Adverse Effects of Praziquantel

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International Journal for Parasitology 32 (2002) 461-471

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0020-7519/02/$20.00 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.PII: S0020-7519(01)00368-X

Adverse effects of praziquantel treatment ofSchistosoma japonicuminfection: involvement of host anaphylactic reactions induced by

parasite antigen release

Jun Matsumoto*

Department of Tropical Medicine and Parasitology, Dokkyo University School of Medicine, Mibu, Shimotsuga, Tochigi, 321-0293, Japan

Received 26 September 2001; received in revised form 21 November 2001; accepted 21 November 2001

Abstract

The present study using a murine model heavily infected with Schistosoma japonicum aimed to elucidate the pathogenesis of adverseeffects of praziquantel treatment of schistosome-infected subjects. Inbred BALB/c mice were infected with S. japonicum (Yamanashi strain)

before being treated with a single dose of praziquantel at 4 or 8 weeks p.i. All the mice treated at 8 weeks p.i. exhibited signs typical of

systemic anaphylaxis until half of them died shortly after praziquantel administration. At autopsy, these mice exhibited remarkable intestinal

alterations characterised by increased mucosal permeability, mucosal oedema and petechial haemorrhage, which are changes typical of

immediate intestinal anaphylaxis. In these mice treated at 8 weeks p.i., degranulation of intestinal mast cells was frequent1y observed, whichwas particularly remarkable around S.japonicum eggs hatched as an effect of praziquantel. Furthermore, the plasma histamine concentration

just after praziquantel treatment was much higher in mice at 8 weeks p.i. than that in uninfected mice or in S. japonicum -infected mice

without drug treatment. In contrast, none of these intestinal changes was observed in untreated or uninfected control mice, or in mice

administered praziquantel at 4 weeks p.i., in which worm pairs had just reached sexual maturation and begun egg-laying. The finding byELISA that serum IgM and IgA levels specific to S. japonicum eggs decreased immediately after praziquantel treatment, together with the

results of immunohistochemistry, revealed the sudden release of parasite antigens from the eggs hatched by praziquantel treatment. The

results of this study demonstrate that adverse effects of praziquantel treatment of schistosomiasis characterised by abdominal signs depend on

anaphylactic reactions due to parasite antigens, especially antigens from eggs hatched as an effect of praziquantel. 2002 Australian Society

for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Praziquantel; Adverse effects; Schistosoma japonicum ;Anaphylaxis

1. Introduction

Schistosomiases, which are caused by blood flukes of thegenus Schistosoma, are estimated to afflict about 200 millionpeople world-wide. After maturing in a final host, schisto-some worm pairs live in their definitive venous habitat andengage in egg-laying for many years. It is widely acceptedthat it is the eggs, rather than the adult worms, that play acrucial role in the pathogenesis of schistosome infection.Chemotherapy remains the major means of controlling schis -tosomiasis because no effective vaccine is available yet.

Praziquantel, a heterocyclic pyrazino-isoquinolinecompound (Gnnert and Andrews, 1977), is the best schis -tosomicidal agent available today for the clinical manage-ment of schistosomiasis and is highly effective against allspecies of schistosomes pathogenic to humans (W ebbe and________* Tel.: +81-282-87-2134; fax: +81-282-86-6431.

E-mail address: [email protected] (J. Matsumoto).

James, 1977). Although clinical trial studies in healthyhuman volunteers revealed no clinically relevant drug-related changes (Frohberg and Schencking, 1981; Frohberg,1984), the history of clinical applications of praziquantel inthe treatment of schistosomiasis patients has included anotable number of cases of adverse effects in patients withschistosomiasis mansoni (Polderman et al., 1984) or schis -tosomiasis japonica (Chen et al., 1983; Noseas et al., 1984;Watt et al., 1986). Most distinguishable side effects reportedpreviously were those related to the gastrointestinal tract,such as abdominal pain and/or discomfort, nausea, vomit-ing, anorexia, loose stools or diarrhoea, and bloody stool.The authors of previous studies noted that these symptomsappeared immediately after treatment and that the frequencyof adverse reactions was correlated with the intensity ofinfection. However, the pathogenesis of these post-treat-ment events is as yet unexplained, though the characteristicsof post-praziquantel adverse effects noted above suggest the


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462 J. Matsumoto / International Journal for Parasitology 32 (2002) 461-471

involvement of immediate host reactions due to parasitesdamaged by the drug.

