(Acta Paediatr Jpn 1992; 34: 5 16
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524)
Possible Role of Streptococcus pyogenes in Mucocutaneous Lymph Node Syndrome. XV. Potential Utility of Streptococcal Pyrogenic Exotoxin Toxoid for the Prophylaxis and Treatment of MCLS Takehisa Akiyama, M.D.’, Kaoru Shimanuki, M.D.2 and Kimio Yashiro, M.D.2 ’SMI Bristol Co. Ltd. and ’Department of Pediatrics, Kitasato University School of Medicine,
Sagamihara, Kanagawa. Japan
Mice made tolerant to streptococcal pyrogenic exotoxin (SPE) by neonatal inoculation with SPE emulsified in incomplete Freund’s adjuvant demonstrated early thrombocytopenia followed by thrombocytosis. This state is the perfect counterpart of patients with mucocutaneous lymph node syndrome (MCLS). We have hypothesized that by inducing tolerance to SPE, the biological activities of the toxin might play leading roles in the pathogenesis of MCLS. In the present investigations, the efficacy of SPE on the prophylaxis and treatment of diseases caused by Streprococcus mogenes (including MCLS) were monitored using the murine model system accompanied with a platelet-counting technique. The mice, rendered tolerant due to neonatal SPE inoculation and followed by immunization with SPE toxoid about 1 month prior to the provocative injections with SPE, demonstrated an almost complete lack of response to the provocation, keeping platelet counts within the normal range of values (except for a marginally significant thrombocytosis 7 days postprovocation). Moreover, anti-SPE titers of the sera from the mice sacrificed on day 35, at which point the observation was terminated, were proved to be markedly elevated when compared with controls. These findings seem to suggest that immunization with the toxoid could overcome tolerance, resulting in the production of an antitoxin. In a second experiment that examined the effect of administration with rabbit antiserum raised against the toxoid, the antiserumtreated mice demonstrated a transitory thrombocytosis on 7 days postprovocation with SPE, followed by an abrupt decrease in the number of platelets from day 10 onward. Such a finding was in complete agreement with that observed in tolerant mice administered with antiserum specific for SPE, suggesting a strong neutralizing activity against SPE of the antitoxoid serum. A third experiment evaluated the effect of F(ab’), fkagments of the rabbit antiserum to the toxoid on platelet activity. The tolerant mice passively administered i.v. with the F(ab’), fractions demonstrated a complete lack of responsiveness, except for a small thrombocytosis on days 7 and 10. Key Words Mucocutaneous lymph node syndrome, MCLS,Srwprocuccus pyogenes, Platelet activation, Streptococcal pyrogenic exotoxin-toxoid
Received February 7, 1992 Revised March 19, 1992 Accepted March 26, 1992
Correspondence address: Dr Takehisa Akiyama, SMI Bristol Co. Ltd., 2-14-1 5 , Kobuchi, Sagamihara, Kanagawa 229, Japan.
Role of Streptococcus pyogenes in MCLS (27) 5 17
Introduction Patients with mucocutaneous lymph node syndrome (MCLS) have demonstrated unequivocally heightened cell-mediated reactivity to Streptococcus pyogenes-associated antigens in spite of a nearly complete lack of antibodyforming activity to the same antigens [l-31. Furthermore, in a subsequent study performed to assess the humoral immunoresponsiveness of peripheral blood lymphocytes from MCLS patients, the cells were cultured with or without the streptococcal antigens, resulting in the reaffirmation of their non-responsiveness to S. pyogenes-associated antigens coupled with normal responsiveness to unrelated antigens [4]. In addition, a high amount of streptococcal pyrogenic exotoxin (SPE) and its presence in 100% of sera on the day of admission from MCLS patients, as well as the association of defervescence with a rapid decline of SPE toward control values following the initiation of treatment with human pglobulin, were proved by the use of our enzyme-linked immunosorbent assay (ELISA) system reinforced by the introduction of monoclonal antibody to SPE [5]. Taken together, these findings strongly suggest the possibility that: (i) the induction of immunological tolerance specific for SPE in the hosts might play an indispensable role in the pathogenesis of MCLS; (ii) the well-known efficacy of intravenous human y-globulin might be accounted for by the putative presence of antibodies to SPE; and (iii) the breakdown of the immunological tolerance to SPE could be attained by the immunization with a toxoid antigenically slightly modified from native SPE, enabling the host to prevent itself from being attacked by the disease. Thus, the probable effects of the toxoid on the prophylaxis and treatment of MCLS were monitored in the current experiments using a model system in which mice were made tolerant to streptococcal antigens by either neonatal inoculation with SPE or infection with group A /?-hemolytic streptococci, immunized approximately 2 months later by multiple courses of injection with the toxoid, and their platelet count recorded in response to the provocative
Vol. 34 No. 5 October I992
infections performed concomitantly with the same species of bacteria. The reliability of the animal model was ascertained in a previous study that indicated a strong correlation between the fluctuation of platelet activation observed in these mice and in patients with MCLS [6].
Materials and Methods Mice Pregnant Std-ddy mice were purchased exclusively from the Shizuoka Agricultural Cooperative Farm a few days before their expected delivery, and thereafter were maintained in the laboratory in order to ascertain the days of birth of their offspring.
Rabbits Rabbits used in the present experiments were all random-bred male New Zealand White strain housed in a closed colony, and weighed approximately 2.5 kg at the beginning of the studies. Needless to say, natural infection in this species of animal with group A /?-hemolyticstreptococci does not occur.
Streptococcuspyogenes In the present studies, two attenuated strains of S. pyogenes, designated as B-346 op and S-43, were employed. Their taxonomical and serobiological characteristics have been described in detail elsewhere [7, 81.
Induction of immunological tolerance Immunological tolerance was induced by neonatal inoculation with either living streptococci of B-346 op strain or SPE emulsified in incomplete Freund’s adjuvant. Provocation was performed approximately 3 months later with four S.C.infections every other day with streptococci of S-43 strain. These methods were the same as those described circumstantially by us previously [3,9].
Extraction of SPE and preparation of toxoid In the present experiments, SPE of type A was employed, and was extracted from the culture supernatant of S. pyogenes strain N Y - 5 accord-
5 I8 (28) Akzyarna et al. ing to the method of Cunningham et al. [lo] and modified by Oyake and Akiyama [ 1 11. The method used for the preparation of the toxoid was modified from that of Wadsworth et al. [ 121. In brief, 640 pg of SPE were dissolved in 2 ml of 0.05 moll1 phosphate buffer solution (pH 7.4) containing formalin at a final concentration of 0.2%, and incubated at 37°C for 5 days. After the period of incubation, the samples were dialysed with continuous stirring against three changes of 3 1 each of phosphate-buffered saline (PBS) for 6 hr at 4°C to eliminate the excess formalin in the solution, and were kept frozen in small volumes and thawed as needed. The results of the following tests established that complete detoxification of the experimental sample took place under the above-mentioned conditions without apparent loss of antigenic activitiy.
