Clin. exp. Immunol. (1980) 40, 496-507.

Trypanosoma brucei brucei:a model for cerebral trypanosomiasis in mice-

an immunological, histological and electronmicroscopic study

A. A. POLTERA, A. HOCHMANN, W. RUDIN*& P. H. LAMBERT WHO ImmunologjResearch and Training Centre, Centre de Transfusion, Department ofMedicine, Hopital Cantonal,

Geneva and * Swiss Tropical Institute, Socinstrasse, Basel, Switzerland

(Acceptedfor publication 30 November 1979)

SUMMARY

The successful induction of cerebral trypanosomiasis in ordinary laboratory mice usingTrypanosoma brucei brucei is reported. Sequential studies demonstrated the presence oftrypanosomes in the interstitium of the choroid plexus at the fourth week after infectionwhich correlated with the appearance of anti-trypanosomal antibodies, a rise of IgM andIgG serum levels and a rise of Clq binding activity as well as a decrease of C3 levels.Electronmicroscopic studies showed that the parasites were flagellated and localizedextracellularly mainly in the interstitium of the choroid plexus. Granular immunofluores-cent deposits of Ig and C3 were most marked in the choroid plexus. Electron-densedeposits suggestive of immune complexes were seen in subendothelial, interstitial andsubependymal areas of the choroid plexus. Since autoantibodies to the brain were foundin the serum of some mice, the possible involvement of autoimmune manifestations in thepathogenesis of cerebral lesions has to be considered. The pattern of inflammatory foci atthe eighth week after infection was very similar to that observed in cerebral Africantrypanosomiasis in man. After treatment with ethidium bromide, trypanosomes persistedin the tissues when circulating parasites could no longer be detected. These observationssuggest a sequential involvement of brain structures during African trypanosomiasis.

Trypanosomes may first migrate from the vascular compartment into the interstitiumof the choroid plexus, possibly favoured by increased vascular permeability. Circulatingimmune complexes and complement activation may be involved at this stage. Trypano-somes localized in the choroid plexus may then trigger a local immunologically mediatedinflammatory reaction favouring the migration of trypanosomes into the CSF and furtherinvasion of other cerebral structures.

INTRODUCTION

Although the ultimate manifestation of human African trypanosomiasis is involvement of thecentral nervous system (CNS), the lack of a good model has limited the experimental investigationof cerebral lesions. Either the experimental host, usually currently available rodents, were notsuitable for human African trypanosomes, or else the commonly used cattle parasite, Trypanosomabrucei brucei (Tbb), killed the rodents before the development of the expected cerebral lesions(Losos & Ikede, 1972). We report on the successful induction of cerebral lesions in experimental

Correspondence: A. A. Poltera, PO Box 30588, Department of Medicine, Kenyatta National Hospital,Nairobi, Kenya.

0099-9104/80/0600-0496$02.00 A) 1980 Blackwell Scientific Publications

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Trypanosoma brucei brucei 497trypanosomiasis using Tbb in mice. The pathogenesis of the lesions was studied using histologicaland immunological methods.

MATERIALS AND METHODS

Animals. Outbred OFQ female mice, 6 weeks old (IFFA CREDO, Centre de Recherche etd'Elevage des Oncins, St Germain sur l'Arbresle, France) were used in these experiments.

Parasite, parasitic antigens and related antibodies. Trjpanosoma brucei brucei LUMP 227 waskindly provided by Professor W. H. R. Lumsden, Department of Medical Protozoology, LondonSchool of Tropical Medicine and Hygiene. The parasite was passed twice through 500-rad-irra-diated mice. After the second passage, blood was collected before killing in tubes containingphosphate saline glucose (PSG) and heparin. After a first centrifugation (5 min, 1,000 r.p.m.) thebuffy coat containing the parasites was resuspended in PSG. After the second centrifugation (10min, 2,500 r.p.m.) the pellet containing the parasites was resuspended in 7.5% glycerine andtransferred into plastic capillaries which were then heat-sealed. The capillaries were stored in aplastic tube containing isopenthan. Up to this stage, the whole procedure was carried out at 40C. Thetubes were packed in cotton wool and stored for 48 hr at - 70'C and then kept in liquid nitrogen.This stabilate was called GUMS 1 (Geneva University Medical School) and was used for all theexperiments in this report. Before freezing and after thawing these capillaries the motility of thetrypanosomes was assessed. For all in vivo experiments, 104 living trypanosomes were inoculatedintraperitoneally. Trypanosome counting was done with an improved Neubauer haemacytometerusing a stain composed of formaldehyde, acetic acid and Ziehl-Neelsen carbol fuchsine diluted indistilled water.

