15.5. 1975
Specialia
been shown to cause liberation of m e p a c r i n e into t h e m e d i u m 1, whereas in T y r o d e their m a j o r i t y remains i n t a c t and retains the drug. This indicates t h a t the described flashing p h e n o m e n o n is p r o b a b l y connected w i t h q u e n c h i n g of t h e fluorescence of m e p a c r i n e accumulated in t h e storage organelles. I r r a d i a t i o n of the platelets p r o b a b l y induces liberation of m e p a c r i n e from these organelles and subsequent dilution of t h e drug (e.g. in the cytoplasm). As a consequence, t h e q u e n c h i n g is diminished, and the fluorescence increases in i n t e n s i t y followed b y a decrease due to fading a n d / o r leaking of t h e m e p a c r i n e out of t h e platelets. Acridine orange and d a u n o m y c i n b e h a v e d like mepacrine. I n fact, platelets loaded w i t h t h e former drugs showed highly fluorescent (yellow-red) granular structures similar in n u m b e r and size to the fluorescent granules
~ -
2b
3b
4b
s~o.ds
Fig. 2. Microfluorimetric measurement of the fluorescence intensity of a single rabbit blood platelet incubated with mepaerine 5 7, 10-5 M. After violet-blue irradiation for 25 see the platelet started to emit flashes.
H. ST~FFE~, personal communication. s Acknowledgment. We thank Drs H. R. BAUMGARTNERand H. STEFFEN, Research Department, F. Hoffmann-La Roche & Co. Ltd, Basel, for advice in performing and interpreting the experiments.
595
seen in platelets stained w i t h mepacrine. I n addition, t h e flashing p h e n o m e n o n was to be seen on exposure of the platelets to violet-blue light. The m e c h a n i s m b y which irradiation acts on 5I-IT storage organelles is not known. The alteration of the organelles Which leads to t h e flashing p h e n o m e n o n seems to be due to the a c t i v a t i n g (violet-blue) and not to the e m i t t e d (green-yellow) light. Thus, mepacrine-loaded platelets, irradiated for 45 min w i t h intense green (546 nm) light, still showed fluorescent granular structures which were flashing on subsequent exposure to violet-blue light. H o w e v e r , the flashing was n o t bound to live cells ; it e v e n t o o k place in mepacrine-loaded platelets fixed for 2 h w i t h 2.5% glutaraldehyde. Therefore, t h e i n v o l v e m e n t of contractile proteins in t h e flashing p h e n o m e n o n is unlikely. B o t h the visualization of t h e 5 H T storage organelles b y fluorescent drugs and the flashing p h e n o m e n o n m a y be of interest for s t u d y i n g these organelles in situ under various conditions, e.g. during t h e release reaction. Such observations will p r o b a b l y not be l i m i t e d to blood platelets since, according to p r e l i m i n a r y experiments, flashing also occurs in other cells which contain subcellular particles (e.g. lysosomes in leucocytes and m a s t cell granules) storing mepacrine. F u r t h e r m o r e , flashing is also observed in isolated chromaffin granules of bovine adrenal medulla loaded in v i t r o w i t h t h e drug.
Zusammen/assung. N a c h B e h a n d l u n g yon Blutpl~ittchen m i t Mepacrin, Acridin-Orange und D a u n o m y c i n , die sich selektiv in den 5 - H y d r o x y t r y p t a m i n - (5HT)-Speicherorganellen anreichern, werden letztere im Fluoreszenzmikroskop sichtbar. U n t e r v i o l e t t - b l a u e r Bestrahlung zeigen die einzelnen P l ~ t t c h e n mehrere sukzessive, b!itzartige Z u n a h m e n der Fluoreszenz-Intensit/it, wobei sie w~hrend einiger Sekunden vollst/indig ausgeleuchtet erscheinen. Die Blitze sind wahrscheinlich durch Freisetzung der fluoreszierenden Stoffe aus je einer 5HTSpeicherorganelle bedingt. H. P. LOREZ, M. DA PRADA and A. PLETSCHER s
Research Department, F. Ho//mann-La Roche & Co. Ltd, CH-4002 Basel (Switzerland), 3 March 1975.
