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Mucosal mast cells in Sprague-Dawley rats infected with Hymenolepis diminuta tapeworms
RESEARCHNOTE MUCOSAL MAST CELLS IN SPRAGUE-DAWLEY RATS INFECTED WITH ~Y~E~~~EPr~ ~~~r~~T~ TAPEWORMS and CLIVE N. COPEMAN
D. WARREN FEATHERSTON
Department of Zoology, University of Otago, P. 0. Box 56, Dunedin, New Zealand (Received 29 January 1990; accepted 5 February 1990) AbstraCt-FEATHERSTON D. W. and COPEMAN C. N. 1990. Mucosal mast cells in Sprague-Dawley rats infected with Hymenolepis diminuta tapeworms. International Journalfor Parasitology 20: 4OlL403. Sixweek-old Sprague-Dawley female rats each infected with 40 Hymenolepis diminuta cysts showed increased mastocytosis from day 30 post-infection (pi.) to day 47 pi. Rats treated on day 40 p.i. with anthelmintic and autopsied 22 days later showed reduced mucosal mast cell (MMC) counts. Other infected rats, treated with anthelminti~ on day 40, challenged with a 10 cysticercoid infection on day 47 and sub~quen~y autopsied between day 8 and 19 post-challenge, maintained a high MMC count. Age of rats in this experiment was not a factor in mastocytosis.
INDEX KEY WORDS: Mucosal mast cells; Hymenolepis diminuta; Sprague-Dawley rats.
~i~p~stro~gyius b~asi~~e~sis-induced mastocytosis of the alimentary tract in rats has been examined extensively (Wells, 1971; Miller & Jarrett, 1971; King & Miller, 1984; Woodbury, Miller, Huntley, Palliser & Wakelin, 1984; King, Miller, Woodbury & Newlands, 1986), and recently reviewed by Rothwell (1989). Woodbury et al. (1984) showed that the number of mucosal mast cells (MMC) reached a peak sometime after the spontaneous expulsion of worms occurred but rat mast cell protease II (RMCPII) concentrations in serum rose to high levels prior to the commencement of expulsion. Hindsbo, Andreassen & Ruitenberg (1982) showed that counts of MMC in rats infected with large numbers of Hymenolepis diminuta cysticercoids (100) differed signi~cantly from both uninfected control and anti-thymocyte serum (ATS) treated rats during a 20 day infection. In a preliminary study (unpublished results) using 40 H. diminuta cysticercoids per rat, only a weak mastocytosis was elicited at 20 days post-infection but significant tapeworm rejection occurred. The work reported here set out to extend these observations to see whether a stronger mucosal mast cell response developed during an extended infection period, if it is was maintained after the removal of the infection and what eff’ect a subsequent small challenge infection (10 cysticercoids) might have on mastocytosis. Sprague-Dawley female rats, 6 weeks old at the commencement of the experiment, were divided into four groups. Group A comprising 35 rats and Group B comprising 21 rats were infected with 40 Hymenolepis diminuta cysticercoids each on day 0. A further 42 control rats were sham infected on day 0 using Hank’s BSS. Seven Group A rats along with all Group B rats on day 40 post-infection fP.i.) were cleared of worms
