mansoni Infections

American Journal of Pathology, Vol. 142, No. 3, March 1993Copyrigbt ) American Societyfor Investigative Pathology

Two Distinct Pathological Syndromes in MaleCBA/J Inbred Mice with Chronic Schistosomamansoni Infections

Gregory Stephen Henderson,* Nancy A. Nix,ttM. Angela Montesano,t Daniel Gold,tGeorge L. Freeman, Jr.,§Thomas L. McCurley,* and Daniel G. Colleyt§Departments ofPathology and Microbiology andImmunology,t Vanderbilt University School ofMedicine, theVeterans Affairs Medical Center;5 and Meharry MedicalCollege5, Nashville, Tennessee

Humans chronicaly infected with Schistosomamansoni most commonly present with therelatively asymptomatic intestinal form of thedisease, whereas a smaUl minority develophepatosplenism characterized by severe hepaticdisease with portal hypertension. Investigationof hypotheses describing the pathogenic mecha-nisms underlying the clinicalforms ofthe humandisease has been limited by the absence of ananimal model that predictably develops such aspectrum ofdisease. We report that inbred maleCBA/J mice that are chronically infected with S.mansoni develop two distinct syndromes, hyper-splenomegaly syndrome (HSS) and moderatesplenomegaly syndrome (MSS). Pathologicalyand immunologicaly, MSS and HSS remarkablyparaUel the intestinal and hepatosplenic clinicalforms, respectively, in humans. HSS affectsapproximately 20% ofthese mice and consists ofmassive splenomegaly, ascites, thymic atrophy,severe anemia, and cachexia. The remainingmajority of mice with MSS develop moderatesplenomegaly only. Histopathologicalfeatures ofHSS include 1) relatively extensive hepaticfibro-sis and granulomatous inflammation, 2) spleniccongestion, 3) lymph node plasmacytosis, and 4)worms and eggs in the pulmonary vasculature.Immunologicaly, the idiotypes present on anti-soluble egg antigen antibodiesfrom HSS mice aredistinct from thosefrom mice with acute infec-tions or the chronic MSS infection. These idio-typic differences are similar to those observed inpatients with intestinal and hepatosplenicformsof the disease and may have regulatory im-

portance. Investigation ofthe celular and molec-ular events that lead to the development ofMSSand HSS may advance current understanding ofthe pathogenesis ofthe clinicalforms ofchronicschistosomiasis in humans. (AmJ Pathol 1993,142:703-714)

Chronic Schistosoma mansoni infection in humans isa spectral illness, and patients usually present withone of several generalized clinical forms.1-3 Themajority of chronically infected patients have anasymptomatic and ambulatory clinical status, whichhas been termed the intestinal (INT) form of thedisease. Less than 10% of chronically infectedpatients exhibit the hepatosplenic (HS) clinical formcharacterized by hepatosplenomegaly, "pipestem"fibrosis, portal hypertension, ascites, esophagealvarices, hematemesis, and often, if untreated, death.The INT and HS ends of the spectrum of humanschistosomiasis are also immunologically distinct.Peripheral blood mononuclear cells (PBMC) fromacutely infected and ambulatory HS patientsrespond more vigorously to some schistosomal anti-gen preparations than do PBMC of INT patients and,after curative chemotherapy, full responsiveness ofINT patients against soluble egg antigens (SEAs) isregained.1'3-5 Patients with the various clinical formsalso have distinct anti-SEA antibody idiotypic reper-toires, which differ in their ability to stimulate in vitro

Supported by the National Institutes of Health [grants Al 11289 andT32-GM-07347 (GSH) and T32-AI-07281 (NAN)], the ConselhoNacional de Pesquisas (MAM), and the Department of VeteransAffairs.

Dr. Montesano's current address: Universidade Federal de Juizde Fora, Juiz de Fora, MG, Brazil.

Dr. Gold's current address: Department of Human Microbiology,Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978,Israel.

Accepted for publication August 28, 1992.Address reprint requests to Dr. Daniel G. Colley, PDB/DPD/NCID/

CDC, Bldg 23, MS F-13, 4770 Buford Highway N.E., Atlanta, GA30341.

