u n d e r s t a n d the g e n e t i c m e c h a n i s m s o f susceptibility to iver- m e c t i n a n d a c q u i r e d refractiveness.
2. M. martini is n o r m a l l y s u s c e p t i b l e to suramin, l e v a m i s o l e a n d a l b e n d a z o l e .
3. Eight drugs p r o v i d e d b y W H O h a v e b e e n tested : C G P 2 0 3 7 6 , C G P 6 1 4 0 , C G I 1 8 0 4 1 , A r t e m e t h e r , T r i c h l o r o m e t h y l t h i a d i a z o l e , T r i f l u o r o m e t h y l g u a n i d i - n o p y r i - m i d i n e , a n d t h e t w o b e n z i m i d a z o l e s , U M F 0 7 8 a n d U M F 2 8 9 . F o u r principal data w e r e s h o w n :
a. T h e macrofilaricidal activity is first d e m o n s t r a t e d b y the a b n o r m a l i t i e s o f t h e e m b r y o g e n e s i s in t h e uteri o f t h e f e m a l e w o r m s ; divided e g g s are the m o s t fragile stages ; uterine microfilariae persist ( C G P 6 1 4 0 ) o r are d e s t r o y e d (UMF 0 7 8 a n d 2 8 9 , e t c . ) .
b. A migration o f the adult w o r m is generally observed. M.
martini filariae are normally in the lymphatic vessels o f the i n t e s t i n e wall. W h e n treated, t h e y are n o m o r e a b l e to maintain themselves in situ and they are drawn passively towards the pulmonary b l o o d system via the thoracic duct and right heart ventricule. This migration induces lymphan- gitis a n d s o m e t i m e s t h r o m b o s i s f o l l o w e d b y i n f a r c t i o n ( a l b e n d a z o l e , CGI 1 8 0 4 1 ) . Similar events may o c c u r in all lymphatic filariae (Brugia, Wuchereria, and ? non nodular Onchocerca volvulus).
c. W h e n a macrofilaricide is a l s o microfilaricide, it m a y i n d u c e a Mazzotti r e a c t i o n , as with D E C : microfilariae are driven from the l y m p h a t i c v e s s e l s , in w h i c h t h e y nor- mally inhabit, a n d they are d e s t r o y e d in the perivascular c o n n e c t i v e tissue b y a multitude o f s y n c h r o n o u s inflam- matory' r e a c t i o n s ( a l b e n d a z o l e , UMF 0 7 8 , U M F 2 8 9 ) . d. A p r o p o r t i o n o f microfilariae and adult filariae d o not migrate a n d are d e s t r o y e d in situ in the l y m p h a t i c v e s - sels, inducing lymphangitis.
O n t h e b a s i s o f t w o trials p e r f o r m e d , U M F 2 8 9 s e e m s t o link high macrofilaricide efficacy a n d m o r e rapid r e c o v e r y o f the side-effects than t h e t w o o t h e r b e n z i m i d a z o l e s .
ACKNOWLEDGEMENTS
T h i s i n v e s t i g a t i o n w a s s u p p o r t e d b y S c i e n c e a n d T e c h n o l o g y for D e v e l o p m e n t P r o g r a m m e o f E u r o p e a n C o m m u n i t y , Contract n ° T S E - 0 0 6 7 - F a n d T S 3 C T 9 - 0 0 3 7 , a n d by U N D P / W o r l d B a n k / W H O p r o g r a m m e for R e s e a r c h and Training in T r o p i c a l D i s e a s e s I D n ° 9 0 0 0 5 3 5 and 9 1 0 3 3 7 .
R E F E R E N C E S
AIMARD L . , W A N J I S . , V I ' O N G P . N . , P E T I T G . , BAIN O . Ophthalmologic^ study of the lesions induced by the filarial worm with dermal microfilariae, Monanema martini, in its murid hosts. Current Eye Research , 1993, / 2 , 8 8 5 - 8 9 1 .
BOUGNOUX M.E. : La filaire à microfilaires dermiques, Monanema martini, modèle expérimental pour l'onchocercose ; complé- ments biologiques et essais thérapeutiques. DEA Parasitologic (Parasitologic : écologie, pathologie), Acad. Montpellier, Univ.