using a dose of 5 mg Praziquantel (DroncitE, Bayer) per 200 g body weight. Group B rats were subsequently challenged with 10 H. dimirtuta cysticercoids on day 47 p.i. Group C comprising 21 rats were infected with 10 cysticercoids each on day 47 p.i. Autopsies were carried out on groups of seven rats at specified times throughout the experimental period with the final groups being examined on day 66 p.i. or day 19 post-challenge (p.c.). At autopsy the small intestine was removed, the worms flushed out with warm Hank’s BSS (38°C) from a 50 ml syringe and counted. Total worm dry weight per rat was determined after heating the tissue in aluminium foil cups in an oven for 48 h at 95°C. Three or four sections of tissue 2-3 cm long were removed from sites O-5,2.530, 50-55 and 75-80% down the length of the small intestine, opened longitudinally, and fixed overnight in Carnoy’s fluid (Enerback, 1966a). Strips of tissue were trimmed, dehydrated and embedded in Paraplast via graded ethanols and xylene. Sections 8 pm thick were cut along the long axis of the gut, stained for 10 min in Astra Blue (Bloom&Kelly, 1960), rinsed in 0.7 t+HCf for 10 min and counterstained with Safranin 0 (Enerback, 1966b) for 1 h. Mast cells were counted in each of 25 villus-crypt units (VCU) (Miller & Jarrett, 1971) and expressed as a mean number per VCU. Statistical analysis, using one way analysis of variance (Statview 512, Apple Macintosh), was used to determine significance where appropriate. Table I summarizes the recovery of worms from infected rats. On day 8 p.i. 91% of worms from Group A rats were recovered showing a high establishment level. Subsequent recoveries from this group showed a decline from 80% at day 20 p.i. to 28% on day 47 p.i. The zero worm recovery from seven Group A rats 401
402
D. W. FEATHERSTON and C. N. COPEMAN TABLE ~-WORM RECOVERY RATES AND DRY WEIGHTS FROM EXPERIMENTAL RATS INFECTED WITH 40 CYSTICERCOIDS (GROUPS A AND B) AND CHALLENGED WITH 10 CYSTKERCOIDS (GROUPS B AND C) OF Hymenolepis diminuia Day*
Group
No. rats infected/ No. rats autopsied.
Mean No.
Mean total worm
WOr"IS f SD
Mean worm
Wt(g) f S.D
Wt(g) f SD
A
8 20 30 47 62
0
f f f f 0
217 717 717
3.0 f 1.41 5.6 f 1.27 6.1 f 2.12
0.1235 f 0.0357 0.3137 f 0.0752
0.0237 zk 0.0095 0.0557 * 0.0169
8 15 19
717 717 717
8.7 f 0.76 9.0 f 0.58 7.3 f 1.8
0.0043 f 0.0023 0.7737 f 0.152 0.7253 f 0.1775
0.0005 f 0.0002 0.0862 f 0.0174 0.1046 f 0.0372
* Group
A days post-infection,
Group
717 616 617 617 O/7
f f f f
1.62
0.1149 1.0433 0.4151 0.2934
I I .49 11.59 15.03
0.0418 0.3662 0.3895 0.5252
0.0031 0.0400 0.0171 0.0197
f f f f 0
0.0011 0.0271 0.0092 0.0122
B
8 15 19 Group
36.4 32.0 20.3 11.3
C
Groups
B and C days post-challenge
after infection
given on day 40.
TABLE ~~MEANNUMBEROFMUCOSALMASTCELLSPERVLLLUSCRYPTUNIT(* SD.)FROMIJNINFECTEDCONTROLS AND SPRAGUE-DAWLEYRATSINFECTEDWITH Hymenolepisdiminuta
Day 20 30 40 47 55(8)* 62(15) 66( 19)
Controls 8.12 f 0.77 9.06 f 0.85” 9.36 f l.06h
Group
A
Group
B
GroupC
_
8.09 f 1.8 12.49 f 2.21”’ 16.93 f 2.91k
8.02 f 0.69” 9.00 f 0.55s 7.98 f 0.76”
9.41 f
1.03’
14.63 f 0.73” 14.42 f 1.41” 15.23 f 0.59h’
8.18 zk 0.84d” 8.44 f 1.61’ 9.71 zk 1.42h”’
Group A, only 40 cysticercoids; Group B, 40 cysticercoids removed at day 40 pi. and subsequently challenged 7 days later with IO cysticercoids; Group C, challenged with 10 cysticercoids only on day 47 p.i. Figures with the same superscript letters are statistically significantly different at the following levels: a,b,c,d,e,f,g,h,i: P < 0.01: x and y: P ~0.05. * Figures in parentheses refer to days post-challenge for Groups B and C.