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704 Henderson et alAJP March 1993, Vol. 142, No. 3

proliferation of regulatory anti-idiotypic T lympho-cytes present in preparations of PBMC from patients6-12 or spleen cells from infected mice.9 These idio-typic differences can also be detected serologicallyby either polyclonal8'9 or monoclonal anti-idiotypicantibodies.10 These differences have led to thehypothesis that the efficacy of immunoregulatorymechanisms, as well as the intensity of infection13and the immunogenetics of the host,3 11'14'15 contrib-utes to the clinical outcome of chronic S. mansoniinfection in humans 1,3,5,11,16,17The direct investigation of this hypothesis has

been hindered by the apparent lack of an animalmodel that represents the spectral presentation seenin chronic human infection. Among inbred and out-bred mouse strains experimentally infected with S.mansoni, a number of differences in the severity ofinfection-induced pathology and in the immuneresponse to worm and egg antigens have beendescribed.18-27 Chronically infected mice areoften referred to as being hepatosplenic23'28-31because of their development of hepatosplenome-galy, increased portal pressure, collateral circulation,and esophageal varices, relative to normal mice andmice in the acute stage of infection.28,29'31'32 How-ever, no clear spectrum of pathology or correlativeimmunology has been defined at any given timepoint in chronically infected mice.We now report that, in groups of male CBA/J mice,

each exposed to an average of 45 cercariae of S.mansoni, a spectrum of morbidity develops as infec-tions become chronic. In approximately 20% of thesemice, a distinct pathological syndrome develops thatgrossly consists of massive splenomegaly, ascites,thymic atrophy, severe anemia, and cachexia. Thehistopathological features of the syndrome aresplenic congestion, lymph node plasmacytosis, andextensive liver fibrosis associated with a relativelylarge percentage of liver mass being occupied byfibrotic granulomatous lesions. These features paral-lel many of those exhibited by HS patients, and thisgeneral similarity is also manifested in parallel immu-nological differences. Immunologically, mice withthis more severe syndrome (hypersplenomegalysyndrome) (HSS) express an anti-SEA antibody idio-typic profile distinct from that of acutely infectedmice and the other 80% of chronically infected micethat do not develop this syndrome but maintain adefinite, but more moderate, splenomegaly (moder-ate splenomegaly syndrome) (MSS). In this report wedescribe the initial parasitological, gross pathologi-cal and histopathological, and immunological char-acteristics that define these two syndromes in maleCBA/J mice with chronic S. mansoni infections.

Materials and Methods

Animals and Infections

Male CBA/J mice (6 to 8 weeks of age) wereobtained from The Jackson Laboratory (Bar Harbor,ME) or through a National Cancer Institute contract[IGA V101 (134A)P-77014] and were maintained inthe American Association for the Accreditation ofLaboratory Animal Care-approved animal care facil-ity of the Veterans Affairs Medical Center. Mice wereinfected by subcutaneous injection of 45 cercariae ofa Puerto Rican strain of S. mansoni maintained inBiomphalaria glabrata snails. Replicate studiesinvolved the use of mice from various shipments,which were infected with different pools of cercariae.

Examination of Gross PathologyMice infected for different lengths of time and age-matched, uninfected, control mice were examined inthe following manner. A blood sample was collectedin a microcapillary tube, from a minimal tail snip, forhematocrit determination. Mice were weighed andanesthetized with ether. A larger blood sample forserum collection was taken from the retro-orbitalplexus, and the mice were sacrificed. Through amedial abdominal incision the presence or absenceof visible ascitic fluid was noted. The liver, spleen,and mesenteric lymph nodes were removed,weighed, and fixed in 10% buffered formalin. Finally,the presence or absence of retroperitoneal fat wasnoted for each mouse, as an indication of cachexia.

Preparation of Tissues for MicroscopicExaminationTissues fixed as described above were embedded inparaffin and sectioned. Liver sections were stainedwith hematoxylin and eosin and with Masson'strichrome stain.33 Spleen and lymph node sectionswere stained with periodic acid/Schiff reagent.Microscopic observations were made and recordedfor tissues from both normal and infected mice.Quantitative measurements of granuloma size andthe overall area of fibrotic tissue were evaluated onhematoxylin/eosin- and Masson's trichrome-stainedtissues, respectively, by using a BioQuant imageanalysis system (Bioquant Inc., Nashville, TN) asdescribed previously.34

Worm Recovery and EnumerationThe method used for perfusion, worm recovery, andenumeration was that described by Colley andFreeman.21'25 Mice were sacrificed by intraperito-

Two Syndromes in Mouse Schistosomiasis 705AJP Marcb 1993, Vol. 142, No. 3

neal injection of 0.5 ml of a mixture containing 3 ml ofsodium pentobarbitol (60 mg/ml; Diamond Labora-tories) and 12 ml of perfusion fluid (0.05 mol/Lsodium citrate, 0.15 mol/L sodium chloride). Perfu-sion fluid (15 to 20 ml) was pumped by a peristalticpump through the left ventricle and exited via the cuthepatic portal vein. Worms were collected by filteringthe perfusate through 73-pm nylon mesh. Wormswere placed in a petri dish in perfusion fluid, exam-ined by stereomicroscopy, and counted.

