultaneous addition of both mitogens, Con A and ET, at the beginning of cultivation led to DNA synthesis suppression if high doses (supraoptimal or optimal) wer e used. The use of suboptima l doses elicited an additive response; in none of the do se combinat ion s tested did the two mitogens act synergisticly. An antagonistic effect of simulta neously added optimal or supraoptimal doses of two lymphocyte-stimulating agent s is well known; the addition of suboptimal con centrations, on the other hand, often leads to a synerg istic effect, less frequently to an additive response (18). An additive effect ha s been observed for staphylococcal enterotoxin Band PHA or Con A (28, 32). From experiments of th is type one cannot draw an y definite conclusion about the identity or diversity of the T -lymphocyte subpopulations responding to the two different mitogens used . Experiments on competitive inhibition of the nonspecific lymphocyte mitogenicity of str eptococcal pyrogenic exotoxins (SPE) and Con A suggested that th ere are differen ces in receptor specificies (27). The mit ogenictiy of th e exo tox ins was inh ibited, but th at o f Can A unaffected, bv gangliosides and sialic acid. a- Methyl-D-mannopyranoside inhibited the stimulatory action of both Ca n A and SPE type A, but not of the other two toxin types, B and C. D-galacto se at a concentrati on of 50 mM partially suppressed the mitogenic activiriy of SPE C, yet not of the other toxin types o r Con A. The aut hors concluded th at some activities of SPE's might be regulated by a ganglioside-binding pro perty whereas others might be contro lled by th e toxin binding to other rece ptors. Binding of ET NY-5 by lymphocytes can be demonstrated by abso rption experiments. When lymphocytes were exp osed to ET for 45 min, a decrease of mitogenic acti vity occured in the supernatants when tested in LTT. Eryth rocytes did not abso rb ET . Whether th e binding was du e to T o r B lymphocytes wa s not investigated. There are man y arguments for T-cell specificity of th e mitogenic effect of ET. Lymphocytes isolated from the th ymu s, but not from the bone mar row, were stimu la ted by ET ; the response was inhibited in rabbits pretreated with ant i-th ymocyte seru m and in mice after th ymectom y (9). T -cells isolated from human cord blood by mean s of spontaneous rosette forma tion with sheep erythrocytes were stimulated by all the toxin type s tested (26); B cells responded poorly. ETstimulated blast cells ha ve the ultrastructural features of T-blasts (30). A T-cellspecific mit ogenic resp on se to ET NY -5 was demonstrated by autoradiographic determination of ET NY- 5-stimulated blasts in combination with E-rosette formati on . E-rosette format ion with sheep red blood cells is a nonspecific marker of hum an T lymphocytes. Collins et al. (2) reported tha t mitogen-transformed T lymphocytes reta in th e ability to form ro sette s with sheep red blo od cells. The influen ce of ET N Y-5 on rose tte form at ion was ruled out by control exp eriments; after 72-h cultivation ET NY-5 did not influence significantly the pr op ortion of rosette-forming cells. Autoradi ographic stu dies revealed tha t 98 Ufo of ET NY-S-stimulated lymphoblasts with incorporated 3H -thymidine formed rosettes with sheep red blood cells. Nonlabelled rosette-forming cells were found in control cultures without ET. ET NY- 5 had prev iou sly been identifi ed as type A in terms of biochemical

Mitogenic Activity of Erythrogenic Toxin

25

and serological characteristics (6). A further purification procedure, viz. thin-layer isoelectric focusing, revealed two peaks of mitogenic activity, their pH values corresponding to the isoelectric points of SPE type A (3) and C (24). Also another biological test, immunologically specific pyrogenic cross tolerance, revealed two antigenically distinct pyrogens in the ET NY-5 preparation (10), one of them cross-reacting with culture filtrate of strain T 28, a producer of toxin A (33), the other with filtrate of strain T 18, which produces toxin C (33, 24). Independently from these biological tests, traces of C toxin were discovered in the ET NY-5 preparation by the precipitation reaction with antiserum to ET type C (6). The study of the interaction of erythrogenic toxins with lymphocytes may be of some importance for defining the pathogenetic role of these toxins in poststreptococcal sequelae; e. g. an immunoregulative function of these toxins might be involved.