Lung Inflammation in I m m u n i t y to Schistosoma mansoni Schistosoma mansoni, the causal agent of a widespread tropical disease, is a parasitic w o r m which lives in the hepatic portal system of man. Infection occurs when cercariae emerge f r o m a w a t e r snail i n t e r m e d i a t e host, p e n e t r a t e exposed h u m a n skin and m i g r a t e via t h e lungs to the portal system. As t h e y o u n g schistosomes, called schistosomula, are m i g r a t i n g t h r o u g h t h e lung capillaries t h e y can be recovered in substantial n u m b e r s b y a simple technique which forms the basis of an assay for m e a s u r i n g i m m u n i t y to reinfection in rats 1 and mice ~ i m m u n i z e d by a p r i m a r y infection w i t h S. mansonl. A d u l t golden h a m s t e r s (Mesocricetus auratus) were i m m u n i z e d w i t h a small p r i m a r y infection of 50 S. mansoni cercariae and challenged at intervals w i t h 250 cercariae. The n u m b e r of schistosomula recovered from t h e lungs of i m m u n e hamsters on d a y 5 after challenge was greatly depressed c o m p a r e d w i t h recoveries from n o r m a l controls given only t h e challenge infection. I m m u n i t y to reinfection develops rapidly and reaches a p l a t e a u representig 70-80% p r o t e c t i o n at a b o u t 6 weeks after the p r i m a r y infection.
M a n y of t h e i m m u n e hamsters suffer from respiratory distress during the period, 4-7 days after challenge w h e n the i n v a d i n g schistosomula are m i g r a t i n g t h r o u g h the lungs, b u t this is n o t observed in n o r m a l hamsters given the same challenge. Histological e x a m i n a t i o n of t h e lungs of i m m u n e h a m s t e r s during this period reveals t h a t the schistosomula s t i m u l a t e an acute i n f l a m m a t o r y reaction. There is a massive infiltration of p o l y m o r p h o n u c l e a r neutrophils into all the lung tissue which persists as more schistosomula enter the lungs. By day 5, when maximum numbers of organisms are present, many macrophages can be seen, fluid exudates containing neutrophils occur in many alveoli and there are extensive haemorrhages which are also visible macroscopically on the lung surface. The histology of control lungs remains essentially
1 H. PEREZ, J. A. CLEGGand S. R. SMITHERS,Parasitology 69, 349 (1974). 2 F. A. SHER, P. E. MACkeNZIE and S. R. SMITHERS,J. infect. Dis. 730, 626 (1974).
596
Speeialia
Protection of hamsters against S. manson{ infection with human serum albumin (HSA) and Maia squinado hemocyanin (MSH) Substance injected on day 3
Schistosomula recovered from lungs on day 5 (%)
Control 1 mg MSH + anti-MSH Soluble MSH 1 mg 5 mg
26.5 + 2.2 11.5 =h 4.6
Control Heat-pptd HSA 1 mg Soluble HSA 1 mg 5 mg
17.1 -t= 4.5 10.0 • 6.1
11.3 ~ 3.5 11.3 =~ 4.3
9.3 ~ 3.6 8.3 • 1.6
Each treatment was given to 10 adult normal male hamsters in 2 experiments. 0.1 ml of each substance, suspended or dissolved in Hanks' saline was given intraeardiae on day 3 after infection with 250 cereariae and lung recoveries were made on day 5. HSA was heat precipitated at 90 ~ for 15 see. 1 mg IRISH was reacted with 1 mi rabbit anti-MSH for 30 min at 37 ~ and the precipitate washed twice with Hanks' saline. The differences between the mean for each treatment and the appropriate control mean were statistically significant at the 1% level (Student's t-test).