Trypanosomal antigens were obtained from LUMP 227 after passage in irradiated mice. Theheparinized blood was applied to a DEAE-cellulose column (Lanham & Godfrey, 1970). Theseparated trypanosomes were recovered, then sonicated and emulsified with an equal volume ofFreunds' incomplete adjuvant and used to immunize rabbits.

The anti-trypanosomal antibodies were conjugated with fluorescein isothiocyanate (FITC,Baltimore, Biological Laboratories, Cockeyville, Maryland) following the method of Johnson &Holborow (1973) and were used for immunohistochemical studies.

The occurrence ofanti-trypanosomal antibodies in the mouse serum was determined by indirectimmunofluorescence (Williams et al., 1963) using smears of trypanosomes of the same stabilate.Anti-trypanosomal antibodies were also assessed by the agglutination test using trypanosomesfrom the same stabilate passed once in irradiated mice, then incubated with the diluted sera ofinfected mice.

Other serological studies. Before killing the animals were bled under anaesthesia from the orbitalsinus. The serum was kept at - 70CC until used. Immunoglobulins (IgM, IgG) and the thirdcomponent of complement (C3) were quantified by radial gel immunodiffusions (Mancini, Car-bonara & Heremans, 1965); for the latter a rabbit anti-rat C3 serum was used (Nordic Labora-tories). The results were expressed as a percentage of the value obtained from a pool of serum fromnormal OF1 mice. Circulating immune complexes were detected in the serum using a modified1251-Clq-binding test (Zubler et al., 1976). Anti-DNA antibodies were assessed by a DNA-bindingradioimmunoassay (Izui et al., 1977). The occurrence of anti-brain antibodies was assessed byindirect immunofluorescence on a cryocut section using a non-infected mouse brain.

Morphological studies. Animals were killed at weekly intervals for histopathology and im-munofluorescence. Certain mice were also studied by electronmicroscopy. For histology, the brainwas separated from the skull and fixed in buffered formalin. A whole-organ block was prepared bytwo parasagittal sections covering the area from the fronto-temporal junction to the medullaoblongata. The tissue was embedded in paraffin wax, serially sectioned at 4 ,um and stained withhaematoxylin and eosin. The sections were screened for the presence of parasites and inflammatorychanges, using a light microscope. For immunofluorescence, the brain was immediately separatedfrom the skull, put in a plastic tube with a screw top and stored in liquid nitrogen until used. A blocksimilar to that used for histology was cut at - 20°C. Frozen sections were cut at 4 ym and stained

498 A. A. Poltera et al.using the direct immunofluorescent technique for trypanosomal antigens, IgM and IgG, C3,fibrinogen and albumin. Sections were examined in a Leitz Othoplan photomicroscope. Fluores-cein-conjugated anti-mouse IgM, anti-mouse IgG and anti-rat C3 were purchased from NordicLaboratories. For electronmicroscopic studies, mice were killed 8 weeks after infection. The brainwas halved sagittally; one portion was used unfixed for immunofluorescent studies and the otherportion was fixed in 2.5% glutaraldehyde. From the fixed portion, the choroid plexus was isolatedby microdissection using a stereomicroscope. After fixation (12-24 hr), the specimen was kept in 0 2M phosphate buffer, pH 7 2. After postfixation in 2% osmium, the specimen was blockstained in 2%U02-acetate and embedded in Epon. The thin sections (about 40 nm) were poststained with leadcitrate and examined in a Philips EM 300.

Therapeutic studies. An attempt to induce therapeutic cure was made using ethidium bromide asa trypanocidal drug. Three groups were studied. Group I represented the natural course of thedisease from the first to the ninth week of infection. Group II comprised animals treated with highdoses of ethidium bromide (10 mg/kg body weight) on days 21, 31 and 36. Group III comprisedanimals treated with minimal doses (1 mg/kg body weight) of ethidium bromide on days 21 and 24.