References 1. Barsumian, E. L., P. M. Schlievert, and D. W. Watson: Nonspecific and specific im-

munological mitogenicity by group A streptococcal pyrogenic exotoxins. Infect. Immun. 22 (1978) 681-688 2. Collins, R. D., ]. L. Smith, G. P. Clein, and C. R. Barker: Absence of B- and T-cell marker on mitogen-transformed T-lymphocytes. Brit. J. Haemat. 26 (1974) 615-625 3. Cunningham, C. M., E. L. Barsumian, and D. W. Watson: Further purification of group A streptococcal pyrogenic exotoxin and characterization of the purified toxin. Infect. Immun. 14 (1976) 767-775 4. Francis, T. C. and]. ]. Oppenheim: Impaired lymphocyte stimulation by some streptococcal antigens in patients with recurrent apthous stomatitis and rheumatic heart disease. Clin. expo ImmunoJ. 6 (1970) 573-586 5. Gerlach, D. und W. Kohler: Untersuchungen zur Heterogenitat von Streptokinasen verschiedener Herkunft. Zbl. Bakt. Hyg., I. Abt. Orig. A 238 (1977) 336-349 6. Gerlach, D., H. Knoll, and W. Kohler: Purification and characterization of eryrhrogen.c toxins. I. Investigation of erythrogenic toxin A produced by Streptococcus pyogenes strain NY-5. Zbl. Bakt. Hyg., I. Abt. Orig. A 247 (1980) 177-191 7. Hhbaloua, V. and M. Posplsil: Lymphocyte-stimulating activity of scarlet fever toxin. Experientia 29 (1973) 704-705 8. Hiibaloua, V.: Biological effects of scarlet fever toxin and the role of activation of lymphocytes. J. Hyg. Epidem. (Praha) 18 (1974) 297-301 9. Hiibaloua, V. and M. Pospisil: The character of mitogenic activity of scarlet fever toxin. 11th Ann. Meet. Czech. Soc. Microbiol., Spindleniv Mlyn 1974. Abstr.: Folia microbiol. (Praha) 20 (1975) 84 10. Hilbaloua, V., H. Knoll, D. Gerlach, and W. Kohler: Purification and characterization of erythrogenic toxins. II. In vivo biological activities of erythrogenic toxin produced by Streptococcus pyogenes strain NY-5. Zbl. Bakt. Hyg., I. Abt. Orig. A 248 (1980) 314-322 11. [ondal, M., G. Holm, and H. Wigzell: Surface markers on human T and B lymphocytes. J. expo Med. 136 (1972) 207-215 12. Keiser, H., I. Kushner, and M. H. Kaplan: "Nonspecific" stimulation of lymphocyte transformation by cellular fractions and acid extracts of group A streptococci. J. Immunol. 106 (1971) 1593-1601 13. Kim, Y. B. and D. W. Watson: Streptococcal exotoxins: Biological and pathological properties. In: Streptococci and streptococcal diseases, Eds. I.. W. Wannamaker and J. M. Matsen, pp. 33-50. Aademic Press, New York (1972) 14. Knoll, Heide, G. Knoll und W. Kohler: Nachweis zellularer Antikorper gegen Streptokokkenantigene bei Kindem. Z. Immun.-Forsch. 151 (1976) 143-152

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H iKnoll , v. Hfibalova, D. Gerlach, and W.Kohler