20 NS %
c m
"~ 10
i
iilii!
ili! 8
0 Hamsters
0 Mice
Protection of hamsters and CBA mice against S. manson{ infection with different numbers of dead E. coti. 3 days after infection with 250 eercariae groups of 5 hamsters or mice were given an intracardiae injection of 0.1 ml Hanks' saline containing 4• 106 -- 4• 106 formalin-killed E. coIi. 5 days after infection schistosomula were recovered from the lungs. Each bar represents the mean recovery of schistosomula from a total of 10 animals in 2 experiments with the standard deviation. The differences between the means and the appropriate control were all statistically significant at the 1% level (Students' t-test) except for treatment of hamsters with 4 • 10~ E. coll.
EXPERIENTIA 31/5
n o r m a l d u r i n g m i g r a t i o n of schistosomula, a l t h o u g h t h e r e are some m i n u t e h a e m o r r h a g e s due t o m e c h a n i c a l d a m a g e to t h e capillaries. I n f l a m m a t o r y r e a c t i o n s a r o u n d i n v a d i n g s c h i s t o s o m u l a h a v e also b e e n o b s e r v e d in t h e lungs of i m m u n e mice 3 a n d r h e s u s m o n k e y s ~. W i t h t h e o b j e c t of d e t e r m i n i n g w h e t h e r t h e a c u t e i n f l a m m a t i o n is t h e cause of t h e g r e a t l y r e d u c e d r e c o v e r y of s c h i s t o s o m u l a f r o m t h e lungs of i m m u n e h a m s t e r s we artificially s t i m u l a t e d i n f l a m m a t i o n in t h e lungs of n o r m a l h a m s t e r s following a n infection, 3 d a y s a f t e r infection w i t h 250 cercariae n o r m a l h a m s t e r s were g i v e n a n i n t r a cardiac i n j e c t i o n of 4 • formalin-killed E s c h e r i c h i a coli (type 0111). L u n g recoveries a t d a y 5 s h o w e d a m a r k e d d e p r e s s i o n e q u i v a l e n t t o 8 1 % p r o t e c t i o n (Figure). Smaller n u m b e r s of d e a d E. coli h a d p r o g r e s s i v e l y less effect. I n t r a p e r i t o n e a l i n j e c t i o n of 4 ?
Specialia
been shown to cause liberation of m e p a c r i n e into t h e m e d i u m 1, whereas in T y r o d e their m a j o r i t y remains i n t a c t and retains the drug. This indicates t h a t the described flashing p h e n o m e n o n is p r o b a b l y connected w i t h q u e n c h i n g of t h e fluorescence of m e p a c r i n e accumulated in t h e storage organelles. I r r a d i a t i o n of the platelets p r o b a b l y induces liberation of m e p a c r i n e from these organelles and subsequent dilution of t h e drug (e.g. in the cytoplasm). As a consequence, t h e q u e n c h i n g is diminished, and the fluorescence increases in i n t e n s i t y followed b y a decrease due to fading a n d / o r leaking of t h e m e p a c r i n e out of t h e platelets. Acridine orange and d a u n o m y c i n b e h a v e d like mepacrine. I n fact, platelets loaded w i t h t h e former drugs showed highly fluorescent (yellow-red) granular structures similar in n u m b e r and size to the fluorescent granules
~ -
2b
3b
4b
s~o.ds
Fig. 2. Microfluorimetric measurement of the fluorescence intensity of a single rabbit blood platelet incubated with mepaerine 5 7, 10-5 M. After violet-blue irradiation for 25 see the platelet started to emit flashes.
H. ST~FFE~, personal communication. s Acknowledgment. We thank Drs H. R. BAUMGARTNERand H. STEFFEN, Research Department, F. Hoffmann-La Roche & Co. Ltd, Basel, for advice in performing and interpreting the experiments.