Se. Techn. Languedoc, 30 septembre 1987, 32 p.
VUONG P.N., WANJI S., SAKKA L., KLAGER S. and BAIN O . : The murid filaria Monanema martini : a model for onchocerciasis. Part I : Description of lesions. Ann. Parasit. Hum. Comp., 1991, 66, 109- 120.
WANJI S., CABARET J . . GANTIER J . - C , BONNAND N . , BAIN O . : The fate
MODELLING HUMAN FILARIASIS
P A R E N T E R A L A D M I N I S T R A T I O N O F C Y T O K I N E S A N D D R U G S I N R O D E N T FILARIASIS V I A OSMOTIC P U M P S
RAPP J . * , DALLAH C.N.**, S C H U L Z - K E Y H.*
KEYWORDS : Interferon-γ, osmotic pump, lemniscomys striatus. Monanema mar
tini.
SUMMARY
Changes in the cytokine production profile are supposed to be respon
sible for the different courses of parasitic infections. Therefore, a big effort is made to understand the roles of cytokines produced by diffe
rent cells during parasitic infections. In the following text we describe first experiments with Interferon-γ done in a natural model for filariasis, the filariae Monanema martini in Lemniscomys striatus. For the applica
tion of this cytokine to the animals we implanted osmotic pumps, which deliver IFN-γ continuously.
I N T R O D U C T I O N
C y t o k i n e s play an important role in the regulation o f the i m m u n e r e s p o n s e c a u s e d b y parasitic infections. T w o sets o f c y t o k i n e s are r e s p o n s i b l e for the activation o f diffe
r e n t e f f e c t o r m e c h a n i s m s ( M o s m a n n et al., 1 9 8 9 ) . I n t e r l e u k i n - 2 ( I L - 2 ) a n d interferon-v (IFN-y), p r o d u c e d b y T h l - l y m p h o c y t e s , are important for the activation o f m a c r o p h a g e s , c y t o t o x i c T - l y m p h o c y t e s a n d natural killer c e l l s , w h e r e a s I L - 4 a n d I L - 5 , p r o d u c e d b y T h 2 - l y m p h o c y t e s , i n d u c e an Ig E r e s p o n s e a n d l e a d to the g e n e r a t i o n o f e o s i - nophilia. T h e type o f c y t o k i n e s p r o d u c e d d e p e n d s o n the host, the parasite, the site a n d t h e p h a s e o f infection. W h i l e T h l r e s p o n s e s a r e u s u a l l y d i r e c t e d a g a i n s t i n t r a c e l l u l a r parasites, the r e s p o n s e s o f T h 2 often act against intestinal helminth infections. T h e s e patterns, h o w e v e r , are not uni
form. Filarial infections often c a u s e T h 2 r e s p o n s e s , but fila
r i a e p o s s e s s d i f f e r e n t e v a s i o n s t r a t e g i e s , e s p e c i a l l y t h e d o w n r e g u l a t i o n o f the i m m u n e d e f e n s e resulting in c h r o n i c infections ( B e h n k e , 1 9 8 7 ) . I m m u n o l o g i c a l hyperreactivity is a s s o c i a t e d with s e v e r e p a t h o l o g y , i.e. s o w d a in the c a s e o f o n c h o c e r c i a s i s .
T h e d i c h o t o m y o f the T h l a n d T h 2 cell population is b a s e d o n multiple, subtle a n d d y n a m i c equilibria. For studies o n t h e s e m e c h a n i s m s a suitable e x p e r i m e n t a l filarial m o d e l is n e e d e d as w e l l as n e w t e c h n i q u e s for t h e c o n s t a n t a n d c o n t i n u o u s application o f c y t o k i n e s a n d o t h e r i m m u n o l o g i cal s u b s t a n c e s into the host. M e t h o d s to m o n i t o r c h a n g e s in c y t o k i n e profiles and cell reactivity are also required.
Monanema martini in t h e s t r i p e d m o u s e , Lemniscomys striatus, is at p r e s e n t t h e b e s t n a t u r a l r o d e n t m o d e l for
* Institut fur Tropenmedizin, University of Tubingen, Germany.