RESULTS

Natural course of the disease

Parasitaemia and serologicalfindingsForty-eight mice were infected and 5 days after peritoneal inoculation trypanosomes appeared inthe peripheral blood. Parasitaemia was determined once or twice a week and the results expressed asthe number of parasites per ml of blood. Fig. 1 shows the progressive increase of the meanparasitaemia with a plateau at the fourth week when there were approximately 107 parasites per mlof blood. The parasitaemia remained high throughout the observation time and reached 108

107 BOO80 b) IgE 1gM - x,

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40 (m) J20 --c,~400 /g

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Fig. 1. Serological features in trypanosome-infected mice.(a) Time of infection correlated with: (I) the mean parasitaemia, (II) antibody tests in the serum: (- *)

direct agglutination, (-o) indirect immunofluorescent (IgM), (- *) indirect immunofluorescent (IgG),and (III) Clq-binding test (o-.o) and C3 concentration in the serum (- *). Standard deviations (±+ 1 s.d.)were calculated for groups with at least four animals/value. For (III) the normal range is represented as thehatched zone (+ 2 s.d.).

(b) The upper graph shows the mean level of serum IgM and IgG during the course of infection. N representsthe normal values in control animals killed at the end of the experiment. The lower graph represents the meanlevel of anti-DNA antibodies in relation to the time of infection. Hatched area is the normal range (±+2 s.d) incontrol mice.

(c) Demonstration of antibody to ganghion cells in the hippocampus by indirect immunofluorescence usingsections of normal mouse brain as a substrate, serum from a mouse at 8 weeks after infection and for staininganti-mouse IgG. (x 40.)

Trypanosoma brucei brucei 499preterminally. Anti-trypanosomal antibodies were detected by indirect immunofluorescence. IgGantibodies rose to a significant titre (1:128) in the second week of infection and IgM antibodiesreached a significant level (1:128) in the fourth week (Fig. la). In both groups the titres remainedhigh after this first peak. Similarly, the direct agglutination test became positive in the fourth week(1:64) and subsequently (1:256). Anti-DNA antibodies were above the normal level from thesecond week, but fell to a high normal level in the sixth and eight weeks (Fig. Ib). Autoantibodiesdirected against the ganglion cells of the thalamus, hippocampus and Purkinje cells of the cerebel-lum were found in four (sixth and eighth weeks of infection) of ten mice examined (Fig. Ic). SerumIgM rose sharply to reach a peak of six times normal during the third week. The mean valueremained high throughout the infection (Fig. Ib). Serum IgG rose later and more progressively asshown in Fig. lb. The increase was about six-fold compared to IgG levels in uninfected mice. Oncethe peak was reached (week 6) it remained high for the remaining time of infection. Circulatingimmune complexes were detected by a modified '251-Clq-binding test. There was an increase inClq-binding activity from the fourth week compared to the level of the control mice. Individualvalues of up to 40% were obtained towards the end of observation time in the untreated group (Fig.1). A progressive decline of C3 in serum was demonstrated, reaching significantly low values fromthe fourth week onwards (Fig. 1). Four mice died during this experiment (weeks 6, 7 and 8).

Morphologic studiesHistopathology. Table I shows the appearance of trypanosomes in the cerebral structures as

observed in the serial whole-organ sections of twenty-two brains. The parasites were first found inthe interstitium of the choroid plexuses (lateral and fourth) during the third or fourth week ofinfection (Fig. 2a). At this stage, the inflammatory lesions of the choroid plexus were characterizedby interstitial oedema and an almost total absence of inflammatory cells. In normal mice theinterstitium is so narrow that it is almost invisible. Subsequently, the trypanosomes increased in

Table 1. Presence of trypanosomes in the CNS

Weeks after infection 1 2 3 4 5 6 7 8 9Total number of mice killed per week 6 6 6 9 2 6 0 4 2

Histology*Choroid plexus (lateral ventricle) 0 0 + + + + + + + + +

0 0 + ++ ++ + +++ ++ +++0 0 0 + + ++

Choroid plexus (fourth ventricle) 0 0 0 + + + 0 + + +0 0 + + + + ++ ++ +++0 00 0 0 0