Effects of praziquantel on adult worms of schistosomesinclude vesication, vacuolisation, and disintegration of thetegumental surface (Becker et al., 1980; Mehlhorn et al.,

1981; Shaw and Erasmus, 1983a,b; Irie et al., 1989),which may present both intestinal metabolites and hidden,inaccessible antigenic sites to the surrounding blood stream.In addition to its effects on adult worms, praziquantel actsboth in vivo and in vitro on mature eggs of schistosomes laidin host tissues to be hatched (Matsuda et al., 1983; Gibodaand Smith, 1994). Matsuda et al. (1988) further presentedevidence for the possibility that egg antigens are releasedfrom hatched eggs into the circulation of treated subjects.Sudden release of schistosome antigens from both adultworms and mature eggs after praziquantel treatmentpresumably evokes extraordinary host immune responses,which are associated with the adverse effects of praziquantelmentioned above.

In this study, we attempted to elucidate the pathogenesisof adverse effects after praziquantel treatment of schisto-some-infected subjects using a murine model heavilyinfected with Schistosoma japonicum. Our observationssuggest that adverse effects of praziquantel treatment ofschistosomiasis patients with marked abdominal symptomsare related to anaphylactic responses due to the parasiteantigens, especially those from schistosome eggs hatchedas an effect of praziquantel.

2. Materials and methods

2.1. Animals, parasites and infection

The Yamanashi strain of S. japonicum, successivelymaintained in ICR mice and Oncomelania nosophora snails,was used in this study. Female BALB/c mice aged 4 weeks(Clea Co.) were divided into four groups: mice administeredpraziquantel at 4 or 8 weeks after S.japonicum infection, S.

japonicum-infected mice at 8 weeks p.i. without praziquan-tel treatment (untreated control), and uninfected mice withpraziquantel administration (uninfected control). At infec-tion, mice were percutaneously infected with S. japonicumby tail immersion for 60 min with 80 cercariae each.Successful infection in each animal was checked by measur-

ing immunoglobulin levels specific to adult worm antigen asdescribed below for mice autopsied at 4 weeks p.i., or bydetecting parasite eggs by stool examination before use inthe following experiments. All animals were autopsiedunder anaesthesia with diethylether, and blood and tissuesamples were obtained.

All experimental animals used in this study were treatedfollowing Guidelines for the Care and Use of LaboratoryAnimals, Dokkyo University School of Medicine,authorised by the Animal Care and Use Committee, DokkyoUniversity School of Medicine.

2.2. Praziquantel treatment

Praziquantel (Biltricide

; Bayer AG) suspended in 2%Cremophor El (Sigma) was perorally administered to eachmouse at a dose of 500 mg/kg body weight at 4 or 8 weeks

p.i. At 8 weeks p.i. 500 l of aqueous Cremophor EI alonewas given to untreated control mice.

2.3. Assessment of anaphylacticresponses

Systemic signs of anaphylaxis at 60 min after praziquan-tel administration were evaluated with a standardised scor-ing system (McCaskill et al., 1984; Poulsen et al., 1987; Liet al., 1999) and scored as follows: 0 = no signs; 1 =scratching and rubbing around the nose and head; 2 =puffiness around the eyes and mouth, pilar erecti, reducedactivity, and/or decreased activity with increased respiratoryrate; 3 = wheezing, laboured respiration, and cyanosis

around the mouth and the tail; 4 = no activity after proddingor tremor and convulsions; 5 = death.