Quantitation of Hepatic SchistosomeEggsLivers from infected mice were removed andweighed. A known weight of liver tissue was thendigested overnight in 5% potassium hydroxide.30The eggs present in an aliquot of the digest werecounted under microscopic visualization, and thetotal number of eggs per liver was calculated foreach mouse.

Preparation of Human Anti-SEAMonoclonal Antibody E5E5 is a human IgG2 monoclonal antibody with anti-SEA specificity that was originally prepared from thePBMC of a patient with the INT clinical form ofschistosomiasis.35 E5 has been shown to stimulatethe in vitro proliferation of anti-idiotypic PBMC frompatients infected with S. mansoni.67 The E5-produc-ing heterohybridoma, generously provided by Dr. A.Goes and Dr. B. Doughty (Texas A & M University,College Station, TX), was grown in Pristane-primednude mice, their ascites fluids were collected, andthe IgG fraction of the ascites was obtained by pre-cipitation with 40% ammonium sulfate. The IgG pre-cipitate was resolubilized, dialyzed, and passed overa GammaBind G-agarose column (Genex, Gaithers-burg, MD) to further purify the IgG fraction containingE5. The final protein concentration of the E5-contain-ing IgG solution was determined with a bicinchoninicacid protein determination kit (Pierce, Rockford, IL).

Preparation of Mouse Anti-SEA Antibodiesfrom Sera of Infected MiceMouse anti-SEA antibodies, termed idiotypicantibodies (Id), were isolated as reported pre-viously. 8-10,36-38 Pools of sera from mice infected foreither 8 weeks or 20 to 30 weeks (further segregatedbased on spleen weight) were collected and storedat -20 C until absorption. SEAs were coupled toCNBr-activated sepharose 4B (Pharmacia, Piscat-away, NJ), and serum pools were mixed with SEA-

sepharose at 4 C overnight, with continuous rotation.SEA-sepharose beads were then washed with 0.01mol/L Na3PO4, 1.0 mol/L NaCI, pH 7.2, to removenonspecifically bound material. Bound, immunoaffin-ity-purified anti-SEA antibodies were eluted with 0.1mol/L glycine-HCI, pH 2.8, and neutralized by col-lection directly into 0.25 volume of 0.1 mol/L sodiumborate, pH 8.0. Fractions were pooled and dialyzedagainst phosphate-buffered saline, pH 7.4.

Preparation of Rabbit Anti-E5 and Anti-IdPolyclonal AntibodiesThe method used for generating anti-E5 and anti-Idpolyclonal antibodies was that described by Monte-sano et al.8-10 Rabbits were immunized subcutane-ously in two sites with 200 pg of E5 or Id purified fromthe various groups of infected mice, diluted 1:1 incomplete Freund's adjuvant (Difco Laboratories,Detroit, Ml). Booster injections of 100 pg of antibodydiluted 1:1 in incomplete Freund's adjuvant weregiven twice at 15-day intervals. Fifteen days after thefinal injection, the rabbits were bled and the serawere obtained. Each rabbit antiserum was precipi-tated with 40% ammonium sulfate. The precipitatewas resuspended in phosphate-buffered saline andexhaustively absorbed (10 to 16 times) on either nor-mal human IgG-coupled or normal mouse immuno-globulin-coupled CNBr-activated sepharose 4B.Absorptions were considered complete when therabbit antibodies were shown to react by enzyme-linked immunosorbent assay (ELISA) with E5 or theimmunizing mouse Ids but not with normal humanimmunoglobulin or normal mouse immunoglobulin,respectively.

Anti-Id ELISA and Competitive ELISAAnti-Id ELISA was performed by reacting variousdilutions of anti-E5, anti-8-week Id, and anti-MSS Idwith purified E5, 8-week Id, or MSS Id antibody,respectively, that had been bound to Immulon 11microtiter plates (Dynatech Laboratories, Inc., Chan-tilly, VA). Specifically bound rabbit anti-Id antibodieswere detected by reaction with biotinylated goat anti-rabbit antibody (Sigma Chemical Co., St. Louis, MO),followed by avidin-peroxidase (Sigma) and 2,2'-azi-no-di(3-ethylbenzthiazoline sulfonate) substrate(Kirkegaard and Perry Laboratories, Inc., Gaithers-burg, MD) addition.39 The resulting optical density ofeach well was measured with a MR 600 microplatereader (Dynatech). The dilutions of rabbit serum atwhich half of maximum specific reactivity wasreached were 1/320 for the rabbit anti-E5 and anti-8-week Id reagents and 1/1 60 for the rabbit anti-MSS


NORM AL

Figure 1. Gross comparison ofspleen sizes ofnormal uninfected, MSS, and HSS mice. The weights (in mg) ofeach ofthe spleens (from top to bottom)are as follows: normal, 60, 110, and 70; MSS, 210, 230, and 270; HSS, 780, 1160, and 1180. The scale bar shows length in mm.