15. Knoll, Heide, M. Gunther, C. Estel und W. Kohler: Lymphozytenrransforrnationstest mit Streptokokkenantigenen be i Neugeborenen und ih ren Miittern. Zbl. Gynak, 100 (1978) 689-695 16. Knoll, Heide, F. Peterm ann und W. Kohler: Untersuchungen zur M itogenitar erythrogener T oxin e. II. Mitteilung: N achwei s erythro gener To xine in Kulturfiltration von Strep tococcus pyogenes. Zbl. Bakt. H yg., I. Abt, Orig, A 240 (1978) 466-473 17. Kno ll, Heide, O. Kiihnemund, and J. Havlicek: Mitogenic and antigenic properties of group A st repto coccal M protein preparations. Immunobiol. 156 (1980) 537-548 18. Moller, G.: Induction of DNA synthesis in human lymphocytes: Interaction between non specifi c mitogens and antigens. Immunology 19 (1970) 583-598 19. Nauciel, C.: Mi togenic activity of purified streptococcal erythrogenic to xin o n lym phocyte s. Ann. Im mun ol. (Inst, Pasteur) 124 C (1973) 383-390 20. Pellegrino, M. A., S. Ferrone, ]. W. Safford Jr., A. B. Hirata, P. I. Terasaki, and R. A . Reisfeld: Stim ula tio n of lym phocyte transformation by strepto coc cal type Ml protein : relationsh ip to HL-A ant igens. J. Immunol. 109 (1972) 97- 102 21. Petermann . F., Heid e Knoll und W. Kohl er: Untersuchungen zur M itogenitat erythroge ner T oxine. I. M itt eilung: Typenspezifische Hemmung der mitogenen Akrivitat erythrogene r Toxine d urch anritox ische Seren von Kaninchen. Zbl. Bakt. H yg., I. Abt, O rig. A 240 (197S) 366-379 22. Plate, ]. M . and B. Amos: Lymphocyte stimulatio n by a glycopeptide isolated from Streptococcus pvog enes C 203 S. I. Isolation and p arti al purificati on. Cell. Immunol. 1 (1971 ) 476-487 23. Plate, ]. M . and B. Amos: Lymphocyte stimulatio n by a glycopeptide isol ated from Stre ptococcus pvogenes C 203 S. II. Kinetics of the response. Cell. Immunol. 1 (1971) 488-499 24. Schlievert, P. M., K. M . Bettin, and D. W. Watson: Purification and characterization of gro up A st reptococcal pyrogenic exo to xin type C. Infect. Immun. 16 (1977) 673-679 25. Schlieuert, P. M ., K. M . Bettin, and D. W. Wa tson: Production of pyrogenic exo toxin by gro ups of streptococci : associati on with group A. J. infect. D is. 140 (1979) 676-681 26. Schlievert, P. M ., D . ]. Schoettl e, and D. W. Wa tson: Nonspe cific T -lymphocyt e mito genesis by pyrogen ic exot oxins from group A streptococci and Staph ylococcus aureus. Infe ct. Imrnun , 25 (1979) 1075-1077 27. Schlieuert, P. M., D. [ . Schoettle, and D. W. Watson: Ganglioside and monosaccharide inhibition of nonspecific lymphocyte mitogenicity by gr oup A streptococcal pyrogenic exotox ins. In fect. Imm un . 27 (1980) 276-279 28. Sham baugh III., G. E. and G. R. Blum enschein : Combined effects of phytohemagglut inin and sta phylococcal enterotoxin B on deo x yrib onucleic acid synthesis during blast tran sform at ion in human lymphocytes. Infect. Immun. 9 (1974) 384- 390 29. Taylor, A . G.: Lymphocyte-trans fo rm ing activity of streptococci bel onging to various Lancefield gro ups. J. M ed. Mi crobiol. 5 (1972) 61-65 30. T rebichausk y, I., V. Hiibaloud, and M. Pospisil: In vitro lymphocyte stimulation with scarlet fever toxin. Foli a bioI. (Praha) 24 (1978) 101-106 31. Wa ite, W. J. and K. Hirschhorn : Chapter 25: The lymphocyte resp onse to acti vators. In: Handbook of Ex pe rim ental Immunolog y, Vol. 2: Cellular Immunology, Ed. D. M . We ir. Blackwell Scienti fic Publ ication s, Oxfo rd - London - Edinburgh - M elbourne (1973) 32. Wa rren, J. R., D . L. Leatherman, and ] . F. Me tzg er: Evidence for cell-receptor act ivity in lymphocyte stim ula tio n by st aphylococcal enterotox in. J. Immunol. 115 (1975) 49-53 33. Watson, D. W.: Host-parasite factors in group A streptococcal infections. Pyrogenic and other effects of immunologic distinct exotoxins related to scarlet fever toxins. .I. expoMed. 111 (1960) 255-284 Dr. Heide Knoll , Zentralinsrirur fiir M ikrobiologie und exp erimenrelle Therapie, Beutenbergstr . 11, DDR-69 Jena