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

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 Streptococcus pyogenes (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 antiserum-treated 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')₂ fragments of the rabbit antiserum to the toxoid on platelet activity. The tolerant mice passively administered i.v. with the F(ab')2 fractions demonstrated a complete lack of responsiveness, except for a small thrombocytosis on days 7 and 10.     

Possible Role of Streptococcus Pyogenes in Mucocutaneous Lymph Node Syndrome

Migration inhibitory factor (M1F)-induced activity of patients with mucocutaneous lymph node syndrome (MCLS) to antigens associated with Streptococcus pyogenes infection was compared to that of control populations consisting of children with illnesses not related to streptococcal infections (group A), and of patients with streptococcal pharyngitis (group B), with the following results. 1. Though a consiaerable number of patients in the acute state of MCLS failed to respond to antigens consisting of (1) a coccus preparation of S. pyogenes (Picibanil), (2) streptococcal pyrogenic exotoxin (SPE) and (3) an extract from cells of a virally transformed human B-cell line, a complete restoration of their responsiveness was observed in parallel with the decrease of fever. 2. While almost all patients of group A were refractory not only to Picibanil and SPE but also to the extract from the transformed human cells, some children with infections due to intracellular microbes showed responsiveness to the extract from the transformed cells. 3. In group B, some patients showed a marked responsiveness and the other a complete refractoriness to these antigens throughout the observation period. These results raise the possibility that S. pyogenes may play a principal role in the pathogenesis of MCLS.     

Possible Role of Streptococcus pyogenes in Mucocutaneous Lymph Node Syndrome XL Immunoelectron Microscopic Observation of Protoplast-like "Spherical Bodies" Detected in Peripheral Blood of MCLS Patients

Protoplast-like "spherical bodies" averaging 0.5-1.5 mU m in diameter and devoid of cell walls were first detected by Ueno et al, in the buffy coat of heparinized venous blood from patients with mucocutaneous lymph node syndrome (MCLS). But the nature of the "bodies" has yet to be clarified because of the absence of convincing evidence pointing to their antigenic characteristics. The present investigations were designed solely to provide a serological identification of the "bodies" by the use of immunoelectron microscopy, with the following results. First, "spherical bodies" bearing a striking resemblance to those observed by the above-mentioned authors were detected in biopsy specimens from challenge sites in mice infected with Streptococcus pyogenes as well as in the buffy coat of peripheral blood from MCLS patients. Second, the "bodies" detected were stained distinctly in both cases by an immunohistochemical technique using, as the primary antibody, a rabbit antiserum raised toward S. pyogenes-derhed protoplasts, which was then absorbed with protoplasts from Staphylococcus aureus and Escherichia coti. Third, the absorbed sera were proved to be not faultless, because complete specificity toward protoplasts from S. pyogenes was not attained due to the presence of a large amount of cross-reactive antigens between protoplasts from the immunizing and absorbing strains of bacteria. The implications of these findings are discussed, particularly in relation to the evaluation of the present serological test for the "spherical bodies."     

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

The absence of seroconversion to streptolysin O in convalescent patients with mucocutaneous lymph node syndrome has been a perplexing problem for investigators who are in favor of a streptococcal etiology of the disease. In the present studies, mice subjected in the newborn period to an infection with a strain of Streptococcus pyogenes, which fails to confer cellular immuno-responsiveness even in adult mice, and then reinfected four to six weeks later with another strain of the same species, which has an intrinsic ability to render mature murine hosts sensitive to streptococcal antigens, expressed an enhanced cell-mediated immunity coupled with a lack of humoral responsiveness to the same antigens. Such immunological characters are an exact counterpart of those of MCLS patients. The involvement of these immunological characters in the pathological process of the disease is discussed.     

Possible Role of Streptococcus pyogenes in Mucocutaneous Lymph Node Syndrome. Ill Electron Microscopic Observations on Group A Hemolytic Streptococci Inoculated into Mice

Because of various circumstantial evidence, we have been asserting the streptococcal etiology of mucocutaneous lymph node syndrome (MCLS), although there has been no recovery of Streptococcus pyogenes from the patients at any site. The present study was designed to provide a satisfactory explanation for the failure to isolate these bacteria from patients with MCLS, by electron microscopic observation of group A ftera-hemolytic streptococci which had been inoculated intramuscularly into mice. “Spherical bodies” were detected in biopsy specimens obtained from the infection sites from 10 – 14 days onward after infection; these were spherical, 0.5–1.5 yua in diameter, devoid of cell walls, perhaps uncultivable on any culture medium, and undistinguishable from those discovered in the buffy coat from peripheral blood of MCLS patients and described as ruined etiological microorganisms by Ueno et al. The implications of these findings, particularly in terms of the etiology of MCLS, were discussed.     