Assays for toxic and antigenic properties of the toxoid The toxic potency of the toxoid was calculated according to the method described by Nakahara (pers. comm.). Briefly, a single 0.05 ml dose each of SPE or the toxoid dilutions containing from 65.5 to 4,OOOng protein and 65.5 to 16,000 ng, respectively, was injected i.c. on the depilated flank of either side of the rabbits. The small wheal that resulted disappeared in a few minutes. Positive reactions usually began to appear 4-6 hr after the inoculation. At first, they consisted of a small circular area of erythema. The red area increased and reached a maximum size and intensity between 18 and 36 hr postinoculation. Soon after reaching their maximum size and intensity, the reactions began to subside. Even the most strongly positive did not persist more than 48 hr. The bright red color became dull and began to fade. The reactions were evaluated as negative or positive at the end of 24 hr. Positive reactions were more than 10 mm in both diameters and bright red, with some swelling of the skin. Subsequently, the reactions due to native SPE were compared with those produced by the stimulation with the toxoid in the same animals. Three rabbits were used for each series of titration, and the results were averaged.
In order to determine the antigenic properties of the toxoid, antisera were raised in three rabbits by primary i.m. immunization with approximately 2 pg of the toxoid emulsified in complete Freund’s adjuvant and five booster i.v. injections of the same amount each of the antigen in saline. Then, the serum collected and pooled from these animals was tested for the presence of antibodies capable of changing the platelet activity when it was inoculated into tolerant mice.
Digestion of antibodies with pepsin to isolate F(ab’), fragments Rabbit antiserum (10 ml) was passed over a column of protein A-Sepharose (AmpureTMPA Kit, Amersham Japan, Tokyo, Japan) and the bound material eluted. The eluate (approximately 41 mg IgG) was dialysed against four changes of 2 1 each of 0.1 mol/l acetate buffer (pH 4.0) for 3.5 hr at 4°C. Following dialysis, pepsin (#70 12, 1:60,000; Sigma, St Louis, MO, USA) was added at a ratio of 0.4mg per 30mg of IgG. Digestion was allowed to proceed for 16 hr at 3 7 T , and was terminated by dialysis against 0.01 mol/l PBS (pH 7.0) at 4’C. The fragments were concentrated by ultrafiltration using a Minicon B 125 Concentrator (Amicon Corp., Danvers, MA, USA), followed by chromatography on the same protein A kit. The great majority of the protein passed straight through the column, although traces of undigested Fc pieces or intact IgG remained. Since the binding of y-chains to protein A involves the Fc piece [ 131, F(ab’), fragments do not bind to the gel. Any residual undigested IgG and Fc fragments were eluted with 0.58% (v/v) glacial acetic acid in 0.15 mol/l NaC1. Platelet counting The number of blood platelets was counted using the method described in detail previously [61. Quantitation of anti-SPE antibodies in the serum ELISA was employed to quantitatively determine the antibodies specific for SPE-A in the
Acta Paediatr Jpn
Role of Streptococcus pyogenes in MCLS (29) 5 19 serum of the mice used in the present studies. In advance, SPE-A was dissolved in 0. I moll1 carbonate buffer solution (pH9.6) so that the amount required (5 pg protein) was contained in 50 p1 of the solution. The portions were added to each well of 96-well flat-bottomed polystyrene microtiter plate (MS-3596F/H; SumitomoBakelite Co, Tokyo, Japan), and the plates were coated by incubation at 4°C overnight. After the plates were washed three times with PBS containing 0.05% Tween 20, non-specific binding sites were blocked by incubation with 1% bovine serum albumin (BSA)-containing PBS at 37°C for 2 hr. After being washed three times with the same buffer solution, murine sera to be tested were diluted 1:1,000 with PBS (50p1), added to each well and incubated at 4% overnight, followed by three washes with 0.05% Tween 20-PBS. Subsequently, the horseradish peroxidaseconjugated F(ab’), fragments of goat antiserum specific for mouse IgG/IgM (Tago, Inc, Burlingame, CA, USA), which were diluted 1:2,000 with PBS containing 0.05% Tween 20 and 1% BSA (1 00 pl), were added to each well, followed by incubation for 135 min at room temperature. After the plates were washed five times with 0.05% Tween 20-PBS, 200p1 of 4.4mmol/l o-pheylendiamine dihydrochloride (Wako Pure Chemical Co, Tokyo, Japan) dissolved in citrate-phosphate buffer (pH 5.0) were added to each well, incubated for 25 min at room temperature, and thereafter the enzyme action in individual wells was stopped by the addition of
50p16N H2S04.In most experiments, the plates were read on a microplate spectrophotometer (MTP-12; Corona Electronics Co, Tokyo, Japan) at a wavelength of 500 nm.