Id antibodies. Competitive ELISAs were performedaccording to the method of Montesano et al.8 10Rabbit anti-E5 or anti-Id reagents at the aforemen-tioned dilutions were preincubated with various dilu-tions of whole serum or various concentrations ofpurified anti-SEA Ids from MSS, HSS, or 8-week-in-fected mice. The preincubated rabbit sera were thentested for their ability to bind the idiotypes expressedon the respective specific antibodies, ie, E5, 8-weekId, or MSS Id. The enzyme/substrate detection sys-tem used in this competitive ELISA was the same asused in the anti-Id ELISA.

ResultsGross Characterization and Incidence ofHSSDuring the course of investigations that required theexamination of large numbers of male CBA/J micewith chronic infections (>20 mice each time), it wasobserved that an appreciable, and relatively predict-able, number of these mice had a distinctive syn-drome, termed HSS, with several grossly observablefeatures. The most striking of these features is mas-sive splenomegaly beyond the level of splenomegaly

Figure 2. HSS at various periods of infection. Ateach infection time point, the indicated ntumberof mice were killed, the spleens were weighed,and the presence or absence of each featuire ofHSS uas noted. The distribution of individualspleen wveights at each infection time point isplotted. The line at 550 mg indicates that miceuwith a spleen weight of 550 mg uniformallyexhibit all otherfeatures ofHSS. The same is tnreof mice in uhich the percentage of body weightofthe spleen is 1.2% (see text). Thepercentage ofmice with a spleen weight of 550 mg at eachinfection time point is indicated in parentheses.

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n=37- (24.3%)

o n=53 n=21(20.8%) (19.1%)

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Two Syndromes in Mouse Schistosomiasis 707AJP March 1993, Vol. 142, No. 3

seen in the majority of mice with chronic infections(Figure 1). Another gross feature characteristic ofHSS is cachexia, which is most readily apparent bythe absence of retroperitoneal fat. HSS mice werealso observed to have 2 to 5 ml of ascites, whereasascites was never seen in mice with MSS. HSS micehave a markedly reduced mass of grossly visiblethymic tissue, compared with MSS mice. In a surveyof >200 mice infected for 8 to 80 weeks, it was foundthat HSS appeared to develop after 10 weeks ofinfection and that there was a critical spleen mass of550 mg and/or 1.2% of total body weight, at or abovewhich all four gross features of HSS were alwayspresent. Approximately 14% of mice infected for 10to 17 weeks had HSS, and in mice infected forbetween 18 and 80 weeks the presence of HSSincreased to between 19 and 24% (Figure 2). HSSmice are also profoundly anemic. In a group of miceinfected for 20 to 25 weeks, a strong inverse corre-lation was observed between spleen weights andhematocrit values (Figure 3; Pearson's r = 0.91; P <0.001). This relationship between mice with severeanemia and those with HSS usually allowed for theidentification of HSS mice before sacrifice by therelative pallor of their muzzles and paws.

Histological Comparison of Spleens,Lymph Nodes, and Lungs from MSSand HSS MiceMicroscopic examination of sections of spleenstaken from MSS and HSS mice showed that bothgroups had white pulp hyperplasia with secondaryfollicle formation and mantle zone expansion (Figure4, A and B). A distinguishing feature of the spleensfrom HSS mice was a greater degree of red pulpexpansion and extensive sinus distention by eryth-rocytes, leukocytes, and megakaryocytes (Figure 4,C and D). Examination of the mesenteric lymphnodes of both MSS and HSS mice showed that both

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HematocritFigure 3. Individual spleen weights versus hematocrit values of micewith infections of20- to 25-week duration. Linear regression analysisrevealed a correlation betuween spleen weight and anemia (Pearson's r= 0.91, P < 0.001).

groups had similar degrees of expansion of primaryand secondary follicles in the cortex. In 100% of thelymph nodes examined from HSS mice (n = 6), themedulla was consistently expanded by sheets ofplasma cells, which often extended into the corticalinterfollicular areas. This finding of extensive medul-lary plasmacytosis was present in only approxi-mately 50% of lymph nodes of MSS mice (n = 6).Histopathological observations of the lungs of HSSmice showed that at least 60% of these animals hadshunted at least one adult worm to their lungs, and allHSS mice had many eggs in their pulmonaryvasculature. In contrast, the lungs of MSS mice didnot contain any worms and only rarely were eggsseen in the lungs.