Meninges 0 00 0 0 + + +0 00 0 0 0 + +0 00 0 0 +

Immunofluorescence*Choroid plexus (lateral ventricle) 0 0 + + + + + + +

0 0 0 + 0 ++ +++ +0 0 + 0 ++

Choroid plexus (fourth ventricle) 0 0 + + + + + + + +0 + 0 + + ++ +0 0 + 0 +

Meninges 0 0 0 0 + +0 0 0 + + + 0 +0 00 0 +

* The degree of parasitic infiltration is shown by the signs 0=nil, + = mild, + + = moderate,+ + + =marked. Results for the individual mice are given in the same order for each histologicalsite.

numbers and inflammatory cells appeared, mainly plasma cells and macrophages, some of whichhad foamy cytoplasm (Fig. 2b). In advanced cases, the choroid villi became thickened, theinterstitium being mainly distended by parasites (Fig. 2c). The cellular response appeared to localizeessentially at the insertion of the plexus. The inflammatory cells consisted primarily of plasma cellsand macrophages, occasionally of giant cells, and rarely of lymphocytes or morular cells. Occa-sional trypanosomes could be detected free in the cerebrospinal fluid (CSF). The invasion of thechoroid plexus by parasites and cells was usually simultaneous in the lateral and fourth plexuses.However, the analysis of serial sections showed that within one plexus there was some variation inthe distribution and composition of the immuno-inflammatory reaction. Small numbers of try-panosomes were found in the meninges but usually not until 2 to 3 weeks after the parasiticinfiltration of the choroid plexus (Table 1). The blood vessels of the fissura lateralis were an early sitefor the meningeal involvement. Perivascular spaces, especially those around veins, occasionallycontained parasites. The initial changes were oedema with few mononuclear cells. Subsequently, thenumber of inflammatory cells increased and these consisted predominantly of plasma cells andmacrophages (Fig. 2c). In advanced cases the meningeal changes became diffuse and could beobserved at the base of the brain, on the lamina quadrigemina, on the anterior and posterior part ofthe pons, and at the base and on the surface of the cerebellum (Fig. 2c). The perivascular infiltrationextended into the white matter and to a lesser degree into the cortex giving the pattern of a diffusemeningoencephalitis.

Immunohistology. Direct staining for trypanosomes in nineteen brains by immunofluoresenceconfirmed that they appeared in the choroid plexus during the third or fourth week after infection(Fig. 3a). It also showed that the parasites occasionally occur free in the CSF at any time afterpenetration of the choroid plexus. Trypanosomes usually appeared in the meninges about 2 to 3weeks after the primary invasion of the choroid plexus. In advanced cases, parasites were found inlarge numbers in the choroid plexuses, in perivascular spaces and occasionally in the white matter(Table 1). Irregular granular deposits of IgM and IgG occurred at 3 to 4 weeks in the choroidplexuses with subependymal, perivascular or interstitial localization (Fig. 3b) in a focal pattern, andpersisted throughout infection. The appearance of Ig deposits in perivascular spaces and on themeninges was related to advanced infection. Direct staining for C3 showed irregular patchydeposits (Fig. 3c) in the choroid plexuses from the fourth week of infection. Plasma cells secretingIgM or IgG were observed in the interstitium of the choroid plexus and later in the meninges. Incontrol animals, similar immunofluorescent studies did not show any significant deposit or staining.

Electronmicroscopic studiesIn mice killed at the eighth week of infection, flagellated trypanosomes were found in the intersti-tium of the choroid plexus (Fig. 4a). No amastigotes could be identified. Occasional parasites were

phagocytized by macrophages which predominated in the inflammatory cellular response togetherwith plasma cells. No parasites were seen inside the choroid ependyma. Deposits consistent withimmune complexes were observed in the choroid plexus with a vascular (Fig. 4b), interstitial andsubependymal (Fig. 4c) localization. They were found between the endothelium and the laminabasalis and/or within the interstitium often in contact either with a lamina basalis or trypanosomesor both.