Results Immunization with SPE or toxoid and platelet functions in tolerant mice In the first experiments, mice were rendered tolerant to either S. pyogenes-associated antigens due to neonatal infection with group A P-hemolytic streptococci and followed by provocation 3 months later with S. pyogenesinfections, or rendered tolerant to SPE by neonatal inoculation with SPE-A and followed by provocation with the same toxin. The purpose of these experiments was to evaluate the effect of immunization carried out, approximately 1 month prior to the provocation, with SPE or the toxoid, on platelet functions as measured by the platelet counting. Beforehand, toxic activity of the toxoid was quantified by means of a rabbit skin test that demonstrated almost perfect detoxification, as determined from the minimum dose required to produce erythema, which was more than 120 times greater than that of intact SPE (Table 1). Figure 1 shows the results of the experiment, in which four groups of five mice rendered tolerant and provoked with living S. pyogenes were employed. The tolerant animals, in either or presence (. 0 *) of the absence (-0-) immunizations with SPE. demonstrated a com-
Table 1. Relative toxic activity of the toxoid quantified by the rabbit skin test compared with that of intact SPE. Size of erythema (rnrn)
Dose of formalin-treated SPE
Dose of SPE Time Postinoculation 62.5 ng
125 ng
250 ng
500 ng
1,000 ng
62.5 ng 250 ng
1,000 ng 4,000 ng 16,000 ng
10 hr
7 x 7 1 2 ~ 1 2 1 2 ~ 1 2 1 2 ~ 1 2 1 4 ~ 1O 4x 0 Ox0 Ox0 Ox0 7 x 7 O x 0 10x10 13x12 O x 0 Ox0 9 x 9 11x11 13x12 13x12 14x12 11 x 10 12 x 11 12 x 12 13 x 13 12 x 11 13 x 13 13 x 12 15 x 15 15 x 15 16 x 15
24 hr
9 x 8 1 2 ~ 1 2 1 2 ~ 1 2 1 2 ~ 1 2 1 3 ~ 51 x2 5 9 x 9 13x12 15x14 15x15 17x17 7 x 6 13x13 13x12 15x15 17x16 17x16 7 x 7
32 hr
6 x 6 9 x 9 8 x 8
7 x 7 3 x 3 10x1013~13 5 x 5 6 x 6 1 2 x 1 1 1 0 x 1 0 1 1 ~ 1 11 5 x 1 5 9 x 9 1 0 x 1 0 1 0 ~ 1 0 1 7 ~ 1 7 7 x 7
Vol. 34 No. 5 October 1992
7 x 7 6 x 6
7 x 7 7 x 5 9 x 9
7 x 6 10x10 10x10 15x15 1 0 x 9 12x12
5 x 5 7 x 7 8 x 6
Ox0 Ox0 9 x 9
Ox0 8 x 8 10x9
5 x 5
7 x 7 12x12 12x12
520 (30) Akiyama et al. those presented in the aforementioned experiment, as illustrated in Fig. 2. On day 35 postprovocation, at which time the observation was terminated, blood was collected from all mice and anti-SPE titers of their sera were assessed by ELISA. The data illustrated in Fig. 3 strongly suggest that immunization with the toxoid could play a decisive role in the breakdown of immunological tolerance, leading to enhanced antibody-formation against SPE.
Provocative infection with 5-43
. ,
1
0
3
7 10
14 17 20
30
35
Time (days after 1st provocative infection)
Fig. 1. Effects of immunization (arrowed) with the toxoid or SPE on platelet activation in mice made tolerant to S. pyogenes-associated antigens by means of neonatal infection with an attenuated strain of group A /I-hemolytic streptococci, and followed by provocation 3 months later with living S. pyogenes of an attenuated strain. Values are given as mean f s.d. of five mice.
pletely uniform behavior in response to the provocation, showing a definite decrease in the number of platelets on days 3 and 7 postprovocation followed by an abrupt increase in number after day 7 onward. The number reached a maximum on day 10 and thereafter waned gradually, but was still elevated over the following weeks. In striking contrast to the results in these groups are those in the remaining two groups, which consisted of five mice that were either rendered tolerant and followed by immunizaor the same tion with the toxoid (-*-A**-), number without neonatal pretreatment that were later immunized (-m-). The immunization with SPE and the toxoid consisted of four S.C. injections, performed on alternate days, with 2 pg each of the preparations. These groups did not respond to the provocation throughout the observation period, except for the former group, which demonstrated a marginally significant increase in platelet number on day 7 postprovocation. On the other hand, in the second series of experiment in which SPE was employed as a substitute for living streptococci to provide the stimuli for tolerance induction and provocation, the results were in complete agreement with
Effects of rabbit antiserum specific for the toxoid on platelet activation The second experiment was devised solely to confirm the aforementioned suggestion, and examined the effect of the administration of rabbit antiserum to the toxoid on the function of platelets. The results of the experiment are summarized in Fig. 4. The antiserum-treated animals that had been provided with neonatal infection with S. pyogenes (- A or SPE inoculation (-0-) exhibited a transitory thrombocytosis on day 7 postprovocation, that concurred with 0)
Provocative inoculation with SPE ,
0
*
,
3
I
7
12
17 20
25
30
35
Time (days alter 1 st provocative inoculation)
Fig. 2: Fluctuation of platelet counts in mice exposed to neonatal and provocative inoculations with SPE, in response to the immunizations with the toxoid or SPE performed approximately 1 month prior to the provopositive tolerance induction but cation. (+), without any immunization; (. 0 *), positive tolerance induction followed by immunization with SPE; (. A *), positive tolerance induction followed by immunization with the toxoid; (-=-), controls subjected to neither neonatal treatment nor immunization.
Acta Paediatr Jpn
Role of Streptococcus pyogenes in MCLS (3 1) 52 1
-5
0.7
1
9
0
-.0
in
0.5.
L
c v1 0
- 0.3,
.-
8
8 ._
0"
0
0.1
,
:
**
0
5
6
0
I 3
2
1
4
7
a
Fig. 3: Anti-SPE titers of sera as quantified by ELISA in mice sacrificed on day 35 postprovocation with (a) multiple infections with S-43 and (b) multiple inoculations with SPE. ( I ) and ( 5 ) were tolerance induced by infection/inoculation (no immunization); (2) and (6) were tolerance induced by infection/inoculation and immunized with SPE; (3) and (7) were tolerance induced by infection/inoculation and immunized with toxoid; and (4) and (8) were not tolerance induced or immunized.
Provocative infection with S-43
The effect of F(ab'), fragments on platelet function
,
1
0
3
7
1012
16
21
20
35
Time (days following 1st provocative infection)
Fig. 4: The effect of passive immunization with rabbit antiserum raised against the toxoid on the platelet functions in mice rendered tolerant to streptococcal antigens due to neonatal treatments and followed by provocative infections.
the period of thrombocytopenia in the other two groups of mice made tolerant by the neonatal infection (. A 0 ) or SPE inoculation (4-) but without any passive immunization. This was followed by an abrupt decrease in platelet number from day 10 onward. Such a result bears a striking resemblance to that observed in tolerant mice administered with antiserum specific for SPE, as reported previously [ 6 ] ,indicating that the antiserum to the toxoid possesses a strong neutralizing activity against SPE.