Gross and Histological Comparison ofLivers of MSS and HSS MiceThe livers of mice with MSS were reddish brown incolor, with multiple yellow-white, pinpoint-sized,punctate areas visible on all serosal and cutsurfaces. The serosal surfaces had a slightly nodulartexture. The livers of mice with HSS had a lighterred-gray color, with multiple dark green areas. Theyellow-white punctate lesions seen in MSS liverswere present at a higher density in HSS livers. Theserosal surfaces of HSS livers were somewhat morenodular than those of MSS livers. The mean volumesof individual hepatic granulomas formed aroundnewly deposited eggs were determined for groups ofMSS and HSS mice, and no significant differencewas found (Figure 5A). However, quantification of themean percentage of granulomatous tissue and asso-ciated fibrosis in the livers of these same groupsshowed that the HSS mice had a significantly greater(P < 0.001) percentage of their liver tissue involvedin granulomatous and fibrotic areas than did the MSSmice (Figure 5B). The qualitative microscopicappearances of MSS and HSS livers were remark-ably distinct, secondary to this difference in thequantity of granulomatous and fibrous tissue. At lowpower, the granulomas in MSS livers were single dis-persed fibrotic lesions (Figure 4C). In contrast, thegranulomas in HSS livers were usually coalescentand only rarely were isolated lesions seen (Figure4D). Thus, predominately intimal fibrosis of largebranches of the portal vein, often without adjacentgranulomas, and marked periportal fibrosis wereconsistently seen in HSS livers but only rarely in MSSlivers (Figure 4, E to H).

Quantification of Worm Burden and Eggsper Liver in MSS and HSS Mice

The numbers of mature worms and eggs per liverwere counted in individual mice with HSS and MSS.

5


Figure 4. Histological comparison of spleens from MSS (A and C) and HSS (B and D) mice. A and B: original magnification, X 40 (periodicacid/Schiffstain); C and D: original magnification, X 400 (hematoxylin and eosin stain). E to H: microscopic appearance of liversfrom mice withMSS and HSS. Granulomas in MSS (E) and HSS (F) livers; original magnification, X 40. Intrahepatic portal veins in MSS (G) and HSS (H) livers;original magnification, x 100. E to H are all stained u'ith Masson's trichrome stain.

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Two Syndromes in Mouse Schistosomiasis 709A/P Mlarch 1993. V'ol. 142, No. 3

TOTAL WORMS MALE WORMS FEMALE WORMS POSSIBLE PAIRS

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Measiirelients fllcisson s trichronic'-stainc'l liver sC'ctio)is. Graniido-n1ias nerc' conisidler'eCl nien' /' the sectionI contciined viable

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tissue C gicgrCaniiloii/ c{-ai.ssociate(Cl/ibr-osiSiS in the liv ers OJ11SS cinld 11SmiceC 7Ihe nivan Percentcage qfJgra inlomcas anclfibrosis in liier tissle'

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mic romi'tric micasuremnicts of( lasson s tricbhrolnie-stained liversections. (alciucitedgOI) iean iales +v SD were 20.2 7.8%Yor-lISS micc (n = 19) carcI 0.0 + 9.2/fr- IISS miiicc (n =19). P <0.001.

No significant difference between MSS (n 20) andHSS (n = 25) groups was found in the number ofmale or female worms or the total number of wormsobtained by perfusion (Figure 6A). The numbers ofmale and female worms present in individual micewere also used to calculate the number of possible,but not necessarily observed, male/female pairsinfecting each mouse. In this case it was found thatthe group of HSS mice had a slightly but significantlygreater mean number of possible pairs than did the

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group (I1SS, n = 30: HSS, n = 19). Gr-oup mlc'anis ar-e inidicate(d by bars

Superimlposecd over the poinits. (Grou/) ieani i'aliies + SD aore as /')ollks.)s

totil i)ors.il JSS 4.2 _ 3-2. 1ISS 4. 7 _ 2. 5; maICle worms( 1155 = 2. 7+ M0HSS = 2.4 1.6; female worms: ISS = .5 + 1. , HSS = 21.2 possible pairs: ISS = 1.0 ±+ 0.7. HSS = 1.8 //.IOnl! the

hi/Terence ill tnhn11{71ber oJ p)ossible pairs is statisticall/ sili/n/iccant (P

< 0 05) B: total eggs per liter ili ISS or4.1SS oinic'. Points corresp)ond

tc) the niin ibcrcj/ 'eggsped? liiver in indidcivi l niice il cacich gr-oup (J1IS= 20; 1ISS15 = 25). Ihe mean liier egg lacdl _ SD/b0)r eacic grouIp,

indiciccitecl hbi bcirs suiperimipc)secd over the incdividcial iacihes, was 25, 5119. 789 eJslIiver for .ISS mice aind 40 14 3 ± 16.240 eggs/liver/for