For the purpose of evaluating vascular permeability inintestinal mucosa due to intestinal anaphylactic response,mice were i.v. injected with 200 l of 0.5% Evans Bluejust after praziquantel administration and autopsied 20min after praziquantel administration. Intestinal organswere removed from each mouse, cut longitudinally, anddye extravasation from blood vessels into the intestinallumen was examined.

2.4. Histologic examination of small intestine

Jejunal samples of 2 cm in length, taken from a location10 cm anal from the pylorus, were obtained from eachmouse and washed gently in 0.15 M PBS. Tissues werefixed in 10% formalin or Carnoys solution, and thenembedded in paraffin. Sections (7 m) were stained withH & E or Toluidine Blue before being examined with anoptical microscope.

2.5. Preparation of parasite antigens

Eggs of S. japonicum were obtained from the intestine ofinfected ICR mice at 7-8 weeks p.i. by the digestion method

(Matsuda et al., 1981). Isolated eggs were lyophilised andstored at 80 C until antigen preparation. Adult wormswere obtained from the portal system and mesentericveins of the infected mice at more than 2 months afterinfection. After collection, adult worms were left in 0.15M PBS for 1-2 h at RT, harvested by filtration through filterpaper and stored at 80 C until use. For ELISA, parasiteantigens were prepared by a technique described previously(Matsuda et al., 1981). The protein concentration of eachantigen was measured using a protein assay kit (Bio RadLaboratories).


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International Journal for Parasitology 32 (2002) 461-471 463

2.6. Measurement of IgG, IgM, IgA and IgE class antibodies

specific to S.japonicum antigens

For measurement of anti-S.japonicum antibodies, micewere bled just before and 60 min after praziquantel admin-

istration at 8 weeks p.i. Serum was separated by centrifuga-tion at RT before being stored at 80 C. The ELISA usedfor measurement of parasite-specific antibodies was essen-tially described by Matsuda et al. (1984). Briefly, microtiterplates (Immulon 200; Greiner) were coated with 100 l ofeither S.japonicum egg (SjE) antigen or S.japonicum adultworm (SjA) antigen (10 g/ml protein concentration, each)at 37 C for 2 h before being kept at 4 C overnight. Plateswere then washed in 0.5% Tween 20 in PBS. Serum sampleswere incubated at 37C for 45 min at a dilution of 1:200 forIgG, IgM, and IgA measurement, or 1:20 for IgE measure-ment, respectively. Horseradish peroxidase (HRP)-labelledgoat anti-mouse IgG, goat anti-mouse IgM, goat anti-mouseIgA (ICN Pharmaceuticals, Inc.) and goat anti-mouse IgE

(Southern Biotechnology Associates, Inc.) were used todetect class-specific responses at a dilution of 1:2,000 forIgG, IgM, and IgA measurement, or 1:500 for IgE measure-ment, respectively. Plates were incubated at 37 C for 60 minbefore being washed five times. Substrate (ABTS; 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (Sigma),H202) was added and incubated for 30 min at RT. Theoptical density of the reactant was measured by a micro-plate reader (Corona) at a wavelength of 415 nm.

2.7. Immunohistochemistry

The paraffin sections of host tissues were treated basicallyas described by Nakane and Pierce (1967) with slight modi-fication. Briefly, the deparaffinised sections were treatedwith methanol containing 1% H2O2 in order to inactivateendogenous peroxidase, followed by washing with PBS.After blocking with 1% BSA in PBS, biotinylated mousemAb reacting to glycoproteins of S.japonicum egg (clone#:48-39-2; Yokoi et al., 1997) was applied and incubated for 1h at RT. After washing, HRP-conjugated streptavidine(Amersham Pharmacia Biotech UK, Ltd.) was reacted for1 h at RT. The sections were washed before reaction withsubstrate containing 0.02% diaminobenzidine (Wako PureChemical Industries, Ltd.) and 0.02% H2O2 in Tris-HClbuffer (pH 7.6) for 10 min. Then the sections were washed

with tap water to stop the reaction, counterstained withhaematoxylin and mounted.