Vol. 34 No. 5 October 1992
Human immune serum globulin prepared by the ethanol method of Cohn et al. [14] and Oncley et al. [I51 produces a high incidence of untoward reactions when given intravenously. The seriousness of the reaction is proportional to the decrease in the serum complement level. There is a need for a safe and effective intravenous preparation of immune globulin so that large volumes of this material can be infused to attain a high titer of circulating antibody rapidly. Therefore, numerous approaches have been utilized to remove the anticomplementary activity: (i) Enzymatic digestion at pH 4 . 0 (ii) ultracentrifugation; and (iii) incubation of the immune globulin at pH 4.0 and 37°C. To date, the most promising method in terms of the removal of anticomplementary activity and retention of long in vivo half-life has been the low pH treatment; however, some of these materials have required additional treatment with pepsin to remove the anticomplementary activity completely [ 161. In the third experiment, the effect of the F(ab'), fraction of the rabbit antitoxoid serum on platelet activation was assessed, and compared with that of the intact whole antiserum. As may be seen from Fig. 5, the platelet counts in mice exposed to neonatal and provocative infections with S. pyogenes (-G)and controls provoked with the infections that did not matched undergo neonatal treatment (4-)
522 (32) Akiyarna el al. Provocative inleciion with 5-43
I
0
3
7
10 12 15
20
25
32
Time (days following 1 st provocative infection)
Fig. 5: Relative effects of F(ab’), fractions and whole unfractionated rabbit antitoxoid serum on platelet functions in mice exposed to neonatal and provocative infections.
our expectations. The tolerant animals passively administrated s.c., four times every other day, with lop1 each of the whole antiserum (. A *) or i.v. with 20 pg each of the F(ab‘), fragments (. A a**), however, demonstrated significant but transitory increases in the number of platelets on days 7 and 10. Taken together, these studies strongly suggest the possibility that intravenous transfusion with F(ab’)z fragments of serum from volunteers hyperimmunized with the toxoid could exert a surprisingly efficacious effect in patients during the early stages of MCLS.
Discussion In the animal model established previously [3, 81, mice exposed to neonatal infection with a strain of S. pyogenes (which fails to confer cell-mediated immunity [CMI] if inoculated into mature murine hosts) and followed by repetitive infections 4-6 weeks later with another strain of the same species of bacteria that is endowed with an intrinsic ability to afford mature mice CMI against streptococcal antigens, demonstrated heightened cellular responses to those antigens in spite of a complete lack of humoral responsiveness to the same stimuli. Such immunological characteristics are
completely identical with those observed in MCLS patients. The results of the current investigations reaffirmed the utility of the animal model by demonstrating early thrombocytopenia followed by thrombocytosis in the SPE-induced tolerant mice in response to the provocative infection, which once again resembles MCLS. In a recent study [5], the incidence of a considerable amount of SPE in 100% of cases combined with its rapid decline during defervescence brought about by treatment with intravenous human y-globulin was demonstrated in the sera of MCLS patients by the use of ELlSA in combination with monoclonal antibodies specific for SPE. In view of these observations, an induction of immunological tolerance against SPE and biological activities of the toxin have been hypothesized as principal factors involved in the initiation of events resulting in MCLS. Needless to say, SPE is also considered to be associated with the development of the skin rash characteristic of so-called streptococcal infection (synonymous with scarlet fever), and this property can be inhibited by antitoxin [ 171. The subsequent discussion will be focused solely on the potential use of the SPE-toxoid for the prophylaxis and treatment of these diseases, the realization of which could be expected in the near future. First, it has already been suggested that the well-known efficacy of intravenous transfusion with human pglobulin in reducing the duration of systemic inflammation in the early course of these diseases can be accounted for by the putative presence in the inoculum of a fair level of antibodies to SPE. Therefore, we are tempted to recommend that the administration of the F(ab’)2 fragments separated from the sera of volunteers that are hyperimmunized with the toxoid should be tried instead of human y-globulin. Second, the monomodal age distribution of MCLS (there is a peak occurrence during the first 2 years of life, and few are affected over the age of 8 years) might be explained exclusively by the maintenance of a special immunological tolerance to 5’. pyogenesassociated antigens, and
Acta Paediatr Jpn
Role of Streptococcus pyogenes in MCLS (33) 523 an induction of the immunological state might be caused by the streptococcal infection(s) occurring perinatally or in the neonatal period [ 81. In addition, in subsequent investigations performed to gauge the responsiveness of the cultured lymphocytes from outpatients attending at regular intervals after recovery from MCLS, we were surprised at the continuation for more than 9 years of the depressed antibodyforming activities to streptococcal antigens [4]. These results will explain the well-known tendency of this disease to relapse, repeatedly in some cases, after apparent recovery from the preceding episode. Taken together, it appears that the immunological tolerance in children less than 4-6 years old whose deficient immunoresponsiveness to SPE and streptolysin-0 was proved, as well as in the above-mentioned outpatients with continuous susceptibility to recurrence of MCLS, could be overcome by an inoculation with the toxoid. Furthermore, pregnant women with a complete lack of antibody formation against the streptococcal antigens should be another target for immunization with the toxoid. Such an immunization will prevent their offspring from being affected with the streptococcal infections during early life, leading to their freedom from the development of the immunological tolerance to S. pyogenes-associated antigens.
4.
5.
6.
7.
8.
9.
10.
Acknowledgements We are grateful to Miss Toshiko Hasobe for her excellent technical assistance.
References 1. Akiyama T, Osawa N, Yashiro K, Hiraishi S . Possible role of Streptococcus pyogenes in mucocutaneous lymph node syndrome. Acta Paediatr Jpn 1985; 27: 205-213. 2. Akiyama T, Osawa N, Yashiro K, Hiraishi S. Possible role of Streptococcus pyogenes in mucocutaneous lymph node syndrome. 11. Heightened cellular reactivitiy to stimulation with mammalian myocardial and arterial antigens in MCLS patients. Acta Paediatr Jpn 1986; 28: 788-796. 3. Akiyama T, Osawa N, Yamaura N, Yashiro K, Hiraishi S. Possible role of Streptococcuspyogenes in mucocutaneous lymph node syndrome. VI. Heightened cellular reactivity to streptolysin-0 in