** Dept. o f Pharmacology/Therapeutics, University o f Nigeria, Enugu.
Parasite, 1994, 1, I S 35
of the filaria Monanema martini in two rodent hosts : recovery rate, migration and localization. Ann. Parasit. Hum. Comp., 1990, 6 5 , 80-88.
WANJI S. : Un nouveau modèle pour la thérapeutique de l'oncho- c e r c o s e : la filaire des muridés à microfilaires dermiques, Monanema martini. T h è s e de Doctorat de l'Université de Montpellier, 18 décembre 1992, 199p.
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/199401s1035
MODELLING HUMAN FILARIASIS
o n c h o c e r c i a s i s d u e to its skin dwelling microfilariae a n d a p a t h o l o g y similar to o n c h o c e r c i a s i s ( V u o n g et al., 1 9 9 1 ) . T h e n e e d for c o n t i n u o u s c y t o k i n e a p p l i c a t i o n c o u l d b e ful
filled b y t h e u s e o f o s m o t i c p u m p s . T h e s e p u m p s , filled prior to implantation, deliver different diluted s u b s t a n c e s at c o n s t a n t rates for several days.
V a r i o u s m u r i n e c y t o k i n e s s p e c i f i c for Mus musculus a r e c o m m e r c i a l l y a v a i l a b l e . H o w e v e r , t h e i r s u i t a b i l i t y for L.
striatus has to b e d e m o n s t r a t e d . First in vitro e x p e r i m e n t s carried out with r e c o m b i n a n t murine IFN-Y s h o w e d a signi
ficant d e c r e a s e in proliferation o f m i t o g e n or antigen stimu
lated s p l e e n c e l l s a n d p e r i t o n e a l m a c r o p h a g e s from naive a n d infected L. striatus ( F i g u r e 1 ) . B u t o t h e r e x p e r i m e n t s in vitro s h o w e d that IFN-Y c a u s e s e n h a n c e d killing o f microfi
lariae ( T a b l e I ) .
T o i n v e s t i g a t e w h e t h e r this interferon-Y is e f f e c t i v e in L.
striatus i n f e c t e d with M. martini w e d e s i g n e d t w o e x p e r i m e n t s , o n e w h e r e IFN-Y w a s administrated to striped m i c e prior to a n d during i n o c u l a t i o n a n d a n o t h e r w h e n it w a s g i v e n during full p a t e n c y .
Fig. 1. - Proliferation of spleen cells and peritoneal macrophages from naive and infected striped mice measured after a 48 h incuba
tion in RPMI 1640 supplemented partially with phytohemagglutinin (PHA) and PHA and interferon-y (IFN).
Table I. - Microfilariae motility assay - microfilariae were incubated in RPMI 1640 with different combination of spleen cells and perito
neal macrophages from naive and infected striped mice and interfe
ron-y. Motility was determined every 24 h.
++ good motility
— no motility.
Similar to o t h e r c y t o k i n e s , IFN-y has a short b i o l o g i c a l half- life, w h e r e a s i m m u n o l o g i c a l effects, s u c h a s m a c r o p h a g e activation, are i n d u c e d w h e n a c o n t i n u o u s s t a b l e level o f this c y t o k i n e is p r e s e n t ( M u r r a y , 1 9 9 0 ) . R e p e a t e d d a i l y i n j e c t i o n s a r e n e c e s s a r y in o r d e r t o p r o v i d e an a d e q u a t e level o f IFN-y in the host. T h i s c o u l d b e a c h i e v e d m o r e effi
ciently by intraperitoneal o r s u b c u t a n e o u s implantation o f o s m o t i c p u m p s ( A l z e t ) , w h i c h d e l i v e r s o l u t i o n s regularly o v e r a p e r i o d o f several days. T h i s m e t h o d a l s o c a n r e d u c e stress p l a c e d o n a n i m a l s b y r e p e a t e d ( d a i l y ) handling.
M A T E R I A L A N D M E T H O D S
•"'"J " h r e e t o f o u r m o n t h s o l d s t r i p e d m i c e w e r e i n f e c t e d I s u b c u t a n e o u s l y with 3 0 infective larvae ( L 3 ) o f M. mar
tini. At different t i m e p o i n t s b l o o d s a m p l e s w e r e t a k e n from the retroorbital vein p l e x u s for b l o o d s m e a r s , l e u c o c y t e c o u n t s a n d i s o l a t i o n o f s e r a f o r s e r o l o g i c a l t e s t s . Additionally, microfilarial densities in the skin w e r e deter
m i n e d by incubating skin b i o p s i e s from the e a r l o b e s in cell culture m e d i u m with c o l l a g e n a s e . After 18 h o u r s the e m e r g e d microfilariae w e r e c o u n t e d .