Evolution of brain lesions after trypanocidal treatment

A group of eighteen mice infected with 104 trypanosomes were treated with ethidium bromide fromweeks 4 to 6. Animals were killed from the fifth to the tenth week. Ethidium bromide induced a

temporary disappearance of the parasites from the circulating blood 5 days after the first injection(10 mg/kg). By day 7, parasites had reappeared in the blood. A second intraperitoneal injection wasgiven on day 10. Five days after the second dose, there were no detectable parasites in the peripheralblood in two of the twelve surviving mice. These two were killed to investigate the possiblepersistence of parasites in the tissues. The remaining ten animals received a third dose of ethidiumbromide on day 15. Relapses occurred in spite of this triple treatment with a relatively high dose ofethidium bromide. Animals started to die from the sixth week of infection. The parasitaemia thenreached similar levels to those in untreated mice. The levels of anti-trypanosomal and anti-DNA

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Fig. 3. Immunofluorescent features in trypanosome-infected mice. (a) Multiple trypanosomes in the villi of thechoroid plexus of the fourth ventricle. Four weeks after infection. ( x 100.) Direct immunofluorescent stainingfor trypanosomal antigens. (b) Villi of the choroid plexus of the fourth ventricle showing interstitial andsubependymal irregular granular deposits for IgM. Eight weeks after infection. ( x 40.) Direct immunofluores-cent staining for IgM. (c) Granular deposits of the third component ofcomplement (C3) in a villus of the choroidplexus of the lateral ventricle showing subependymal and interstitial arrangement. Eight weeks after infection.( x 100.) Direct immunofluorescent staining for C3.

antibodies, of serum Ig (M and G), of circulating immune complexes and of C3 were notsignificantly changed by this trypanocidal treatment. Autoantibodies directed against cerebralganglion cells were observed in five of the thirteen cases examined. In the two mice in which therewere no detectable parasites in the circulating blood 15 days after the start of chemotherapy,parasites were found in the choroid plexuses by both histology and immunohistology (Fig. Sa).Similarly, many trypanosomes were detected in the choroid plexuses in the two parasitaemic micekilled 4 days after the third injection (Fig. 5a). In relapsed animals killed at weeks 8 or 10, parasiteswere observed in large numbers in the choroid interstitium and to a lesser extent also in themeninges. They were seen only rarely in perivascular spaces in the white matter. Deposits of Ig (Mand G) and C3 could be demonstrated in the choroid plexuses throughout the infection. Inadvanced cases, Ig was also present in the perivascular spaces of the meninges and the brain.Immunoglobulin-secreting plasma cells were found in the choroid plexus (Fig. 5b), the meningesand less regularly in the perivascular spaces of the grey or white matter. Once relapse had occurred,the pattem of distribution for parasites and inflammatory cells reappeared as described for theuntreated mice. There was cellular infiltration in the plexus and meninges and early encephalitis wasevident. Four animals died spontaneously during the experiment (weeks 6, 8 and 10).

Fig. 2. Histological features in trypanosome-infected mice.(a) Trypanosomes in a vein (arrowhead) and in the interstitium (arrows) of the choroid plexus of the lateral

ventricle. Four weeks after infection. (H & E x 150.)(b) The choroid plexus of the lateral ventricle filled with trypanosomes and macrophages. Note inflammatory

cells at insertion of plexus (short arrow) and more rarely in meningeal sulcus. Six weeks after infection. (H & Ex 12). Inset: high power view of the pointed area (long arrow,) depicting interstitial trypanosomes andmacrophages with foamy cytoplasm. (H & E x 250.)

(c) The choroid plexus of the fourth ventricle with enlarged villi and inflammatory cells. Note cellularinfiltration of the meninges and in perivascular spaces. Eight weeks after infection. (H & E x 9) Inset. arrowedarea of the choroid villi containing trypanosomes (small points) and inflammatory cells of mononuclear type.(H&Ex 60.)

(d) Meningeal vessels near the hippocampus and the choroid plexus of the lateral ventricle surrounded bymononuclear cells and occasional trypanosomes. Eight weeks after infection. (H & E x 60). Inset: magnificationof the arrowed zone showing one trypanosome and plasma cells. (H & E x 250.)

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Trypanosoma brucei bruceien'

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Fig. 4. Electronmicroscopic studies of the choroid plexus of the fourth ventricle in trypanosome-infected mice(a) Tip of a choroid villus with trypanosomes (t) in the interstitium (i) mostly bearing flagella (arrow heads).c = Capillary, ep= ependyma. ( x 1,700.) (b) Electron-dense deposits in the lamina basalis (arrow s) of a capillaryof the choroid plexus. Trypanosome with flagella in the choroid interstitium. Part of a red blood cell in thevascular lumen. ( x 5,850.) (c) Electron-dense deposits (arrows s) suggesting immune complexes on the interstitialside of the lamina basalis of the ependyma. Note trypanosome in the interstitium. ( x 17,000.)