2.8. Determination of plasma histamine level

In order to evaluate the plasma histamine level, bloodsamples were obtained from mice with or without prazi-quantel administration at 8 weeks after S.japonicum infec-tion. Uninfected mice with praziquantel administration werealso used. These animal groups were prepared in addition tothose used in other experiments mentioned above. Thirtyminutes after praziquantel treatment, blood was collected

into chilled tubes containing 40 l of 7.5% potassium-ethy-lenediamine-tetraacetic acid. After centrifugation, plasmaaliquots were collected and frozen at 80 C until use.Plasma histamine levels were determined using an immu-noassay kit (IBL) following the manufacturers specifica-

tions.

3. Results

3.1. Characterisation of systemic signs after praziquantel

treatment

All the mice (n = 12) administered praziquantel at 8weeks after S.japonicum infection exhibited signs typicalin rodent models for systemic anaphylaxis (DInca et al.,1990; Levine and Saltzman, 1998; Li et al., 1999). Theseverity of systemic signs was scored at 60 min after prazi-quantel treatment as described in Section 2 (Fig. 1).

Systemic anaphylactic signs appeared within 10 min afterpraziquantel treatment, and were characterised by reducedactivity with increased respiratory rate. Subsequently, theanimals exhibited laboured respiration, cyanosis and no

Fig. 1. Systemic sign scores. Schistosoma japonicum -infected mice weretreated with praziquantel at 4 or 8 weeks p.i. Sixty minutes after adminis-

tration, signs were scored on a scale from 0 (no sign) to 5 (death), asdescribed in Section 2.


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activity until six of the 12 mice died within 60 min afterpraziquantel administration. In contrast, mice treated withpraziquantel at 4 weeks p.i. (n = 14) exhibited no severesigns and recovered completely within an hour, althoughsome initially exhibited slight signs of anaphylaxis. Neither

the uninfected control mice (n = 15) nor the untreatedcontrol mice (n = 13) exhibited any systemic sign ofanaphylaxis.

3.2. Characterisation of intestinal reactions

At autopsy of mice administered praziquantel at 8 weeksp.i., we found large amounts of mucous secreted into theintestinal lumen, mucosal oedema, and petechial haemor-rhage in the intestinal mucosa. Such changes were absent inmice administered praziquantel at 4 weeks p.i., in theuntreated control mice and in the uninfected control mice.

Altered vascular permeability in intestinal organs was

assessed by Evans Blue dye extravasation. Intestines fromS. japonicum-infected mice administered praziquantel at 8weeks p.i. exhibited dark blue discoloration (Fig. 2),whereas untreated control mice receiving dye injection didnot, although slight blue discoloration was observed aroundegg granulomas. These results indicate that vascular perme-ability was markedly increased in the intestine of S.japoni-cum-infected mice after praziquantel administration.

Histological examination revealed that some S. japoni-cum eggs laid in the intestinal tissues of mice administeredpraziquantel at 8 weeks p.i. had hatched (Fig. 3), whereas allof the eggs in untreated mice remained unhatched. Thesefindings show that miracidia hatched after praziquanteladministration, consistent with the result describedpreviously (Matsuda et al., 1984). No eggs were found in

the intestine from S. japonicum-infected mice administeredpraziquantel before autopsy at 4 weeks p.i., with someexceptions in which S. japonicum eggs were quite rarelyobserved on histological examination. It was confirmed byan increased level of anti-SjA immunoglobulins that all of

the mice at 4 weeks p.i. were infected withS.japonicum.On histopathological observation, the small intestine ofmice administered praziquantel at 8 weeks p.i. exhibitedobvious oedema, defined as separation of the epithelialcell layer from the lamina propria, and profound dilatationof microvessels (Fig. 4A-D). Some of the mice exhibitedsloughing of epithelial cells at the tips of villi (Fig. 4C) andhaemorrhage (Fig. 4D). These findings were essentially thesame as those described for intestinal anaphylaxis in experi-mental animal models (DInca et al., 1990; Levine andSaltzman, 1998; Li et al., 1999), which were absent inmice administered praziquantel at 4 weeks p.i. (Fig. 4E),in the untreated control mice (Fig. 4F) and in the uninfectedcontro l mice.