autopsied on day 62 p.i. confirmed the efficacy of the anthelmintic treatment given on day 40 pi. Recoveries of the IO cysticercoid challenge infections in Group B rats on day 8 p.c. showed a strikingly different result from the day 8 p.i. recoveries of the primary infection. Whereas in the primary infection all rats were infected at day 8 p.i. only two rats out of seven were infected at day 8 p.c., one with two worms and the other with four. The 30% recovery from the challenge infection, compared to 91% in rats carrying a primary infection, was extremely low when compared with rats autopsied subsequently at day IS p.c. and day 19 p.c. Rats from Group C, infected only on day 47 with 10 cysts and acting as age controls for the Group B challenged rats, showed significantly higher recovery rates when compared with the equivalent experimental rats. As there was no significant difference in the number of MMC per VCU taken from the different sections
along the gut, results shown in Table 2 are total counts from each rat. Counts of MMC from control rats ranging from between 7.98 and 9.36 per VCU throughout the experiment were not significantly different. There were significant differences between a number of infected groups as well as differences between infected groups and their equivalent control groups. Group A rats on day 20 p.i. showed no significant difference in MMC per VCU from the controls but at day 30, counts of 12.49 were significantly different (P
403
Research Note anthelmintic on day 40 pi. Group B rats showed that a small challenge infection of 10 cysticercoids can maintain the higher level of MMC per VCU through to day 66 p.i., i.e. 19 days post-challenge. Counts of MMC in Group C rats were not significantly different from day 20 p.i. control rats. The MMC response of Sprague-Dawley female rats 6 weeks old at infection to 40 cysticercoids takes at least 4 weeks to develop. While this experiment showed a significant difference at 30 days pi. a previous smaller preliminary experiment did show a slight difference at day 20. Such variations could be host mediated as the strain is outbred, or the response may be dependent on the number of cysticercoids used in the infection. Hindsbo et al. (1982), using 100 cysticercoid infections reported significant differences in MMC numbers between controls and infected rats from day 3 pi. onwards as well as increased MMC counts with age in the control groups. There was no significant difference in MMC numbers between groups of control rats in this experiment. Again, when rejection occurred between day 20 and day 47 in Group A rats and the recovery rates dropped from 80 to 28%, the MMC counts rose significantly. This is similar to the results obtained by Woodbury et cd. (1984) who showed that MMC numbers reached a maximum after N. brffsiliensis had been rejected. Whether or not rejection is related to the increase in MMC numbers is not clear. What is clear is that when worms are removed with anthelmintic, MMC numbers quickly return to the control group levels suggesting that an elevated mastocytosis requires the presence of the parasite. Group B results show that MMC numbers can be maintained after removal of the primary infection provided an early challenge infection is established. The results indicate that the number of cysticercoids used in the challenge infection does not have to be great. There is also a hint that an elevated mastocytosis may interfere with a subsequent challenge infection as the numbers of challenge worms recovered from Group B rats were fewer and significantly smaller than those from the control rats in Group C. This reduction in secondary worms confirms similar results obtained by Andreassen & Hopkins (1980). Further work is also needed to show the relationship between the size of challenge infection and the time between the removal of the primary infection and the initiation of the challenge infection. These results suggest that the time period is somewhere between 8 and 22 days. Age of the host
does not have any challenge infection difference between and the uninfected
effect on the MMC response to the of 10 cysts as there is no significant MMC counts of the Group C rats controls.