Fig. 5. Parasite persistence during and after treatment. (a) The upper part shows several trypanosomes persistingin villus of the choroid plexus of the lateral ventricle during treatment (4 days after the second dose when therewere no parasites in the circulating blood). Six weeks after infection. Direct immunofluorescent staining fortrypanosomal antigens. ( x 250.) The lower part depicts multiple trypanosomes in the interstitium of the villi ofthe choroid plexus of the fourth ventricle 4 days after the end of treatment (day 19). Six weeks after infection.(H & E x 150.) (b) IgM-secreting plasma cells in a choroid plexus of the lateral ventricle. Four days after thetermination of treatment. Six weeks after infection. Direct immunofluorescent staining for IgM. ( x 150.)

In another experiment, eighteen animals were infected as before and treated on days 21 and 24with small doses of ethidium bromide (1 mg/kg). No significant differences occurred in parasitae-mia, or in the serological and morphological findings as compared to the untreated mice. Six micedied spontaneously during the experiment (weeks 6, 7 and 8).

DISCUSSION

Ever since Mott (1906) described the chronic meningoencephalitis which characterized sleepingsickness in man, the demonstration of parasites in lesions in the CNS has been a major difficulty. It isknown that the choroid plexus may be involved in the cellular response in human autopsy cases(Calwell, 1937; Poltera, Owor & Cox, 1977), but the significance of this finding is difficult to assess.Trypanosomes have also been demonstrated in the choroid plexus in various experimentallyinfected animals (Peruzzi, 1928; Ormerod & Venkatesan, 1971 a, b; Losos & Ikede, 1972; Stevens &Moulton, 1977; Fink & Schmidt, 1979).

In the present study, an experimental model for the induction of chronic meningoencephalitis isreported using ordinary laboratory mice and a freshly isolated trypanosome from cattle (TbbLUMP 227). The sequential studies showed that the parasites were detectable in the circulatingblood from the end of the first week of infection. By morphological studies (histology andimmunohistology), the presence of intravascular and interstitial parasites was assessed in thechoroid plexuses starting during the third or fourth week of infection, at a time when trypanosomeshave been rarely observed free in the CSF or in the meninges. While the number of parasitesincreased in the choroid plexuses, trypanosomes usually appeared in the meninges in the sixth week,particularly in the fissura lateralis, the perivascular spaces at the base of the brain and thepontecerebellar area. Electronmicroscopy confirmed that parasites within the choroid plexuseswere localized predominantly in the choroid interstitium in the lumen and wall of the vessels butnever within the ependyma. In all these sites the trypanosomes were flagellated. This does notaccord with the report of Ormerod & Venkatesan (1971 a, b) suggesting the presence of amastigoteforms in the choroid plexus. This sequential distribution of the trypanosomes in the CNS could beconsistent with penetration of the choroid plexus from the vascular compartment to the intersti-tium. An increased permeability of peripheral blood vessels has been reported to favour themigration of the parasites to the interstitium in experimental trypanosomiasis (Goodwin, 1971).The vessels of the choroid plexus are structurally similar to the non-cerebral vessels, unlike those ofthe CNS (Johnson, Griffin & Hirsch, 1979). This difference could account for the apparentlyprimary invasion of the choroid plexus by the parasite. Serologically, an increase of the Clq-bind-ing activity, suggesting the formation of circulating immune complexes, and a decreased level of C3,were observed from the fourth week of infection. Granular deposits ofIgM and IgG and C3appeared usually in the choroid plexus from the fourth week and their distribution is consistent witha localization of immune complexes in/or around the lamina basalis (vascular or subependymal)and in the interstitium. Indeed, electronmicroscopy localized such complexes in a subendothelia,interstitial and subependymal position.