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mice infected with S. pyogenes and in MCLS patients. Acta Paediatr Jpn 1988; 30: 68-72. Osawa N, Yamaura N, Ikewaki N, Yashiro K, Akiyama T. Possible role of Streptococcus pyogenes in mucocutaneous lymph node syndrome. VIII. Immunological tolerance to S. pyogenesassociated antigens seems essential to induction of MCLS. Acta Paediatr Jpn 1989; 3 1: 462-468. Kawai M, Osawa N, Yamaura N et al. Possible role of Streptococcus pyogenes in mucocutaneous lymph node syndrome. IX. Quantitation by ELISA of streptococcal pyrogenic exotoxin in the serum of MCLS patients. Acta Paediatr Jpn 1989; 3 1: 529-536. Akiyama T, Osawa N, Shimanuki K, Yashiro K, Oyake T. Possible role of Streptococcus pyogenes in mucocutaneous lymph node syndrome. XII. Variable responses of platelets in MCLS seem to be explainable by streptococcal pyrogenic exotoxin. Acta Paediatr Jpn 1991; 33: 20-26. Akiyama T, Osawa N, Nakasato K, Yamashina S , Yamamoto N, Tamaki H, Yashiro K. Possible role of Streptococcus pyogenes in mucocutaneous lymph node syndrome. 111. Electron microscopic observations on group A hemolytic streptococci inoculated into mice. Acta Paediatr Jpn 1987; 29: 417-425. Akiyama T, Osawa N, Yashiro K. Possible role of Streptococcus pyogenes in mucocutaneous lymph node syndrome. IV. Immunological tolerance following exposure of neonatal mice to live attenuated strains of Streptococcus pyogenes. Acta Paediatr Jpn 1987; 29: 426-432. Akiyama T, Osawa N, Yamaura N, Yashiro S , Watanabe M. Possible role of Streptococcus pyogenes in mucocutaneous lymph node syndrome. VII. A weak response to Epstein-Barr viral capsid antigen in mice infected neonatally with S. pyogenes. Acta Paediatr Jpn 1988; 30: 654-661. Cunningham CM, Barsumian EL, Watson DW. Further purification of group A streptococcal pyrogenic exotoxin and characterization of the purified toxin. Infect Immun 1976; 14: 767-715. Oyake T, Akiyama T. Possible role of Streptococcus pyogenes in mucocutaneous lymph node syndrome. X. Effects of streptococcal pyrogenic exotoxin on the development of immunological abnormalities in tolerant mice. Nihon Shonikagakkai Zasshi 1990; 94: 2320-2327 (in Jpn). Wadsworth A, Quigley JJ, Sickles GR. Preparation of diphtheria toxoid. The action of formaldehyde: Precipitation by calcium. J Infect Dis 1937; 61: 237-250. Kronvall G, Frommel D. Definition of staphylococcal protein A reactivity for human immunoglobulin G fragments. Immunochemistry 1977; 7: 124-127. Cohn EJ, Strong LE, Hughes WL, Mulford DJ, Ashworth JN, Melin M, Taylor HL. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. J Am Chem SOC1946; 68: 459-475. Oncley JL, Melin M, Richert DA, Cameron JW, Gross PM Jr. The separation of the antibodies,
524 (34) Akiyama et al. isoagglutinins, prothrombin, plasrninogen, and B,-lipoprotein into subfractions of human plasma. J Am Chem Soc 1949; 71: 541-550. 16. World Health Organisation. The use of human immunoglobulin. World Health Organisation
Technical Report Series No. 327. 1966. 17. Ginsburg I. Mechanisms of cell and tissue injury induced by group A streptococci: Relation to poststreptococcal sequelae. J Infect Dis 1972; 126: 294-340.
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524)
Possible Role of Streptococcus pyogenes in Mucocutaneous Lymph Node Syndrome. XV. Potential Utility of Streptococcal Pyrogenic Exotoxin Toxoid for the Prophylaxis and Treatment of MCLS Takehisa Akiyama, M.D.’, Kaoru Shimanuki, M.D.2 and Kimio Yashiro, M.D.2 ’SMI Bristol Co. Ltd. and ’Department of Pediatrics, Kitasato University School of Medicine,
Sagamihara, Kanagawa. Japan
Mice made tolerant to streptococcal pyrogenic exotoxin (SPE) by neonatal inoculation with SPE emulsified in incomplete Freund’s adjuvant demonstrated early thrombocytopenia followed by thrombocytosis. This state is the perfect counterpart of patients with mucocutaneous lymph node syndrome (MCLS). We have hypothesized that by inducing tolerance to SPE, the biological activities of the toxin might play leading roles in the pathogenesis of MCLS. In the present investigations, the efficacy of SPE on the prophylaxis and treatment of diseases caused by Streprococcus mogenes (including MCLS) were monitored using the murine model system accompanied with a platelet-counting technique. The mice, rendered tolerant due to neonatal SPE inoculation and followed by immunization with SPE toxoid about 1 month prior to the provocative injections with SPE, demonstrated an almost complete lack of response to the provocation, keeping platelet counts within the normal range of values (except for a marginally significant thrombocytosis 7 days postprovocation). Moreover, anti-SPE titers of the sera from the mice sacrificed on day 35, at which point the observation was terminated, were proved to be markedly elevated when compared with controls. These findings seem to suggest that immunization with the toxoid could overcome tolerance, resulting in the production of an antitoxin. In a second experiment that examined the effect of administration with rabbit antiserum raised against the toxoid, the antiserumtreated mice demonstrated a transitory thrombocytosis on 7 days postprovocation with SPE, followed by an abrupt decrease in the number of platelets from day 10 onward. Such a finding was in complete agreement with that observed in tolerant mice administered with antiserum specific for SPE, suggesting a strong neutralizing activity against SPE of the antitoxoid serum. A third experiment evaluated the effect of F(ab’), fkagments of the rabbit antiserum to the toxoid on platelet activity. The tolerant mice passively administered i.v. with the F(ab’), fractions demonstrated a complete lack of responsiveness, except for a small thrombocytosis on days 7 and 10. Key Words Mucocutaneous lymph node syndrome, MCLS,Srwprocuccus pyogenes, Platelet activation, Streptococcal pyrogenic exotoxin-toxoid
Received February 7, 1992 Revised March 19, 1992 Accepted March 26, 1992
Correspondence address: Dr Takehisa Akiyama, SMI Bristol Co. Ltd., 2-14-1 5 , Kobuchi, Sagamihara, Kanagawa 229, Japan.
Role of Streptococcus pyogenes in MCLS (27) 5 17
Introduction Patients with mucocutaneous lymph node syndrome (MCLS) have demonstrated unequivocally heightened cell-mediated reactivity to Streptococcus pyogenes-associated antigens in spite of a nearly complete lack of antibodyforming activity to the same antigens [l-31. Furthermore, in a subsequent study performed to assess the humoral immunoresponsiveness of peripheral blood lymphocytes from MCLS patients, the cells were cultured with or without the streptococcal antigens, resulting in the reaffirmation of their non-responsiveness to S. pyogenes-associated antigens coupled with normal responsiveness to unrelated antigens [4]. In addition, a high amount of streptococcal pyrogenic exotoxin (SPE) and its presence in 100% of sera on the day of admission from MCLS patients, as well as the association of defervescence with a rapid decline of SPE toward control values following the initiation of treatment with human pglobulin, were proved by the use of our enzyme-linked immunosorbent assay (ELISA) system reinforced by the introduction of monoclonal antibody to SPE [5]. Taken together, these findings strongly suggest the possibility that: (i) the induction of immunological tolerance specific for SPE in the hosts might play an indispensable role in the pathogenesis of MCLS; (ii) the well-known efficacy of intravenous human y-globulin might be accounted for by the putative presence of antibodies to SPE; and (iii) the breakdown of the immunological tolerance to SPE could be attained by the immunization with a toxoid antigenically slightly modified from native SPE, enabling the host to prevent itself from being attacked by the disease. Thus, the probable effects of the toxoid on the prophylaxis and treatment of MCLS were monitored in the current experiments using a model system in which mice were made tolerant to streptococcal antigens by either neonatal inoculation with SPE or infection with group A /?-hemolytic streptococci, immunized approximately 2 months later by multiple courses of injection with the toxoid, and their platelet count recorded in response to the provocative
Vol. 34 No. 5 October I992
infections performed concomitantly with the same species of bacteria. The reliability of the animal model was ascertained in a previous study that indicated a strong correlation between the fluctuation of platelet activation observed in these mice and in patients with MCLS [6].