HSS miiice. These niean ialles ar-e signfiJccaitl cl// eret (<<) 0.5).

group of MSS mice (1.8 + 1.1 versus 1.0 ± 0.7, P<0.05; Figure 6A). The group of HSS mice was alsofound to have a signficantly higher mean number ofeggs per liver than did the group of MSS mice (P <0.05, Figure 6B), although, as with worm burdens,considerable overlap existed between individualmembers of the two groups.

Competitive ELISA Analysis of Sera andPurified Id Antibodies from HSS, MSS,and Acute-Phase Mice

Idiotypic competitive ELISAs, as used by Montesanoet al,8 were initially performed to test the ability ofdilutions of pooled sera from uninfected mice, micein the acute phase of infection (8 weeks), or micewith chronic MSS or HSS infections to competitively

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inhibit the binding of Id-specific polyclonal rabbitanti-E5 antibodies to E5, a cross-reactive Id-bearing,human monoclonal anti-SEA antibody. At dilutions of1/10 and 1/20 sera from mice either infected for 8weeks or with chronic MSS infections efficientlyinhibited the binding of anti-E5 to E5 (Figure 7A). In

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Id Concentration (mcgmr/mi)Figure 7. A: 851anti-E5 competitive ELISA utsinig senin pools ascompetitors. The humelani anti-SEA monoclonal anitibody, E5 was iused tocoat the twells ofmicrotiterplates. The ability, of rabbit anti-E5 sertu ata dilutioni of 1/320 to binid to the plate-boutnd k5 uwas evaluated by

FLLISA, after preitmctubationi with serial diluitionis of pooled sera fromneither niormal uniin1fected mice, mice infected Jbr 8 ueeks (acuttepbase), chrontically inifected MSS mice, or chronically^ intfcectced HSSmiiice. B: E51anti-E5 competitive ELISA u(sinlg anti-SELA anitibodies (Id)

puritriedfrom the senrun pools used in A (Id) as competitors. The abilityof rabbit anti-E5 senum at a diluitioni o) 1/ 320 to bintd to plate-bound15 was evaluated bv ELISA, after preinicutbationi with the indicatedconcentrations ofamiti-SEAantibodies puoifiedfrorn eitbera siniglepoolofsera ftoro nice infectedfor 8 uweeks or cbronically injccted 155 miiceor tuo separate pools of sera firom cbronically ilkctdcl HSS miiice. Allclata are reported as the inean optical d/enlsity cat 4/0 71)i u/'triplicateivells.

contrast, inhibition of the E5/anti-E5 ELISA was notseen with any dilution of sera from mice with HSS orfrom uninfected mice. To more accurately control forvariable amounts of antibody in the serum pools,specific anti-SEA antibodies were immunoaffinitypurified from pools of sera from mice infected for 8weeks, pools of sera from mice with chronic MSSinfections, or two separate pools of sera from micewith chronic HSS infections. Protein concentrationsof each antibody preparation were assayed for inhi-bition of the E5/anti-E5 ELISA system. As with wholeserum dilutions, immunoaffinity-purified anti-SEAantibodies from mice with either 8-week infections orchronic MSS infections both contained Ids capableof inhibiting the binding of anti-E5 antibodies to E5.Also, neither preparation of anti-SEA antibodies fromtwo distinct pools of chronically infected HSS micehad detectable inhibitory ability (Figure 7B), ie, theydid not contain the cross-reactive, E5-expressed Idsshared by chronically MSS mice and acutely 8-week-infected mice.

To establish further the breadth of differencesapparently present between the Ids expressed byanti-SEA antibodies from MSS and HSS mice, sepa-rate purified anti-SEA antibody preparations fromthese sources were tested for their inhibitory ability inpolyclonal 8-week Id/anti-8-week Id and MSS Id/anti-MSS Id competitve ELISAs. The results of the 8-weekId/anti-8-week Id competitive ELISA (Figure 8A)showed that MSS Id and 8-week Id inhibited bindingequally, with each preparation apparently sharingIds in the same proportions. In this polyclonal sys-tem, the two HSS Id preparations also inhibited bind-ing (in contrast to the results seen in the monoclonalE5/anti-E5 system) but required approximately16-fold higher concentrations of HSS Id than of MSSId or 8-week Id to achieve 50% inhibition. A similardegree of differential inhibitory activity between thetwo HSS Id preparations and the 8-week Id and MSSId preparations was seen in the MSS Id/anti-MSS Idcompetitive ELISA (Figure 8B).