Possible Role of Streptococcus pyogenes in Mucocutaneous Lymph Node Syndrome. V. Further Electron Microscopic Observations on Group A Beta-Hemolytic Streptococci Inoculated into Immune Mice

In the preceding studies, protoplast-like “spherical bodies” were detected electron microscopically in mice which were challenged intramuscularly, approximately 15 days before, with an attenuated strain of Streptococcus pyogenes. The “spherical bodies” were 0.5–1.5 Mm in diameter, poor in ultra-structural preservation of the cytoplasm, devoid of cell walls, and indistinguishable from those discovered in the buffy coat of peripheral blood from patients with mucocutaneous lymph node syndrome (MCLS). The present investigation was intended to elucidate the intracellular localization of the “spherical bodies” and to understand better the mechanisms responsible for the elimination of viable streptococci from the infected sites. Accordingly, a larger amount of bacterial inoculum was injected i.m. into mice immunized, in advance, against streptococcal infection, and every biopsy specimen was obtained on day 3 postchallenge. Subsequently, ingested “spherical bodies” were detected under an electron microscope in the cytoplasm of phagocytic cells, including polymorphonuclears, eosinophils and fibroblasts, situated in the perimysium.     

Possible Role of Streptococcus Pyogenes in Mucocutaneous Lymph Node Syndrome XIII. No Recovery of β-Hemolytic Streptococci from Pharynx of MCLS Patients

Lack of recovery of β-hemolytic streptococci from the throat of 80 patients finally diagnosed as mucocutaneous lymph node syndrome was again confirmed, although α-hemolytic streptococci were consistently isolated from all patients but one. The implications of these findings were discussed, particularly in terms of the possible role of Streptococcus pyogenes in the pathogenesis of this disease.     

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

A negligible, if not entirely negative, production of antibody to Epstein-Bart virus(EBV)-associated antigens in patients with mucocutaneous lymph node syndrome(MCLS) has been a perplexing problem for investigators who are in favor of a streptococcal etiology of the disease. In the present investigations, antibody formation to EB viral capsid antigen (VCA) was assessed by the use of the indirect immunofluorescence technique in mice which had been subjected to neonatal infection with an attenuated strain of Streprococcus pyogenes or inoculation with either heat-killed streptococci or streptolysin-O, both of which were emulsified in Freund's complete adjuvant (FCA). Each mouse of these groups was inoculated 30 days or so later with EBV dilution in FCA, blood was collected about three weeks after virus infection, and antibody titers were assessed individually. According to the results of these experiments, no apparent difference was detected among the mice, irrespective of the methods of their neonatal treatment concerning their refractoriness to EB-VCA; the majority of the animals showed titers of less than 1:8, no case having a titer above 1:30. In contrast, all sera from control mice yielded positive responses showing antibody titers of 1:250 and over. Such an immunological character is an exact counterpart of that of MCLS patients. Accordingly, the recent research focus on EBV as a possible etiological agent of MCLS seems to be too restrictive.     

Possible Role of Streptococcus pyogenes in Mucocutaneous Lymph Node Syndrome IX. Quantitation by ELISA of Streptococcal Pyrogenic Exotoxin in the Serum of MCLS Patients