Materials and Methods Mice Pregnant Std-ddy mice were purchased exclusively from the Shizuoka Agricultural Cooperative Farm a few days before their expected delivery, and thereafter were maintained in the laboratory in order to ascertain the days of birth of their offspring.
Rabbits Rabbits used in the present experiments were all random-bred male New Zealand White strain housed in a closed colony, and weighed approximately 2.5 kg at the beginning of the studies. Needless to say, natural infection in this species of animal with group A /?-hemolyticstreptococci does not occur.
Streptococcuspyogenes In the present studies, two attenuated strains of S. pyogenes, designated as B-346 op and S-43, were employed. Their taxonomical and serobiological characteristics have been described in detail elsewhere [7, 81.
Induction of immunological tolerance Immunological tolerance was induced by neonatal inoculation with either living streptococci of B-346 op strain or SPE emulsified in incomplete Freund’s adjuvant. Provocation was performed approximately 3 months later with four S.C.infections every other day with streptococci of S-43 strain. These methods were the same as those described circumstantially by us previously [3,9].
Extraction of SPE and preparation of toxoid In the present experiments, SPE of type A was employed, and was extracted from the culture supernatant of S. pyogenes strain N Y - 5 accord-
5 I8 (28) Akzyarna et al. ing to the method of Cunningham et al. [lo] and modified by Oyake and Akiyama [ 1 11. The method used for the preparation of the toxoid was modified from that of Wadsworth et al. [ 121. In brief, 640 pg of SPE were dissolved in 2 ml of 0.05 moll1 phosphate buffer solution (pH 7.4) containing formalin at a final concentration of 0.2%, and incubated at 37°C for 5 days. After the period of incubation, the samples were dialysed with continuous stirring against three changes of 3 1 each of phosphate-buffered saline (PBS) for 6 hr at 4°C to eliminate the excess formalin in the solution, and were kept frozen in small volumes and thawed as needed. The results of the following tests established that complete detoxification of the experimental sample took place under the above-mentioned conditions without apparent loss of antigenic activitiy.
Assays for toxic and antigenic properties of the toxoid The toxic potency of the toxoid was calculated according to the method described by Nakahara (pers. comm.). Briefly, a single 0.05 ml dose each of SPE or the toxoid dilutions containing from 65.5 to 4,OOOng protein and 65.5 to 16,000 ng, respectively, was injected i.c. on the depilated flank of either side of the rabbits. The small wheal that resulted disappeared in a few minutes. Positive reactions usually began to appear 4-6 hr after the inoculation. At first, they consisted of a small circular area of erythema. The red area increased and reached a maximum size and intensity between 18 and 36 hr postinoculation. Soon after reaching their maximum size and intensity, the reactions began to subside. Even the most strongly positive did not persist more than 48 hr. The bright red color became dull and began to fade. The reactions were evaluated as negative or positive at the end of 24 hr. Positive reactions were more than 10 mm in both diameters and bright red, with some swelling of the skin. Subsequently, the reactions due to native SPE were compared with those produced by the stimulation with the toxoid in the same animals. Three rabbits were used for each series of titration, and the results were averaged.
In order to determine the antigenic properties of the toxoid, antisera were raised in three rabbits by primary i.m. immunization with approximately 2 pg of the toxoid emulsified in complete Freund’s adjuvant and five booster i.v. injections of the same amount each of the antigen in saline. Then, the serum collected and pooled from these animals was tested for the presence of antibodies capable of changing the platelet activity when it was inoculated into tolerant mice.
Digestion of antibodies with pepsin to isolate F(ab’), fragments Rabbit antiserum (10 ml) was passed over a column of protein A-Sepharose (AmpureTMPA Kit, Amersham Japan, Tokyo, Japan) and the bound material eluted. The eluate (approximately 41 mg IgG) was dialysed against four changes of 2 1 each of 0.1 mol/l acetate buffer (pH 4.0) for 3.5 hr at 4°C. Following dialysis, pepsin (#70 12, 1:60,000; Sigma, St Louis, MO, USA) was added at a ratio of 0.4mg per 30mg of IgG. Digestion was allowed to proceed for 16 hr at 3 7 T , and was terminated by dialysis against 0.01 mol/l PBS (pH 7.0) at 4’C. The fragments were concentrated by ultrafiltration using a Minicon B 125 Concentrator (Amicon Corp., Danvers, MA, USA), followed by chromatography on the same protein A kit. The great majority of the protein passed straight through the column, although traces of undigested Fc pieces or intact IgG remained. Since the binding of y-chains to protein A involves the Fc piece [ 131, F(ab’), fragments do not bind to the gel. Any residual undigested IgG and Fc fragments were eluted with 0.58% (v/v) glacial acetic acid in 0.15 mol/l NaC1. Platelet counting The number of blood platelets was counted using the method described in detail previously [61. Quantitation of anti-SPE antibodies in the serum ELISA was employed to quantitatively determine the antibodies specific for SPE-A in the
Acta Paediatr Jpn
Role of Streptococcus pyogenes in MCLS (29) 5 19 serum of the mice used in the present studies. In advance, SPE-A was dissolved in 0. I moll1 carbonate buffer solution (pH9.6) so that the amount required (5 pg protein) was contained in 50 p1 of the solution. The portions were added to each well of 96-well flat-bottomed polystyrene microtiter plate (MS-3596F/H; SumitomoBakelite Co, Tokyo, Japan), and the plates were coated by incubation at 4°C overnight. After the plates were washed three times with PBS containing 0.05% Tween 20, non-specific binding sites were blocked by incubation with 1% bovine serum albumin (BSA)-containing PBS at 37°C for 2 hr. After being washed three times with the same buffer solution, murine sera to be tested were diluted 1:1,000 with PBS (50p1), added to each well and incubated at 4% overnight, followed by three washes with 0.05% Tween 20-PBS. Subsequently, the horseradish peroxidaseconjugated F(ab’), fragments of goat antiserum specific for mouse IgG/IgM (Tago, Inc, Burlingame, CA, USA), which were diluted 1:2,000 with PBS containing 0.05% Tween 20 and 1% BSA (1 00 pl), were added to each well, followed by incubation for 135 min at room temperature. After the plates were washed five times with 0.05% Tween 20-PBS, 200p1 of 4.4mmol/l o-pheylendiamine dihydrochloride (Wako Pure Chemical Co, Tokyo, Japan) dissolved in citrate-phosphate buffer (pH 5.0) were added to each well, incubated for 25 min at room temperature, and thereafter the enzyme action in individual wells was stopped by the addition of
50p16N H2S04.In most experiments, the plates were read on a microplate spectrophotometer (MTP-12; Corona Electronics Co, Tokyo, Japan) at a wavelength of 500 nm.