The choroid plexus has been recognized experimentally as constituting one of the targetstructures in the CNS in immune complex diseases, such as chronic virus infections, and inautoimmune diseases (Lampert & Oldstone, 1973). In autopsies of patients with lupus erythema-tosus, the choroid plexus has been shown to contain deposits of immune complexes by immuno-fluorescence and electronmicroscopy (Gershwin, Hyman & Steinberg, 1975; Brentjens et al., 1977).Since circulating immune complexes have been reported to occur in human (Fruit et al., 1977) andexperimental trypanosomiasis (Lambert & Houba, 1974; Galvao Castro, Hochmann & Lambert,1978) our combined results suggest that these complexes may represent deposits of trappedcirculating immune complexes or that they represent antigens released from the parasites in theinterstitium with antibodies formed locally or diffusing from the blood vessels. Indeed, it has beendemonstrated in rabbits that serum IgG may filter through the choroid plexus and appear in theCSF (Harbeck et al., 1979). In man, IgG-secreting plasma cells (Greenwood et al., 1976), anti-try-panosomal antibodies (Fr&il & Coulm, 1977) high concentrations ofIgM (Greenwood & Whittle,

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Trypanosoma brucei brucei 5051973) and of IgM and IgG (Giordano et al., 1977) have been demonstrated. Local formation ofantibodies to trypanosomes therefore cannot be ruled out since experimentally we observed plasmacells secreting IgM and IgG in the choroid plexuses and meninges. Inflammatory cells of themononuclear type appeared in this model in response to the parasite invasion in the CNS with asimilar localization; however, a noticeable infiltration by inflammatory cells was not usually seenbefore the sixth week. In late infection, the perivascular cuffing in the white matter was similar to theclassical picture of human meningoencephalitis in sleeping sickness (Mott, 1906; Poltera et al.,1977).

These observations suggest a sequential involvement of brain structures during African try-panosomiasis.

Trypanosomes may first migrate from the vascular compartment into the interstitium of thechoroid plexus, possibly favoured by an increase in vascular permeability. Circulating immunecomplexes and complement activation may be involved at this stage. Trypanosomes localized in thetissues may then trigger a local immunologically mediated inflammatory reaction favouring themigration of trypanosomes into the CSF and further invasion of other cerebral structures.

The occurrence of an autoimmune response has been demonstrated in American trypanoso-miasis (Khoury et al., 1979). The present experiment showed anti-DNA antibodies in the serum andthere was also evidence that some mice developed autoantibodies directed against ganglion cells ofthe CNS. The significance of these antibodies is not yet clear. However, since polyclonal B cellactivation has been reported in experimental trypanosomiasis (Hudson et al., 1976) and shown to beassociated with autoantibodies (Kobayakawa et al., 1979), and since rheumatoid factor has alsobeen found in human trypanosomiasis (Houba, Brown & Allison, 1969), the association of anautoimmune response with polyclonal B cell activation has to be considered. The increased serumlevels of IgM and IgG in our experiment also indicate a polyclonal B cell activation. T cells havebeen shown to be necessary for the induction of an autoimmune encephalitis (Bernard, Leyden &Mackay, 1976). It has to be established whether T cells are essential for the induction of cerebraltrypanosomiasis in mice.

Our therapeutic trial, although unsuccessful since relapses occurred, demonstrated the persist-ence of trypanosomes in the choroid plexuses when the parasites were no longer detectable in theblood after drug injection. The choroid plexuses may therefore act as a reservoir in our model. Asimilar mechanism has been postulated for neonatal bacterial meningitis (Gilles, Jammes &Berenberg, 1977). Jennings, Whitelaw & Urquhart (1977) reported relapses after drug treatment inexperimental murine trypanosomiasis without formal drug resistance; it appeared that cure couldonly be obtained if treatment was started within the first week of infection, although arsenical drugswere not used by these authors. Arsenical drugs should therefore be applied in this model to test thepossible occurrence of relapses as well as toxic effects. Such studies should be of interest sincerelapses after drug treatment occur in human African trypanosomiasis (Buyst, 1975; Kazyumba,1979) and since arsenical encephalopathy is still a major problem in the treatment of sleepingsickness patients (Sina et al., 1977).

The contribution of the following departments is gratefully acknowledged: Department of Medical Illustration(Mr G. Rapin), Department of Pathology (Miss G. Leyvraz) and Department of Radiotherapy. We are indebtedto Mrs M. Devouge, Mrs A. Genz and Mrs J. Ringrose for secretarial work and to Mr P. Bauer for technicalassistance (EM preparations).

This work was supported by WHO and by the Swiss National Foundation (Grant No. 3.847.177).

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