A. : In a first e x p e r i m e n t L. striatus w e r e treated 4 0 w e e k s p o s t infection with r e c o m b i n a n t m u r i n e IFN-Y, to investi
g a t e w h e t h e r IFN-Y m e d i a t e s an effect o n the parasites, at t h e p e a k o f microfilaridermia. E a c h a n i m a l r e c e i v e d 1 06U IFN-Y o v e r a p e r i o d o f 14 d a y s a n d t h e n all t h e a n i m a l s w e r e d i s s e c t e d .
B . : In the s e c o n d e x p e r i m e n t t h e s a m e d o s e o f IFN-Y w a s given b e t w e e n o n e w e e k b e f o r e to o n e w e e k post infection to verify if IFN-Y treatment is a b l e to i n f l u e n c e the c o u r s e o f infection.
O s m o t i c p u m p s w e r e i m p l a n t e d s u b c u t a n e o u s l y o n t h e b a c k o f the a n i m a l s slightly p o s t e r i o r to the s c a p u l a e . First t h e skin w a s s h a v e d , disinfected a n d a short incision w a s m a d e . F o r c e p s w e r e inserted to c r e a t e a p o c k e t o f sufficient size, the p u m p s w e r e i m p l a n t e d a n d the incision w a s c l o s e d with sutures.
B o t h e x p e r i m e n t s w e r e c a r r i e d o u t w i t h o s m o t i c p u m p s (Alzet m o d e l 2 0 0 2 ) delivering a total d o s e o f 1 06U r-murine IFN-Y o v e r a p e r i o d o f 14 days ( = 3 x l 06U / h ) . Animals with p u m p s filled w i t h P B S a n d u n t r e a t e d a n i m a l s s e r v e d a s c o n t r o l s . S i n c e t h e r e w a s n o d i f f e r e n c e b e t w e e n the t w o c o n t r o l g r o u p s w e put t h e m t o g e t h e r to a s i n g l e c o n t r o l g r o u p .
R E S U L T S
\ l t h o u g h w e w e r e u s i n g r e c o m b i n a n t IFN-Y d e r i v e d 1 V from m u r i n e origin, c l e a r effects in L. striatus c o u l d b e d e m o n s t r a t e d . In t h e a n i m a l s d i s s e c t e d 1 4 d a y s a f t e r implantation o f the p u m p ( e x p . A ) the size o f the s p l e e n w a s s i g n i f i c a n t l y e n l a r g e d . A b o u t e i g h t t i m e s m o r e c e l l s w e r e f o u n d in s p l e e n s o f t h e s e a n i m a l s than in untreated c o n t r o l s . H o w e v e r , t h e s e s p l e e n cells w e r e not a b l e to p r o liferate in r e s p o n s e to m i t o g e n or antigen stimulation.
T w o w e e k s after implantation o f o s m o t i c p u m p s with IFN-Y a d e c r e a s e in the l e u c o c y t e c o u n t s in the peripheral b l o o d o f b o t h treated g r o u p s w a s o b s e r v e d (significant in e x p . B ) .
36 Parasite, 1994, 7, I S
In e x p e r i m e n t B , w h e r e t h e p u m p s w e r e r e m o v e d , t h e leu
c o c y t e c o u n t s i n c r e a s e d again.
T h e s e animals, treated with interferon-y prior to infection, s h o w e d h i g h e r i m m u n o g l o b u l i n titers ( I g G a n d I g M ) in the early p h a s e o f infection. After o n e w e e k o f treatment, at t h e d a y o f i n f e c t i o n , t h e n e u t r o p h i l c o u n t s w e r e significantly i n c r e a s e d , w h e r e a s t h e e o s i n o p h i l s w e r e r e d u c e d d u r i n g t h e first 14 days. U p t o w e e k 2 0 p.i. microfilaridermia rea
c h e d t i t e r s w h i c h w e r e t w i c e a s h i g h as in t h e c o n t r o l g r o u p , but this difference w a s not significant. Fotir o f t h e treated a n d t w o o f t h e c o n t r o l a n i m a l s w e r e d i s s e c t e d 3 5 w e e k s p o s t i n f e c t i o n in o r d e r t o c a r r y o u t l y m p h o c y t e transformation tests. No differences w e r e s e e n b e t w e e n t h e treated a n d t h e u n t r e a t e d c o n t r o l g r o u p .