3.3. Parasite-specific antibody levels

Parasite-specific antibody levels were determined byELISA using antigen prepared from eggs (SjE) or adultworms (SjA) of S. japonicum. In mice treated at 8 weeksp.i., SjE-specific IgM and IgA class antibody levels weremarkedly reduced just after praziquantel administration,whereas SjE-specific IgG or any class of anti-SjA antibodylevel were almost unchanged (Fig. 5). Both SjA-specific andSjE-specific IgE levels in sera from S. japonicum-infectedmice at 8 weeks p.i. were as low as those in uninfectedcontrol mice, and did not show any apparent change after

Fig. 2. Vascular leakage in intestinal mucosa was examined by dye extravasation 20 min after i.v. injection of Evans Blue dye. (Upper) Small intestine

from a S. japonicum-infected mouse at 20 min after praziquantel treatment at 8 weeks p.i. (Lower) Sample from a non-treated control mouse with S.japonicum infection (at 8 weeks p.i.). Representative samples are presented.


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Fig. 3. Schistosoma japonicum eggs in the intestinal tissues of mice treated

with praziquantel at 8 weeks p.i. Some eggs were hatched as an effect ofpraziquantel (A, arrows; B). H & E stain. Bar: 60 m in (A); 30 m in (B).

praziquantel treatment. In untreated control mice, no changewas observed in any parasite-specific antibody level.

3.4.Release ofS. japonicum egg antigens into intestinaltissue

The results of immunohistochemical examination usingS. japonicum egg-specific mAb demonstrate that S. japoni-cum egg antigen was released from eggs hatched after prazi-quantel administration at 8 weeks p.i.: the parasite eggantigen was detected in host intestinal tissues aroundhatched eggs, which were observed frequently in intestine

of praziquantel-administered mice (Fig. 6A-C). The reac-tion was confined to within the eggshell in unhatched eggs(Fig. 6D).

3.5.Increased mast cell degranulation after praziquanteladministration

Histologic analysis of intestinal mucosa revealed thatmast cells were present in intestinal submucosa and serosaof S. japonicum-infected mice at 8 weeks p.i., whereas onlya few mast cells were found in mice administered prazi-quantel at 4 weeks p.i. and in uninfected control mice.The number of mast cells observed in a transverse sectionof a jejunal sample from mice at 8 weeks post S. japonicum

infection was 89.4 73.2 (average SD), while those ofmice at 4 weeks p.i. and uninfected control mice were

2.2 2.2 and 4.8 1.9, respectively (n = 5, each). Mastcell degranulation, characterised by a reduced number of

intracellular granules, was observed more frequently inthe intestine of praziquantel-treated mice than in that ofuntreated mice. In addition, mast cell degranulation wasmost remarkable around hatched eggs in intestinal mucosa(Fig. 7A,B). Non-degranulated mast cell was recognised byits cytoplasm full of metachromatic granules (Fig. 7C).

3.6. Elevated plasma histamine level in praziquantel-treatedmice

The plasma histamine concentration was measured at 30min after praziquantel treatment. The histamine level was

214.9

67.0 ng/ml (average

SD) in mice treated at 8weeks p.i., 41.5 18.5 ng/ml in uninfected control miceand 54.7 16.1 ng/ml in mice at 8 weeks after S. japonicuminfection without drug treatment (Fig. 8).