DiscussionThis study demonstrates that male CBA/J micechronically infected with small worm burdens of S.mansoni develop two distinct syndromes, MSS andHSS, which differ pathologically and immunologi-cally and appear to be generally analogous to theINT and HS clinical forms, respectively, of chronicschistosomiasis in humans. This is the first report ofthe predictable occurrence of such syndromes inchronically infected mice; however, pathologicalvariance in infected mice is not without precedent.Cheever55 found that a minority of chronically

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Id Concentration (mcgm/mI)

Figure 8. A: Eight-week Idlaoti-8-week Id competitive FIISA utsinigpurified anti-SEA antibody, (Id) preparations as cornpetitors. Id aniti-body purifiedfrom serum pooledfroI 8-u'eek-infected mnice u'as used

to coat the wells ofmicrotiter plates. The ability ofrabbit anti-8-week Idseruim at a diluitioni of 1/320 to bind to the plate-bounlld 8-u'eek Id wasevaluated by EI.ISA, afterpreincubation uwith the inidicated cotncenttra-tions of Id anztibodies purified fromn either a sinzgle pool of sera fronimice infected for 8 weeks or chrontically inzfected MSS mice or tuwoseparate pools of sera froni chronically in?fected HSS niice. B: MSSId/lnti-MSSId competitive ELISA uisinigputrified anti-SEAan4tibody (Id)preparationis as competitors. Id antibody purified from senim pooledfrom chronically infected MSS mice u'as used to coat the u'ells ofmicrotiter plates. The ability of rabbit anti-MSS Id senrm at a dilutionof 11160 to bind to the plate-boun1d MSS Id uwas evaluated byIEIISA,afterpreinicuibatioi u'ith the indicated concentrations ofId an tibodiespturified eitherfrom a sinigle pool ofserafrom mice injected for 8 weeks

or chrotnically infected MSS mice or two separate pools of sera fromcbronically infected IISS mice. Data are reportced as the mean opticaldensity, at 410 nin of triplicate wells.

infected C3H mice develop a relatively higherdegree of hepatic fibrosis but apparently do notdevelop any of the other pathological features of HSSseen in CBA/J mice. The degree of splenomegaly,

anemia, and portal hypertension that develops as aresult of chronic S. mansoni infection also varies con-siderably between genetic strains of mice but hasnot heretofore been described to vary significantlywithin a given strain of mice with similar infectionintensities. 18-20,22-24,28,29,31,40-42,55 Other laborato-ries have extensively used the CBA/J strain in stud-ies of S. mansoni infection18'19'24'43 and none havereported a HSS-like syndrome in their chronicallyinfected populations. This could be related to differ-ences in the routine infection intensity established inthese laboratories and/or to the need to sacrificerelatively large numbers of mice to easily observe thespectrum of gross pathology we have noted inchronic infection in CBA/J mice. It could also berelated to our use of male CBA/J mice, in that wehave observed major differences in the morbidityand mortality seen in male versus female mice,44and essentially all other laboratories exclusively usefemales.

The massive splenomegaly in HSS appears toresult from a combination of a relatively greaterdegree of lymphoproliferation and congestion than inMSS mice. HSS mice have a significantly greaterpercentage of the liver given over to granulomatousand fibrotic tissue than do MSS mice. Also, there is aremarkably distinct pattern to this fibrosis, consistingof both large bands of granulomatous and fibrotictissue running throughout the parenchyma and inti-mal fibrosis of large branches of portal vein. Thisfibrotic pattern, particularly the portal vein fibrosis, isreminiscent of the Symmers' "clay pipestem" fibrosisseen in the livers of humans with HS schisto-somiasis.13,45-47 A similar pattern of fibrosis hasbeen noted by Andrade26 in outbred mice withchronic S. mansoni infections. As in HS patients,such fibrosis could be expected to cause increasedportal hypertension. Although no direct measure-ment of portal pressures was undertaken in thisstudy, strong indirect evidence of increased portalpressure in HSS mice consisted of the presence ofsplenic congestion, 1819'24'42 ascites, and portal-sys-temic collaterals via which worms and eggs wereshunted to the pulmonary vasculature. These condi-tions were not seen in MSS mice.