Results Immunization with SPE or toxoid and platelet functions in tolerant mice In the first experiments, mice were rendered tolerant to either S. pyogenes-associated antigens due to neonatal infection with group A P-hemolytic streptococci and followed by provocation 3 months later with S. pyogenesinfections, or rendered tolerant to SPE by neonatal inoculation with SPE-A and followed by provocation with the same toxin. The purpose of these experiments was to evaluate the effect of immunization carried out, approximately 1 month prior to the provocation, with SPE or the toxoid, on platelet functions as measured by the platelet counting. Beforehand, toxic activity of the toxoid was quantified by means of a rabbit skin test that demonstrated almost perfect detoxification, as determined from the minimum dose required to produce erythema, which was more than 120 times greater than that of intact SPE (Table 1). Figure 1 shows the results of the experiment, in which four groups of five mice rendered tolerant and provoked with living S. pyogenes were employed. The tolerant animals, in either or presence (. 0 *) of the absence (-0-) immunizations with SPE. demonstrated a com-
Table 1. Relative toxic activity of the toxoid quantified by the rabbit skin test compared with that of intact SPE. Size of erythema (rnrn)
Dose of formalin-treated SPE
Dose of SPE Time Postinoculation 62.5 ng
125 ng
250 ng
500 ng
1,000 ng
62.5 ng 250 ng
1,000 ng 4,000 ng 16,000 ng
10 hr
7 x 7 1 2 ~ 1 2 1 2 ~ 1 2 1 2 ~ 1 2 1 4 ~ 1O 4x 0 Ox0 Ox0 Ox0 7 x 7 O x 0 10x10 13x12 O x 0 Ox0 9 x 9 11x11 13x12 13x12 14x12 11 x 10 12 x 11 12 x 12 13 x 13 12 x 11 13 x 13 13 x 12 15 x 15 15 x 15 16 x 15
24 hr
9 x 8 1 2 ~ 1 2 1 2 ~ 1 2 1 2 ~ 1 2 1 3 ~ 51 x2 5 9 x 9 13x12 15x14 15x15 17x17 7 x 6 13x13 13x12 15x15 17x16 17x16 7 x 7
32 hr
6 x 6 9 x 9 8 x 8
7 x 7 3 x 3 10x1013~13 5 x 5 6 x 6 1 2 x 1 1 1 0 x 1 0 1 1 ~ 1 11 5 x 1 5 9 x 9 1 0 x 1 0 1 0 ~ 1 0 1 7 ~ 1 7 7 x 7
Vol. 34 No. 5 October 1992
7 x 7 6 x 6
7 x 7 7 x 5 9 x 9
7 x 6 10x10 10x10 15x15 1 0 x 9 12x12
5 x 5 7 x 7 8 x 6
Ox0 Ox0 9 x 9
Ox0 8 x 8 10x9
5 x 5
7 x 7 12x12 12x12
520 (30) Akiyama et al. those presented in the aforementioned experiment, as illustrated in Fig. 2. On day 35 postprovocation, at which time the observation was terminated, blood was collected from all mice and anti-SPE titers of their sera were assessed by ELISA. The data illustrated in Fig. 3 strongly suggest that immunization with the toxoid could play a decisive role in the breakdown of immunological tolerance, leading to enhanced antibody-formation against SPE.
Provocative infection with 5-43
. ,
1
0
3
7 10
14 17 20
30
35
Time (days after 1st provocative infection)
Fig. 1. Effects of immunization (arrowed) with the toxoid or SPE on platelet activation in mice made tolerant to S. pyogenes-associated antigens by means of neonatal infection with an attenuated strain of group A /I-hemolytic streptococci, and followed by provocation 3 months later with living S. pyogenes of an attenuated strain. Values are given as mean f s.d. of five mice.
pletely uniform behavior in response to the provocation, showing a definite decrease in the number of platelets on days 3 and 7 postprovocation followed by an abrupt increase in number after day 7 onward. The number reached a maximum on day 10 and thereafter waned gradually, but was still elevated over the following weeks. In striking contrast to the results in these groups are those in the remaining two groups, which consisted of five mice that were either rendered tolerant and followed by immunizaor the same tion with the toxoid (-*-A**-), number without neonatal pretreatment that were later immunized (-m-). The immunization with SPE and the toxoid consisted of four S.C. injections, performed on alternate days, with 2 pg each of the preparations. These groups did not respond to the provocation throughout the observation period, except for the former group, which demonstrated a marginally significant increase in platelet number on day 7 postprovocation. On the other hand, in the second series of experiment in which SPE was employed as a substitute for living streptococci to provide the stimuli for tolerance induction and provocation, the results were in complete agreement with
Effects of rabbit antiserum specific for the toxoid on platelet activation The second experiment was devised solely to confirm the aforementioned suggestion, and examined the effect of the administration of rabbit antiserum to the toxoid on the function of platelets. The results of the experiment are summarized in Fig. 4. The antiserum-treated animals that had been provided with neonatal infection with S. pyogenes (- A or SPE inoculation (-0-) exhibited a transitory thrombocytosis on day 7 postprovocation, that concurred with 0)
Provocative inoculation with SPE ,
0
*
,
3
I
7
12
17 20
25
30
35
Time (days alter 1 st provocative inoculation)
Fig. 2: Fluctuation of platelet counts in mice exposed to neonatal and provocative inoculations with SPE, in response to the immunizations with the toxoid or SPE performed approximately 1 month prior to the provopositive tolerance induction but cation. (+), without any immunization; (. 0 *), positive tolerance induction followed by immunization with SPE; (. A *), positive tolerance induction followed by immunization with the toxoid; (-=-), controls subjected to neither neonatal treatment nor immunization.
Acta Paediatr Jpn
Role of Streptococcus pyogenes in MCLS (3 1) 52 1
-5
0.7
1
9
0
-.0
in
0.5.