4. Discussion

This study demonstrates that the pathogenesis of adverseeffects of praziquantel treatment of S. japonicum infectioninvolves host anaphylactic reactions, which are provoked byantigens from the parasite damaged by the drug. The S.

japonicum-infected mice used in this study exhibitedsystemic signs typical of immediate hypersensitivity, until

half of the animals died within 60 min after praziquanteladministration. The responses were particularly remarkablein the small bowel and characterised by increased intestinalpermeability and mucous secretion, together with petechialhaemorrhage. Histologic examinations revealed alterationsin the jejunum of praziquantel-treated mice, such as markedvascular congestion, oedema of the lamina propria, and insome mice sloughing of epithelial cells at the tips of micro-villi. These findings are essentially the same as those forrodent models of intestinal anaphylaxis (DInca et al., 1990;Levine and Saltzman, 1998; Li et al., 1999). The intestinalalterations observed in this study seem to be strongly corre-lated with abdominal symptoms associated with post-prazi-


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Fig. 4. Histopathologic alterations in small intestine of S. japonicum-infected mouse after praziquantel administration. (A-D) Vascular congestion andoedema in lamina propria (A,B), sloughing of epithelial cells at the tip of villi (C), and haemorrhage (D; arrow) were obvious in the jejunum of miceadministered praziquantel at 8 weeks p.i. (E) Jejunum from a mouse administered praziquantel at 4 weeks p.i. (F) Jejunum from a mouse withoutpraziquantel administration at 8 weeks post -S. japonicum infection. H & E stain. Bar: 300 m in (A); 120 m in (B-F).


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Fig. 5. Changes in serum antibody levels specific to S. japonicum antigens

before and after praziquantel treatment in three groups of mice: mice withpraziquantel administration at 8 weeks post S. japonicum infection (closedsquare; n = 6); mice at 8 weeks p.i. without praziquantel administration

(open square; n = 5); uninfected control mice treated with praziquantel(open circle; n = 5), IgG, IgM, IgA and IgE class antibodies to adultworm antigen (SjA) or to egg antigen (SjE) were determined by ELISA.Values are presented as the average SD.

quantel adverse effects previously reported in humanpatients with Schistosoma mansoni (Polderman et al., 1984) orwith S. japonicum (Chen et al., 1983; Noseas et al.,1984; Watt et al., 1986) infection. However, the patho-physiology of unfavourable reactions has not beenexplained, and this is the first report describing the patho-

physiology of adverse effects of praziquantel in hostsinfected with schistosomes.

Anaphylactic responses generally occur shortly aftercontact with foreign antigens, referred to as allergens,

with which we have been sensitised in advance. The ques-tion is what played the role of allergen in S.japonicum-infected mice after praziquantel administration? Bothadult worms and eggs ofS.japonicum existed in the infectedmice at 8 weeks p.i. Praziquantel acts both on adult wormsand eggs of schistosomes causing disruption of the outersurface tegument of adult worms (Becker et al., 1980; Mehl-horn et al., 1981; Shaw and Erasmus, 1983a,b; Irie et al.,1989) and immediate hatching of mature eggs laid in hosttissues (Matsuda et al., 1983; Giboda and Smith, 1994)These post-praziquantel alterations in parasites undoubtedlyinduce a sudden release of antigenic components from bothadult worms and eggs damaged by praziquantel, which initi-ates a series of responses in praziquantel-treated hosts. As

determined by ELISA for parasite-specific immunoglobu-lins, anti-S.japonicum egg IgM and IgA levels remarkablydecreased just after praziquantel treatment, suggesting thatantigenic components from the parasite eggs, rather thanthose from adult worms, were abundantly released andthen bound to specific immunoglobulins in the blood circu-lation. In addition, the release of S.japonicum eg g antigenfrom hatched eggs into surrounding tissues was clearlydemonstrated by the results of immunohistochemistryusing a mAb specific for S.japonicum egg antigen.