Acknowle~ge~enls-Technical
assistance from Karen Judge, is gratefully Gerald Stokes and Robin Baldwin acknowledged. We also wish to thank Mr M. Roberts of Bayer New Zealand Ltd, for the supply of Droncit used in this experiment. REFERENCES J. & HOPKINS C. A. 1980. Immunologically mediated rejection of Hymenolepis diminuta by its normal host, the rat. Journal of Parasitology 66:898-903. BLOOMG. & KELLY J. W. 1960. The copper phthalocyanine
ANDREASSEN
dye ‘Astrablau’ and its staining properties, especially the staining of mast cells. Histochemie 2: 48-57. ENERBACKL. 1966a. Mast cellsin rat gastrointestinal mucosa. I. Effects of fixation. Acta Pathologies et Microbio~o~ic~ Scandinavica 66: 289-302. ENERBACK L. 1966b.. Mast cells in rat gastrointestinal mucosa. 2. Dye-binding and metachromatic properties. Acta Pathologica et Microbiologica Scandinavica 66: 303312. HINDSBO O., ANDREASSEN J. & RUITENBERG J. 1982. Immunologi~l and histopathological reactions of the rat against the tapeworm Hymenolepis diminuta and the effects of anti-thymocyte serum. Parasite Immunology 4: 59-76. KING S. J. & MILLER H. R. P. 1984. Anaphylactic release of mucosal mast cell protease and its relationship to gut permeability in Nippostrongylus-primed rats. Immunology 51: 653-660. KING S. J., MILLER H. R. P., WOODBURYR. G. & NEWLANDS G. F. J. 1986. Gut mucosal mast cells in N~ppostrong,vlusprimed rats are the major source of secreted rat mast cell protease II following systemic anaphylaxis. European Journal of Immunology 16: 151-155. MILLER H. R. P. &JARRE~ W. F. H. 1971. Immune reactions in mucous membranes. I. Intestinal mast cell response during helminth expulsion in the rat. ~mmunofogy 20: 277288. ROTHWELL T. L. W. 1989. Immune expulsion of parasitic nematodes from the alimentary tract. International Journul for Parasitology 19: 139-168. WELLS P. D. 1971. Nippostrongylus brasiliensis: mast cell populations in rats. Experimental Parasitology 30: 30-35. WOODFIURYR. G., MILLER H. R. P., HUNTLEYJ. F., PALLISER A. C. & WAKELIN D. 1984. Mucosal mast cells are functionally active during spontaneous expulsion of intestinal nematode infections in rats. Nufure (London) 312: 45&452.
D. WARREN FEATHERSTON
Department of Zoology, University of Otago, P. 0. Box 56, Dunedin, New Zealand (Received 29 January 1990; accepted 5 February 1990) AbstraCt-FEATHERSTON D. W. and COPEMAN C. N. 1990. Mucosal mast cells in Sprague-Dawley rats infected with Hymenolepis diminuta tapeworms. International Journalfor Parasitology 20: 4OlL403. Sixweek-old Sprague-Dawley female rats each infected with 40 Hymenolepis diminuta cysts showed increased mastocytosis from day 30 post-infection (pi.) to day 47 pi. Rats treated on day 40 p.i. with anthelmintic and autopsied 22 days later showed reduced mucosal mast cell (MMC) counts. Other infected rats, treated with anthelminti~ on day 40, challenged with a 10 cysticercoid infection on day 47 and sub~quen~y autopsied between day 8 and 19 post-challenge, maintained a high MMC count. Age of rats in this experiment was not a factor in mastocytosis.
INDEX KEY WORDS: Mucosal mast cells; Hymenolepis diminuta; Sprague-Dawley rats.