595
seen in platelets stained w i t h mepacrine. I n addition, t h e flashing p h e n o m e n o n was to be seen on exposure of the platelets to violet-blue light. The m e c h a n i s m b y which irradiation acts on 5I-IT storage organelles is not known. The alteration of the organelles Which leads to t h e flashing p h e n o m e n o n seems to be due to the a c t i v a t i n g (violet-blue) and not to the e m i t t e d (green-yellow) light. Thus, mepacrine-loaded platelets, irradiated for 45 min w i t h intense green (546 nm) light, still showed fluorescent granular structures which were flashing on subsequent exposure to violet-blue light. H o w e v e r , the flashing was n o t bound to live cells ; it e v e n t o o k place in mepacrine-loaded platelets fixed for 2 h w i t h 2.5% glutaraldehyde. Therefore, t h e i n v o l v e m e n t of contractile proteins in t h e flashing p h e n o m e n o n is unlikely. B o t h the visualization of t h e 5 H T storage organelles b y fluorescent drugs and the flashing p h e n o m e n o n m a y be of interest for s t u d y i n g these organelles in situ under various conditions, e.g. during t h e release reaction. Such observations will p r o b a b l y not be l i m i t e d to blood platelets since, according to p r e l i m i n a r y experiments, flashing also occurs in other cells which contain subcellular particles (e.g. lysosomes in leucocytes and m a s t cell granules) storing mepacrine. F u r t h e r m o r e , flashing is also observed in isolated chromaffin granules of bovine adrenal medulla loaded in v i t r o w i t h t h e drug.
Zusammen/assung. N a c h B e h a n d l u n g yon Blutpl~ittchen m i t Mepacrin, Acridin-Orange und D a u n o m y c i n , die sich selektiv in den 5 - H y d r o x y t r y p t a m i n - (5HT)-Speicherorganellen anreichern, werden letztere im Fluoreszenzmikroskop sichtbar. U n t e r v i o l e t t - b l a u e r Bestrahlung zeigen die einzelnen P l ~ t t c h e n mehrere sukzessive, b!itzartige Z u n a h m e n der Fluoreszenz-Intensit/it, wobei sie w~hrend einiger Sekunden vollst/indig ausgeleuchtet erscheinen. Die Blitze sind wahrscheinlich durch Freisetzung der fluoreszierenden Stoffe aus je einer 5HTSpeicherorganelle bedingt. H. P. LOREZ, M. DA PRADA and A. PLETSCHER s
Research Department, F. Ho//mann-La Roche & Co. Ltd, CH-4002 Basel (Switzerland), 3 March 1975.
Lung Inflammation in I m m u n i t y to Schistosoma mansoni Schistosoma mansoni, the causal agent of a widespread tropical disease, is a parasitic w o r m which lives in the hepatic portal system of man. Infection occurs when cercariae emerge f r o m a w a t e r snail i n t e r m e d i a t e host, p e n e t r a t e exposed h u m a n skin and m i g r a t e via t h e lungs to the portal system. As t h e y o u n g schistosomes, called schistosomula, are m i g r a t i n g t h r o u g h t h e lung capillaries t h e y can be recovered in substantial n u m b e r s b y a simple technique which forms the basis of an assay for m e a s u r i n g i m m u n i t y to reinfection in rats 1 and mice ~ i m m u n i z e d by a p r i m a r y infection w i t h S. mansonl. A d u l t golden h a m s t e r s (Mesocricetus auratus) were i m m u n i z e d w i t h a small p r i m a r y infection of 50 S. mansoni cercariae and challenged at intervals w i t h 250 cercariae. The n u m b e r of schistosomula recovered from t h e lungs of i m m u n e hamsters on d a y 5 after challenge was greatly depressed c o m p a r e d w i t h recoveries from n o r m a l controls given only t h e challenge infection. I m m u n i t y to reinfection develops rapidly and reaches a p l a t e a u representig 70-80% p r o t e c t i o n at a b o u t 6 weeks after the p r i m a r y infection.