L
c v1 0
- 0.3,
.-
8
8 ._
0"
0
0.1
,
:
**
0
5
6
0
I 3
2
1
4
7
a
Fig. 3: Anti-SPE titers of sera as quantified by ELISA in mice sacrificed on day 35 postprovocation with (a) multiple infections with S-43 and (b) multiple inoculations with SPE. ( I ) and ( 5 ) were tolerance induced by infection/inoculation (no immunization); (2) and (6) were tolerance induced by infection/inoculation and immunized with SPE; (3) and (7) were tolerance induced by infection/inoculation and immunized with toxoid; and (4) and (8) were not tolerance induced or immunized.
Provocative infection with S-43
The effect of F(ab'), fragments on platelet function
,
1
0
3
7
1012
16
21
20
35
Time (days following 1st provocative infection)
Fig. 4: The effect of passive immunization with rabbit antiserum raised against the toxoid on the platelet functions in mice rendered tolerant to streptococcal antigens due to neonatal treatments and followed by provocative infections.
the period of thrombocytopenia in the other two groups of mice made tolerant by the neonatal infection (. A 0 ) or SPE inoculation (4-) but without any passive immunization. This was followed by an abrupt decrease in platelet number from day 10 onward. Such a result bears a striking resemblance to that observed in tolerant mice administered with antiserum specific for SPE, as reported previously [ 6 ] ,indicating that the antiserum to the toxoid possesses a strong neutralizing activity against SPE.
Vol. 34 No. 5 October 1992
Human immune serum globulin prepared by the ethanol method of Cohn et al. [14] and Oncley et al. [I51 produces a high incidence of untoward reactions when given intravenously. The seriousness of the reaction is proportional to the decrease in the serum complement level. There is a need for a safe and effective intravenous preparation of immune globulin so that large volumes of this material can be infused to attain a high titer of circulating antibody rapidly. Therefore, numerous approaches have been utilized to remove the anticomplementary activity: (i) Enzymatic digestion at pH 4 . 0 (ii) ultracentrifugation; and (iii) incubation of the immune globulin at pH 4.0 and 37°C. To date, the most promising method in terms of the removal of anticomplementary activity and retention of long in vivo half-life has been the low pH treatment; however, some of these materials have required additional treatment with pepsin to remove the anticomplementary activity completely [ 161. In the third experiment, the effect of the F(ab'), fraction of the rabbit antitoxoid serum on platelet activation was assessed, and compared with that of the intact whole antiserum. As may be seen from Fig. 5, the platelet counts in mice exposed to neonatal and provocative infections with S. pyogenes (-G)and controls provoked with the infections that did not matched undergo neonatal treatment (4-)
522 (32) Akiyarna el al. Provocative inleciion with 5-43
I
0
3
7
10 12 15
20
25
32
Time (days following 1 st provocative infection)
Fig. 5: Relative effects of F(ab’), fractions and whole unfractionated rabbit antitoxoid serum on platelet functions in mice exposed to neonatal and provocative infections.
our expectations. The tolerant animals passively administrated s.c., four times every other day, with lop1 each of the whole antiserum (. A *) or i.v. with 20 pg each of the F(ab‘), fragments (. A a**), however, demonstrated significant but transitory increases in the number of platelets on days 7 and 10. Taken together, these studies strongly suggest the possibility that intravenous transfusion with F(ab’)z fragments of serum from volunteers hyperimmunized with the toxoid could exert a surprisingly efficacious effect in patients during the early stages of MCLS.
Discussion In the animal model established previously [3, 81, mice exposed to neonatal infection with a strain of S. pyogenes (which fails to confer cell-mediated immunity [CMI] if inoculated into mature murine hosts) and followed by repetitive infections 4-6 weeks later with another strain of the same species of bacteria that is endowed with an intrinsic ability to afford mature mice CMI against streptococcal antigens, demonstrated heightened cellular responses to those antigens in spite of a complete lack of humoral responsiveness to the same stimuli. Such immunological characteristics are
completely identical with those observed in MCLS patients. The results of the current investigations reaffirmed the utility of the animal model by demonstrating early thrombocytopenia followed by thrombocytosis in the SPE-induced tolerant mice in response to the provocative infection, which once again resembles MCLS. In a recent study [5], the incidence of a considerable amount of SPE in 100% of cases combined with its rapid decline during defervescence brought about by treatment with intravenous human y-globulin was demonstrated in the sera of MCLS patients by the use of ELlSA in combination with monoclonal antibodies specific for SPE. In view of these observations, an induction of immunological tolerance against SPE and biological activities of the toxin have been hypothesized as principal factors involved in the initiation of events resulting in MCLS. Needless to say, SPE is also considered to be associated with the development of the skin rash characteristic of so-called streptococcal infection (synonymous with scarlet fever), and this property can be inhibited by antitoxin [ 171. The subsequent discussion will be focused solely on the potential use of the SPE-toxoid for the prophylaxis and treatment of these diseases, the realization of which could be expected in the near future. First, it has already been suggested that the well-known efficacy of intravenous transfusion with human pglobulin in reducing the duration of systemic inflammation in the early course of these diseases can be accounted for by the putative presence in the inoculum of a fair level of antibodies to SPE. Therefore, we are tempted to recommend that the administration of the F(ab’)2 fragments separated from the sera of volunteers that are hyperimmunized with the toxoid should be tried instead of human y-globulin. Second, the monomodal age distribution of MCLS (there is a peak occurrence during the first 2 years of life, and few are affected over the age of 8 years) might be explained exclusively by the maintenance of a special immunological tolerance to 5’. pyogenesassociated antigens, and
Acta Paediatr Jpn
Role of Streptococcus pyogenes in MCLS (33) 523 an induction of the immunological state might be caused by the streptococcal infection(s) occurring perinatally or in the neonatal period [ 81. In addition, in subsequent investigations performed to gauge the responsiveness of the cultured lymphocytes from outpatients attending at regular intervals after recovery from MCLS, we were surprised at the continuation for more than 9 years of the depressed antibodyforming activities to streptococcal antigens [4]. These results will explain the well-known tendency of this disease to relapse, repeatedly in some cases, after apparent recovery from the preceding episode. Taken together, it appears that the immunological tolerance in children less than 4-6 years old whose deficient immunoresponsiveness to SPE and streptolysin-0 was proved, as well as in the above-mentioned outpatients with continuous susceptibility to recurrence of MCLS, could be overcome by an inoculation with the toxoid. Furthermore, pregnant women with a complete lack of antibody formation against the streptococcal antigens should be another target for immunization with the toxoid. Such an immunization will prevent their offspring from being affected with the streptococcal infections during early life, leading to their freedom from the development of the immunological tolerance to S. pyogenes-associated antigens.
4.
5.
6.
7.
8.
9.
10.
Acknowledgements We are grateful to Miss Toshiko Hasobe for her excellent technical assistance.
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