The relationship between the intensity of the gran-ulomatous response and the development of patho-genic fibrosis in infected humans and mice18'48 haslong been enigmatic. Colley et a1l have hypothe-sized that the more intense in vitro anti-SEA respon-siveness of PBMC from HS patients is likely to bereflected in vivo by more exuberant egg-inducedgranulomatous lesions (perhaps with differentialcytokine profiles), which may in turn lead to the char-acteristic HS hepatic fibrosis. In this study, a signifi-

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cant difference between the volumes of new granu-lomas in HSS and MSS mice was not seen.Extrapolation of this result to pathogenesis inhumans is not possible. With regard to pathogenesisin mice, this result suggests that the intensity of thegranulomatous response, as measured by lesion vol-ume, is not related to, or causal in, the developmentof HSS in mice. However, a possibility raised bythese results and supported by other stud-ies18,19,24,49,55 is that lesion volume is not an accu-rate measure of the degree of fibrogenic and patho-genic potential of a granulomatous response. Anexample might be that differential immunoregulatoryenvironments may allow the cells within granulomasof HSS mice to produce a battery of cytokines thatpromote a greater degree of hepatic fibrosis withoutforming a lesion significantly larger than that seen inMSS mice.

Anti-SEA antibodies from INT and HS patientshave distinct idiotypic repertoires and anti-idiotypic Tlymphocyte-stimulatory abilities. These differencesin anti-SEA antibodies have been interpreted asstrong correlative evidence that anti-idiotypic T lym-phocytes, which are activated by certain cross-reac-tive idiotypes on anti-SEA antibodies, have a regula-tory role in the immunopathogenesis of chronicinfection in humans. 6,8-10,12,50-52 The possibility thatdistinct immunoregulatory environments may con-tribute to the development of MSS and HSS is sup-ported by the results of the competitive ELISA stud-ies, which show that HSS and MSS mice, like HS andINT patients, have distinct shared anti-SEA idiotypicrepertoires. This establishes an immunological, aswell as pathological, similarity between chronicallyinfected CBA/J mice and humans and potentiallyallows for direct investigation of the role of humoral/cellular network interactions in regulating diseasepathogenesis. It is of note that, without segregationof serum pools based on the MSS versus HSS dif-ferential, pooled sera from total chronically infectedmale CBA/J populations fail to inhibit binding in theE5/anti-E5 competitive ELISA in a manner analogousto that seen with HSS sera or anti-SEA antibodies,9which have an apparent overall loss of 8-week Ids. Itis now clear that this is not so when only sera fromMSS mice are pooled.The number of egg-producing adult worm pairs is

known to vary somewhat between individual miceinfected with an equal average number of cercariae.The relationship of infection intensity to the develop-ment of MSS and HSS was investigated. Data inter-pretation is complicated in part by the presence ofworms and eggs in the lungs of HSS mice, and con-clusions could not be firmly established by enumer-ating eggs per liver and worms in the portal vascu-

lature in each group. The lack of significantdifferences between the groups in the numbers oftotal, male, or female worms is offset by the signifi-cantly higher number of "possible worm pairs" in thegroup of HSS mice. Although statistically significant,this difference may be of little biological significance,considering that the difference is less than one pair,the groups show extensive overlap, and the calcu-lated number of possible pairs does not necessarilyreflect the true number of worms paired in vivo. Thegroup of HSS mice was found to have a significantlylarger mean number of eggs per liver than the MSSgroup. A relatively large number of eggs per livercould be due to a larger number of egg-producingworm pairs, to more fecund worm pairs (for example,perhaps due to higher tumor necrosis factor-a lev-els53 in HSS mice), and/or to a slower rate of eggdestruction and clearance from the liver. The wormpair data do not conclusively rule out any of thesepossibilities. It should also be noted that the individ-ual eggs per liver values in each group overlapappreciably. When the number of eggs is less thanapproximately 50,000, it is not a reliable predictor ofthe development of HSS.

Cheever13 found that at autopsy chronicallyinfected patients with HS have statistically moreintense infections, compared with patients with theINT clinical form; however, a significant number ofpatients with HS have infection intensities compara-ble to those of their INT counterparts. Hence, infec-tion intensity is not, on a one to one basis, predictiveof HS in humans. The existence of this "gray area" inthe association of clinical status with infection inten-sity led to studies that implied that immuno-regulatory1-3'54 and/or immunogenetic3,1114,15mechanisms are also important determinants of theclinical outcome of chronic S. mansoni infection inhumans. Considered in light of the distinct patholog-ical and immunological states of HSS and MSS mice,the less than decisive differences in the worm countsand eggs per liver data suggest that, as in humans,infection intensity may be but one of many factorsaffecting the development of each syndrome. Theexistence of these distinct syndromes in an inbredanimal model may now allow for identification andinvestigation of further pathogenic factors related tothe development of each syndrome. This will hope-fully lead to testable hypotheses describing patho-genic mechanisms in human schistosomiasis.

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