In order to clarify the contribution of S.japonicum eggantigen originating from hatched eggs to the pathogenesisof post-praziquantel anaphylaxis, we administered prazi-quantel to the infected mice at 4 weeks p.i. At this periodof S.japonicum infection in mice, the worm pairs have justreached sexual maturation and are about to begin egg-laying(Moloney and Webbe, 1983). Therefore, there exist few, ifany, mature eggs ofS.japonicum in infected mice at 4 weeksp.i. When treated with praziquantel at 4 weeks p.i., mice didnot exhibit the severe systemic signs observed in mice givenpraziquantel at 8 weeks p.i. Furthermore, histopathologicobservation revealed that intestinal lesions were completelyabsent in mice treated at 4 weeks p.i. These observationsrevealed that antigenic components from hatched eggs,rather than those from adult worms , play a major role asallergens in the pathogenesis of adverse effects in praziquan-tel-treated mice, particularly in that of intestinal injuries.

Mast cells induce profound anaphylactic reactionsthrough vigorous release of a wide variety of inflammatorychemical mediators, arachidonic acid metabolites, and anumber of immuno-regulatory cytokines (Schwartz andAusten, 1984; Stevens and Austen, 1989; Gordon et al.,1990; Galli, 1993). In this study, histologic examinationrevealed that mucosal mast cells had infiltrated into intestinal mucosa of mice by 8 weeks after S.japonicum infec-tion, whereas mast cells were nearly absent in mice at 4weeks after S.japonicum infection and in uninfected controlmice. On the basis of these findings, together with the fact


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Fig. 6. The result of immunohistochemistry using a monoclonal antibody specific to S. japonicum egg antigen. The Antigen was released from hatched

eggs in intestinal tissues of mice treated with praziquantel at 8 weeks post -S.japonicum infection (A-C). The reaction was confined to within the eggshellin unhatched eggs (D). Bar: 120 m in (A); 30 m in (B,D); 20 m in (C).

that mature S.japonicum eggs were present in bowel tissuesat 8 weeks p.i. but absent at 4 weeks p.i., we speculate thatmature eggs, but not adult worms, induce mast cell infiltra-tion in the bowel of infected mice. Mature eggs of S. japo-nicum secrete ES proteins originating from a miracidiumdwelling in eggs (Kawanaka and Carter, 1992). Theseproteins secreted from mature eggs probably stimulate thehost immune system, including mast cell infiltration into theintestine of S. japonicum-infected subjects. In the presenceof intestinal mast cell infiltration, sudden release of antigensfrom a number of mature S. japonicum eggs laid in hostbowel tissues appears to provoke large-scale activation ofintestinal mast cells and subsequent release of chemicalmediators, resulting in immediate anaphylactic reactions.

Actually, mast cell degranulation was observed frequentlyin the intestinal mucosa of praziquantel-treated mice, andwas particularly remarkable in tissues around hatched eggs.

IgE class antibody is characterised by its high activity toinduce degranulation of mast cells and basophils despite itsextremely low concentration in serum. Therefore, parasite-specific IgE is highly likely to have played a pivotal role inthe development of anaphylactic reactions observed in thisstudy although its serum level measured by ELISA wasquite low at 8 weeks post-S. japonicum infection, whenpost-praziquantel adverse effects were observed. Gaubertet al. (1999) measured anti-egg IgE in serum from S.mansoni-infected BALB/c mice, with the result that theIgE level did not increase until 60 days p.i., but began to


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Fig. 7. Mucosal mast cells in a jejunal sample from mice with or withoutpraziquantel treatment at 8 weeks after S. japonicum infection. (A,B)Degranulated mast cells (arrow heads) around a hatched egg in jejunumfrom a mouse after treatment with praziquantel. (C) Non-degranulated

mast cells (arrows) in jejenum from an untreated mouse. Toluidine Bluestain. Bar: 30 m in (A); 20 m in (B,C).

elevate thereafter. In S. japonicum-infected mice also, theparasite-specific IgE level measured by ELISA may begin toincrease around 8 weeks p.i. or later.