M a c r o p h a g e s (McJ>) isolated from t h e p e r i t o n e u m o f patent a n i m a l s s h o w e d a h i g h b a s e l i n e p r o l i f e r a t i o n , w h e r e a s c o i n c u b a t i o n o f M<D's with s p l e e n cells resulted in a l o w e r proliferation. In b o t h c a s e s proliferation c o u l d b e i n c r e a s e d b y t h e a d d i t i o n o f filarial a n t i g e n o r P h y t o h e m a g g l u t i n i n ( P H A ) . H o w e v e r , t h e p r e s e n c e o f IFN-y r e d u c e d t h e prolife
ration to b a s e l i n e o r l o w e r levels. D e s p i t e this proliferation inhibiting effect, IFN-y e n a b l e s m a c r o p h a g e s to kill microfi
lariae in vitro within o n e to t h r e e days. T h e p r o d u c t i o n o f nitric o x i d e b y IFN-y activated M O ' s c o u l d b e s h o w n .
D I S C U S S I O N A N D C O N C L U S I O N
I
m p l a n t a t i o n o f o s m o t i c p u m p s is a sui ta b le m e t h o d for t h e a p p l i c a t i o n o f s h o r t lived s u b s t a n c e s l i k e I F N- y o r o t h e r c y t o k i n e s . D r u g s c a n b e d e l i v e r e d satisfactorally as w e l l . Drugs a n d c y t o k i n e s a d m i n i s t e r e d in this w a y h a d t h e s a m e o r an e v e n b e t t e r efficacy than injected o n e s (Murray, 1 9 9 0 , o w n o b s e r v a t i o n ) . O t h e r implantation sites in t h e animal c a n b e u s e d , if t h e r e is sufficient s p a c e for t h e p u m p . T h e p o c k e t s h o u l d b e large e n o u g h a n d a b o u t 1 c m l o n g e r t h a n t h e p u m p . T h i s p r e v e n t s t h e p u m p f r o m r e s t i n g directly o n t h e w o u n d . In addition, t h e p u m p s h o u l d b e inserted with its delivery portal first to m i n i m i z e interaction b e t w e e n t h e d e l i v e r e d c o m p o u n d a n d t h e h e a l i n g o f t h e w o u n d . Normally t h e incision h e a l s within t h r e e days, a n d t h e a n i m a l s s h o w n o sign o f discomfort. T h e r e f o r e , it is a u s e f u l a l t e r n a t i v e t o r e p e a t e d i n j e c t i o n s e s p e c i a l l y for r o d e n t s w h i c h a r e s e n s i t i v e t o frequent m a n i p u l a t i o n a n d are not well a d a p t e d to l a b o r a t o r y m a i n t e n a n c e .
T h i s investigation a l s o s h o w s that r e c o m b i n a n t m u r i n e IFN- y is effective in L. striatus. T h e i n c r e a s i n g cell c o u n t s in t h e s p l e e n o f IFN-y treated a n i m a l s d o not c o r r e s p o n d with in vitro a s s a y s c a r r i e d o u t in parallel, w h e r e t h e proliferation o f s p l e e n c e l l s isolated from i n f e c t e d a n d n a i v e a n i m a l s w a s s u p p r e s s e d in t h e p r e s e n c e o f IFN-y, e v e n w h e n stimulated with filarial antigen.
A l t h o u g h t h e r e w a s n o great effect o n t h e c o u r s e o f t h e fila
rial i n f e c t i o n , s o m e o f t h e r e s u l t s s u g g e s t that c y t o k i n e w o r k in t h i s m o d e l s h o u l d b e c o n t i n u e d . E n c o u r a g i n g results i n c l u d e t h e r e d u c e d l e u c o c y t e c o u n t s , t h e c h a n g e s b e t w e e n t h e different t y p e s o f l e u c o c y t e s a n d t h e r a i s e d microfilaridermia. T h e s e results a l s o s u p p o r t t h e h y p o t h e s i s that IFN-y, a T h l - c y t o k i n e , c o u n t e r a c t s a T h 2 r e s p o n s e , w h i c h is s u p p o s e d to b e i n v o l v e d in p r o t e c t i o n .