~i~p~stro~gyius b~asi~~e~sis-induced mastocytosis of the alimentary tract in rats has been examined extensively (Wells, 1971; Miller & Jarrett, 1971; King & Miller, 1984; Woodbury, Miller, Huntley, Palliser & Wakelin, 1984; King, Miller, Woodbury & Newlands, 1986), and recently reviewed by Rothwell (1989). Woodbury et al. (1984) showed that the number of mucosal mast cells (MMC) reached a peak sometime after the spontaneous expulsion of worms occurred but rat mast cell protease II (RMCPII) concentrations in serum rose to high levels prior to the commencement of expulsion. Hindsbo, Andreassen & Ruitenberg (1982) showed that counts of MMC in rats infected with large numbers of Hymenolepis diminuta cysticercoids (100) differed signi~cantly from both uninfected control and anti-thymocyte serum (ATS) treated rats during a 20 day infection. In a preliminary study (unpublished results) using 40 H. diminuta cysticercoids per rat, only a weak mastocytosis was elicited at 20 days post-infection but significant tapeworm rejection occurred. The work reported here set out to extend these observations to see whether a stronger mucosal mast cell response developed during an extended infection period, if it is was maintained after the removal of the infection and what eff’ect a subsequent small challenge infection (10 cysticercoids) might have on mastocytosis. Sprague-Dawley female rats, 6 weeks old at the commencement of the experiment, were divided into four groups. Group A comprising 35 rats and Group B comprising 21 rats were infected with 40 Hymenolepis diminuta cysticercoids each on day 0. A further 42 control rats were sham infected on day 0 using Hank’s BSS. Seven Group A rats along with all Group B rats on day 40 post-infection fP.i.) were cleared of worms
using a dose of 5 mg Praziquantel (DroncitE, Bayer) per 200 g body weight. Group B rats were subsequently challenged with 10 H. dimirtuta cysticercoids on day 47 p.i. Group C comprising 21 rats were infected with 10 cysticercoids each on day 47 p.i. Autopsies were carried out on groups of seven rats at specified times throughout the experimental period with the final groups being examined on day 66 p.i. or day 19 post-challenge (p.c.). At autopsy the small intestine was removed, the worms flushed out with warm Hank’s BSS (38°C) from a 50 ml syringe and counted. Total worm dry weight per rat was determined after heating the tissue in aluminium foil cups in an oven for 48 h at 95°C. Three or four sections of tissue 2-3 cm long were removed from sites O-5,2.530, 50-55 and 75-80% down the length of the small intestine, opened longitudinally, and fixed overnight in Carnoy’s fluid (Enerback, 1966a). Strips of tissue were trimmed, dehydrated and embedded in Paraplast via graded ethanols and xylene. Sections 8 pm thick were cut along the long axis of the gut, stained for 10 min in Astra Blue (Bloom&Kelly, 1960), rinsed in 0.7 t+HCf for 10 min and counterstained with Safranin 0 (Enerback, 1966b) for 1 h. Mast cells were counted in each of 25 villus-crypt units (VCU) (Miller & Jarrett, 1971) and expressed as a mean number per VCU. Statistical analysis, using one way analysis of variance (Statview 512, Apple Macintosh), was used to determine significance where appropriate. Table I summarizes the recovery of worms from infected rats. On day 8 p.i. 91% of worms from Group A rats were recovered showing a high establishment level. Subsequent recoveries from this group showed a decline from 80% at day 20 p.i. to 28% on day 47 p.i. The zero worm recovery from seven Group A rats 401
402
D. W. FEATHERSTON and C. N. COPEMAN TABLE ~-WORM RECOVERY RATES AND DRY WEIGHTS FROM EXPERIMENTAL RATS INFECTED WITH 40 CYSTICERCOIDS (GROUPS A AND B) AND CHALLENGED WITH 10 CYSTKERCOIDS (GROUPS B AND C) OF Hymenolepis diminuia Day*
Group
No. rats infected/ No. rats autopsied.
Mean No.
Mean total worm
WOr"IS f SD
Mean worm
Wt(g) f S.D
Wt(g) f SD
A
8 20 30 47 62
0
f f f f 0
217 717 717
3.0 f 1.41 5.6 f 1.27 6.1 f 2.12
0.1235 f 0.0357 0.3137 f 0.0752
0.0237 zk 0.0095 0.0557 * 0.0169
8 15 19
717 717 717
8.7 f 0.76 9.0 f 0.58 7.3 f 1.8
0.0043 f 0.0023 0.7737 f 0.152 0.7253 f 0.1775
0.0005 f 0.0002 0.0862 f 0.0174 0.1046 f 0.0372
* Group
A days post-infection,
Group
717 616 617 617 O/7
f f f f
1.62
0.1149 1.0433 0.4151 0.2934
I I .49 11.59 15.03
0.0418 0.3662 0.3895 0.5252
0.0031 0.0400 0.0171 0.0197
f f f f 0
0.0011 0.0271 0.0092 0.0122
B
8 15 19 Group
36.4 32.0 20.3 11.3
C
Groups
B and C days post-challenge
after infection
given on day 40.