M a n y of t h e i m m u n e hamsters suffer from respiratory distress during the period, 4-7 days after challenge w h e n the i n v a d i n g schistosomula are m i g r a t i n g t h r o u g h the lungs, b u t this is n o t observed in n o r m a l hamsters given the same challenge. Histological e x a m i n a t i o n of t h e lungs of i m m u n e h a m s t e r s during this period reveals t h a t the schistosomula s t i m u l a t e an acute i n f l a m m a t o r y reaction. There is a massive infiltration of p o l y m o r p h o n u c l e a r neutrophils into all the lung tissue which persists as more schistosomula enter the lungs. By day 5, when maximum numbers of organisms are present, many macrophages can be seen, fluid exudates containing neutrophils occur in many alveoli and there are extensive haemorrhages which are also visible macroscopically on the lung surface. The histology of control lungs remains essentially
1 H. PEREZ, J. A. CLEGGand S. R. SMITHERS,Parasitology 69, 349 (1974). 2 F. A. SHER, P. E. MACkeNZIE and S. R. SMITHERS,J. infect. Dis. 730, 626 (1974).
596
Speeialia
Protection of hamsters against S. manson{ infection with human serum albumin (HSA) and Maia squinado hemocyanin (MSH) Substance injected on day 3
Schistosomula recovered from lungs on day 5 (%)
Control 1 mg MSH + anti-MSH Soluble MSH 1 mg 5 mg
26.5 + 2.2 11.5 =h 4.6
Control Heat-pptd HSA 1 mg Soluble HSA 1 mg 5 mg
17.1 -t= 4.5 10.0 • 6.1
11.3 ~ 3.5 11.3 =~ 4.3
9.3 ~ 3.6 8.3 • 1.6
Each treatment was given to 10 adult normal male hamsters in 2 experiments. 0.1 ml of each substance, suspended or dissolved in Hanks' saline was given intraeardiae on day 3 after infection with 250 cereariae and lung recoveries were made on day 5. HSA was heat precipitated at 90 ~ for 15 see. 1 mg IRISH was reacted with 1 mi rabbit anti-MSH for 30 min at 37 ~ and the precipitate washed twice with Hanks' saline. The differences between the mean for each treatment and the appropriate control mean were statistically significant at the 1% level (Student's t-test).
20 NS %
c m
"~ 10
i
iilii!
ili! 8
0 Hamsters
0 Mice
Protection of hamsters and CBA mice against S. manson{ infection with different numbers of dead E. coti. 3 days after infection with 250 eercariae groups of 5 hamsters or mice were given an intracardiae injection of 0.1 ml Hanks' saline containing 4• 106 -- 4• 106 formalin-killed E. coIi. 5 days after infection schistosomula were recovered from the lungs. Each bar represents the mean recovery of schistosomula from a total of 10 animals in 2 experiments with the standard deviation. The differences between the means and the appropriate control were all statistically significant at the 1% level (Students' t-test) except for treatment of hamsters with 4 • 10~ E. coll.
EXPERIENTIA 31/5
n o r m a l d u r i n g m i g r a t i o n of schistosomula, a l t h o u g h t h e r e are some m i n u t e h a e m o r r h a g e s due t o m e c h a n i c a l d a m a g e to t h e capillaries. I n f l a m m a t o r y r e a c t i o n s a r o u n d i n v a d i n g s c h i s t o s o m u l a h a v e also b e e n o b s e r v e d in t h e lungs of i m m u n e mice 3 a n d r h e s u s m o n k e y s ~. W i t h t h e o b j e c t of d e t e r m i n i n g w h e t h e r t h e a c u t e i n f l a m m a t i o n is t h e cause of t h e g r e a t l y r e d u c e d r e c o v e r y of s c h i s t o s o m u l a f r o m t h e lungs of i m m u n e h a m s t e r s we artificially s t i m u l a t e d i n f l a m m a t i o n in t h e lungs of n o r m a l h a m s t e r s following a n infection, 3 d a y s a f t e r infection w i t h 250 cercariae n o r m a l h a m s t e r s were g i v e n a n i n t r a cardiac i n j e c t i o n of 4 • formalin-killed E s c h e r i c h i a coli (type 0111). L u n g recoveries a t d a y 5 s h o w e d a m a r k e d d e p r e s s i o n e q u i v a l e n t t o 8 1 % p r o t e c t i o n (Figure). Smaller n u m b e r s of d e a d E. coli h a d p r o g r e s s i v e l y less effect. I n t r a p e r i t o n e a l i n j e c t i o n of 4 ?