In the present paper we describe the use of an enzyme-linked immunosorbent assay reinforced with an introduction of monoclonal antibody, for the detection and quantitation of streptococcal pyrogenic exotoxin (SPE) in the serum of patients with mucocutaneous lymph node syndrome (MCLS). The amount of SPE was usually at a high level, and its 100% incidence in patients' sera was proved whenever the assay was made on the day of admission, thereby showing a marked contrast to carefully matched control sera which failed to mediate any positive result. As for the change in detected amount of the toxin, a clear dichotomy was observed between the serum of gammaglobulin-treated patients and that of infants given aspirin; in the former the positive result turned to negative rapidly following the initiation of treatment coupled with a defervescence, while in the latter the reduction of SPE levels was scarcely monitored for as long as 17 days after the onset of illness. Quantitation of SPE might be an auxiliary test for the diagnosis of MCLS, because a considerable amount of SPE was assessed in a patient who developed characteristic huge coronary artery aneurysms following an illness which did not fulfill the diagnostic criteria. These findings support our speculation in relation to the certain role of S. pyogenes as an etiological agent for MCLS. The possible mechanisms of gammaglobulin treatment in reducing the prevalence of cardiovascular lesions and the duration of systemic inflammation are discussed.     

The Effects of a Japanese Traditional Medicine on Mucocutaneous Lymph Node Syndrome

Huang-lien-chieh-tu-tang (HLCT), an anti-inflammatory agent used in Japan since the seventh century, was given to six patients with acute mucocutaneous lymph node syndrome (MCLS) together with aspirin. Compared with six patients treated with aspirin alone (Group A) or six patients with aspirin and steroid (Group B), these HLCT treated patients (Group C) had significantly faster clinical and laboratory improvement. The periods required for resolution of the clinical signs in Group A, B and C were an average of 4.5, 6.4 and 2.0 days for skin rashes, 5.3, 5.0 and 3.2 days for conjunctivitis, and 6.3, 7.0 and 3.2 days for oral mucosa congestion, respectively. [Fibrinogen level decreased to less than 400 mg/dl in 29.5, 25.3 and 15.2 days, and sGOT and sGPT became less than 50 u in 13.3, 11–0 and 7.3 days in each group. In Group C patients, cardiovascular side effects often seen in steroid therapy have not been observed and heart murmurs and EKG abnormalities were less frequent than in the other groups. (Acta Paediatr Jpn 23(2): 177–184 1981)     

Possible Role of Streptococcus pyogenes in Mucocutaneous Lymph Node Syndrome VIII. Immunological Tolerance to S. pyogenes-Associated Antigens Seems Essential to Induction of MCLS

In our previous studies, we attempted to show that new-born mice infected with an attenuated strain of Streptococcus pyogenes , and exposed to streptococcal antigens approximately one month later, failed to develop a humoral response to those antigens but were able to respond normally to other unrelated antigens. Since such immunological characteristics are identical to those of patients with the mucacutaneous lymph node syndrome (MCLS), the present studies were performed with the aim of assessing the antibody-forming activities of peripheral blood lymphocytes collected from MCLS patients. These lymphocytes were cultured in the presence of pokeweed mitogen, streptolysin-O and streptococcal C-polysaccha-ride, leading to their refractoriness to S. pyogenes-associated antigens coupled with normal responsiveness to unrelated antigens. The implication of such immunological responsiveness elicited from neonatal exposure to streptococcal antigens is discussed, particularly with reference to the induction of MCLS.     

Possible Role of Streptococcus pyogenes in Mucocutaneous Lymph Node Syndrome VI. Heightened Cellular Reactivity to Streptolysin-O in Mice Infected with S. pyogenes and in MCLS Patients

The present investigations were performed to determine, by using the macro-phage migration inhibition test (MIT), whether cell-mediated hypersensitivity to streptolysin-O (SLO) was developed in mice infected with Streptococcus pyogenes as well as in patients with mucocutaneous lymph node syndrome (MCLS), with the following results: 1. The splenic lymphocytes from murine hosts infected with B-346 op strain of S. pyogenes, which fails to induce cellular immunity in infected mice, showed a complete lack of responsiveness to SLO. In contrast, the mice infected with streptococci of Sv and S-43 strains, both of which possess an intrinsic activity of eliciting enhanced cellular hypersensitivity in the same species of animals, exhibited positive responses to the hemolysin from strep-tococcal culture supernatant. 2. Although a considerable number of MCLS patients in the acute phase of the illness failed to respond to SLO, a complete restoration of their responsiveness was observed in parallel with recovery from the acute illness. Conversely, all patients in the subacute phase of the disease reacted well in vitro to SLO, followed by a gradual decline of their responsiveness over a period of 1–2 weeks. Furthermore, the implications of these findings are discussed in association with the possible role of S. pyogenes as an etiological agent for MCLS.