TABLE ~~MEANNUMBEROFMUCOSALMASTCELLSPERVLLLUSCRYPTUNIT(* SD.)FROMIJNINFECTEDCONTROLS AND SPRAGUE-DAWLEYRATSINFECTEDWITH Hymenolepisdiminuta
Day 20 30 40 47 55(8)* 62(15) 66( 19)
Controls 8.12 f 0.77 9.06 f 0.85” 9.36 f l.06h
Group
A
Group
B
GroupC
_
8.09 f 1.8 12.49 f 2.21”’ 16.93 f 2.91k
8.02 f 0.69” 9.00 f 0.55s 7.98 f 0.76”
9.41 f
1.03’
14.63 f 0.73” 14.42 f 1.41” 15.23 f 0.59h’
8.18 zk 0.84d” 8.44 f 1.61’ 9.71 zk 1.42h”’
Group A, only 40 cysticercoids; Group B, 40 cysticercoids removed at day 40 pi. and subsequently challenged 7 days later with IO cysticercoids; Group C, challenged with 10 cysticercoids only on day 47 p.i. Figures with the same superscript letters are statistically significantly different at the following levels: a,b,c,d,e,f,g,h,i: P < 0.01: x and y: P ~0.05. * Figures in parentheses refer to days post-challenge for Groups B and C.
autopsied on day 62 p.i. confirmed the efficacy of the anthelmintic treatment given on day 40 pi. Recoveries of the IO cysticercoid challenge infections in Group B rats on day 8 p.c. showed a strikingly different result from the day 8 p.i. recoveries of the primary infection. Whereas in the primary infection all rats were infected at day 8 p.i. only two rats out of seven were infected at day 8 p.c., one with two worms and the other with four. The 30% recovery from the challenge infection, compared to 91% in rats carrying a primary infection, was extremely low when compared with rats autopsied subsequently at day IS p.c. and day 19 p.c. Rats from Group C, infected only on day 47 with 10 cysts and acting as age controls for the Group B challenged rats, showed significantly higher recovery rates when compared with the equivalent experimental rats. As there was no significant difference in the number of MMC per VCU taken from the different sections
along the gut, results shown in Table 2 are total counts from each rat. Counts of MMC from control rats ranging from between 7.98 and 9.36 per VCU throughout the experiment were not significantly different. There were significant differences between a number of infected groups as well as differences between infected groups and their equivalent control groups. Group A rats on day 20 p.i. showed no significant difference in MMC per VCU from the controls but at day 30, counts of 12.49 were significantly different (P
403
Research Note anthelmintic on day 40 pi. Group B rats showed that a small challenge infection of 10 cysticercoids can maintain the higher level of MMC per VCU through to day 66 p.i., i.e. 19 days post-challenge. Counts of MMC in Group C rats were not significantly different from day 20 p.i. control rats. The MMC response of Sprague-Dawley female rats 6 weeks old at infection to 40 cysticercoids takes at least 4 weeks to develop. While this experiment showed a significant difference at 30 days pi. a previous smaller preliminary experiment did show a slight difference at day 20. Such variations could be host mediated as the strain is outbred, or the response may be dependent on the number of cysticercoids used in the infection. Hindsbo et al. (1982), using 100 cysticercoid infections reported significant differences in MMC numbers between controls and infected rats from day 3 pi. onwards as well as increased MMC counts with age in the control groups. There was no significant difference in MMC numbers between groups of control rats in this experiment. Again, when rejection occurred between day 20 and day 47 in Group A rats and the recovery rates dropped from 80 to 28%, the MMC counts rose significantly. This is similar to the results obtained by Woodbury et cd. (1984) who