Biogenic amines are on a molar basis the dominantcomponents of the secretory granules in rodent mast cells,

and a major vasoactive mediator released by activated mastcells is histamine (Schwartz and Austen, 1984). Therefore,we measured plasma histamine levels in S. japonicum-infected mice at 30 min after praziquantel administration.The plasma histamine level was much higher in S.japoni-cum-infected mice just after praziquantel administrationthan in the infected mice without praziquantel treatment.This result strongly suggests that mast cells, by releasingvarious chemical mediators, play an important role in thepathogenesis of the adverse effects of praziquantel treatmentof schistosomiases.

Our observations in this study have led us to the conclu-sion that intestinal mast cells, which have in advance infil-trated into the intestine of S. japonicum-infected mice, are

activated by the sudden release of antigens from eggshatched as an effect of praziquantel, and that these mastcells, by releasing a variety of chemical components, playa pivotal role in the pathogenesis of post-praziquantelanaphylactic signs in S. japonicum-infected mice. However,studies have revealed the existence of novel pathways ofimmediate allergic reactions including non-mass cell-mediated pathways (Oettgen et al., 1994; Korsgren et al.,1997; Takeda et al., 1997), although it is generally believedthat mast cells are central to host anaphylactic re actions.Blood-dwelling basophils are also the major source of hista-mine and are involved in the development of allergic

Fig. 8. Plasma histamine levels in S. japonicum-infected mice with (n =

13) or without (n = 13) praziquantel administration at 8 weeks p.i. Data forthe uninfected control mice with praziquantel treatment (n = 12) are alsoshown. Plasma samples were collected at 30 min after praziquantel admin-

istration. Values are presented as the average SD.


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diseases (Costa et al., 1997). Accordingly, it is likely thatbasophils were, at least in part, responsible for an increase ofplasma histamine and subsequent anaphylactic reactions inmice showing post-praziquantel adverse effects althoughthis possibility has not been investigated. The precise

mechanism(s) of host reactions involved in the pathogenesisof post-praziquantel adverse effects therefore remains unex-plained and must be clarified in further detailed studies.

In praziquantel treatment of human schistosomiases,intense abdominal symptoms appear immediately aftertreatment, and the frequency of adverse reactions is corre-lated with the intensity of infection (Chen et al., 1983; Nose-as et al., 1984; Polderman et al., 1984; Watt et al., 1986). Inaddition, severe symptoms occurred more frequently inpatients given high dosages of praziquantel than in thosegiven divided doses at relatively low dosages (Noseas etal., 1984). These characteristics of post-praziquanteladverse effects suggest that immediate reactions due to anti-gens from damaged parasites are involved in the pathogen-

esis of post-praziquantel adverse effects in human patients,as is the case for the murine model used in this study.Accordingly, murine schistosomiasis japonica is a promis-ing animal model for the determination of the immunologi-cal and pathophysiological aspects of adverse effects ofpraziquantel in human schistosomiases, and findings inthis murine model are expected to provide valuable infor-mation concerning the clinical use of praziquantel. Forexample, as proposed by Noseas et al. (1984) on thebasis of their clinical observations, chemotherapy withtwo or more praziquantel administrations each at a lowdosage, which will reduce the amount of parasite antigensreleased after treatment, is recommended to minimise theoccurrence of severe adverse effects, although single admin-istration is more convenient.

Acknowledgements

The author is very grateful to Professor H. Matsuda ofDokkyo University School of Medicine for constructiveadvice and critical reading of the manuscript. Many thanksare expressed to Mr Y. Hirose, Dr M. Kirinoki, Dr S. Kawai,Dr Y. Chigusa, Professor Y. Kamikawa and other staff ofDokkyo University School of Medicine for technicalsupport and/or fruitful discussion. Thanks to Dr H. Yokoifor providing a mAb used in this study. This study was

supported in part by grants for Emerging and re-emerginginfectious diseases from the Ministry of Health, Labor andWelfare, Japan, and from the Japan-US Cooperative Medi-cal Research Program.

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Adverse Effects of Praziquantel

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