T h e different effects o f IFN-y t o l y m p h o c y t e s in vivo a n d in vitro s h o w that this c y t o k i n e has different features, d e p e n -
MODELLING HUMAN FILARIASIS
R E F E R E N C E S
BEHNKEJ.M. Adv. Parasitol., 1987, 26. 1-71.
MOSMANN T.R. and COFFMANN R.L. Adv. Immunol., 1989, 46, 11-147.
MURRAY H . W . / . Inf. Disease: 1990, 161, 992-994.
VUONG P . N . , WANJI S., SAKKA L., KLAGER S., BAIN O . Ann. Parasitol.
Hum. Comp.. 1991. 3, 109-120.
T H E EFFICACY O F U M F 0 7 8 O N ACANTHO-
CHEILONEMA VITEAE A N D LlTOMOSOIDES SIGMO- DONTIS I N MERIONES UNGUICULATUS
DALLAH C.N.**, HOFFMANN W.*, RAPP J.*, OKONKWO P.O.**, SCHULZ-KEY H.*
KEYWORDS : UMF 078. Acanthocheilonema viteae. Lilomosoides sigmodontis.
SUMMARY
UMF 078, a new benzimidazole analogue of Flubendazole, was tes
ted on jirds infected with the filarial parasites Acanlhocheilonema viteae and Lilomosoides sigmodontis respectively. A total of 19 jirds, eight subcutaneously inoculated with infective larvae of A. viteae and eleven with L. sigmodontis infective larvae, were used in this study.
UMF 078 had both microfilaricidal and macrofilaricidal activities against both :«irasites. Blood microfilariae were completely eliminated in the two treated groups by day 56 post-treatment. Very few adult worms were recovered, and in both groups all the worms recovered were abnormal. In L. sigmodontis however, microfilariae were detec
ted in various organs/fluids (peritoneal fluid, pleural cavity fluid, lungs) on autopsy of the jirds.
I N T R O D U C T I O N
A viteae in jirds is o n e (if t h e a c c e p t e d filarial m o d e l s for t h e s c r e e n i n g o f drugs targeted against Onchocerca volvulus. Adult O. volvulus has p r o v e d a big c h a l l e n g e t o r e s e a r c h e r s , b e c a u s e o f its host specificity a n d the a b s e n c e o f an effective macrofilaricide. T h e r e is still an urgent n e e d for a n o n - t o x i c drug with a macrofilaricidal o r a p e r m a n e n t sterilizing effect o n t h e adult O. volvulus. U M F 0 7 8 ( d e v e l o -
* Institut für Tropenraedizin, University of Tubingen, Germany.
** Dept. o f Pharmacology/Therapeutics University of Nigeria, Enugu.
Parasite, 1 9 9 4 , J , I S 37
ding o n different factors like o t h e r c y t o k i n e s a n d i m m u n e c e l l s . In an o r g a n i s m c y t o k i n e s as well as i m m u n e c e l l s b e l o n g t o a h i g h l y c r o s s l i n k e d i m m u n o l o g i c a l n e t w o r k . M o r e significant results c o u l d p e r h a p s b e o b t a i n e d by using c o m b i n a t i o n s o f c y t o k i n e s o r with d e p l e t i o n o f different c y t o k i n e s with t h e h e l p o f a n t i b o d i e s against t h e m .
For further investigations, in addition to the administration o f c y t o k i n e s , it is n e c e s s a r y t o m o n i t o r the c h a n g e s o f c y t o k i n e profiles d u r i n g t h e c o u r s e o f filarial infection. T e s t s b a s e d o n PCR a n d ELISA are b e i n g e s t a b l i s h e d .
ACKNOWLEDGEMENTS
T h i s w o r k is s u p p o r t e d b y CEC grant ( T S * C T 9 - 0 0 3 7 ) a n d B o e h r i n g e r I n g e l h e i m .