showed that MMC numbers reached a maximum after N. brffsiliensis had been rejected. Whether or not rejection is related to the increase in MMC numbers is not clear. What is clear is that when worms are removed with anthelmintic, MMC numbers quickly return to the control group levels suggesting that an elevated mastocytosis requires the presence of the parasite. Group B results show that MMC numbers can be maintained after removal of the primary infection provided an early challenge infection is established. The results indicate that the number of cysticercoids used in the challenge infection does not have to be great. There is also a hint that an elevated mastocytosis may interfere with a subsequent challenge infection as the numbers of challenge worms recovered from Group B rats were fewer and significantly smaller than those from the control rats in Group C. This reduction in secondary worms confirms similar results obtained by Andreassen & Hopkins (1980). Further work is also needed to show the relationship between the size of challenge infection and the time between the removal of the primary infection and the initiation of the challenge infection. These results suggest that the time period is somewhere between 8 and 22 days. Age of the host
does not have any challenge infection difference between and the uninfected
effect on the MMC response to the of 10 cysts as there is no significant MMC counts of the Group C rats controls.
Acknowle~ge~enls-Technical
assistance from Karen Judge, is gratefully Gerald Stokes and Robin Baldwin acknowledged. We also wish to thank Mr M. Roberts of Bayer New Zealand Ltd, for the supply of Droncit used in this experiment. REFERENCES J. & HOPKINS C. A. 1980. Immunologically mediated rejection of Hymenolepis diminuta by its normal host, the rat. Journal of Parasitology 66:898-903. BLOOMG. & KELLY J. W. 1960. The copper phthalocyanine
ANDREASSEN
dye ‘Astrablau’ and its staining properties, especially the staining of mast cells. Histochemie 2: 48-57. ENERBACKL. 1966a. Mast cellsin rat gastrointestinal mucosa. I. Effects of fixation. Acta Pathologies et Microbio~o~ic~ Scandinavica 66: 289-302. ENERBACK L. 1966b.. Mast cells in rat gastrointestinal mucosa. 2. Dye-binding and metachromatic properties. Acta Pathologica et Microbiologica Scandinavica 66: 303312. HINDSBO O., ANDREASSEN J. & RUITENBERG J. 1982. Immunologi~l and histopathological reactions of the rat against the tapeworm Hymenolepis diminuta and the effects of anti-thymocyte serum. Parasite Immunology 4: 59-76. KING S. J. & MILLER H. R. P. 1984. Anaphylactic release of mucosal mast cell protease and its relationship to gut permeability in Nippostrongylus-primed rats. Immunology 51: 653-660. KING S. J., MILLER H. R. P., WOODBURYR. G. & NEWLANDS G. F. J. 1986. Gut mucosal mast cells in N~ppostrong,vlusprimed rats are the major source of secreted rat mast cell protease II following systemic anaphylaxis. European Journal of Immunology 16: 151-155. MILLER H. R. P. &JARRE~ W. F. H. 1971. Immune reactions in mucous membranes. I. Intestinal mast cell response during helminth expulsion in the rat. ~mmunofogy 20: 277288. ROTHWELL T. L. W. 1989. Immune expulsion of parasitic nematodes from the alimentary tract. International Journul for Parasitology 19: 139-168. WELLS P. D. 1971. Nippostrongylus brasiliensis: mast cell populations in rats. Experimental Parasitology 30: 30-35. WOODFIURYR. G., MILLER H. R. P., HUNTLEYJ. F., PALLISER A. C. & WAKELIN D. 1984. Mucosal mast cells are functionally active during spontaneous expulsion of intestinal nematode infections in rats. Nufure (London) 312: 45&452.