EXPERIMENTAL PARASITOLOGY 47, 81-90

(1979)

Nipposfrongyhs brasiliensis: Response in Adoptively

Intestinal Goblet-Cell Immunized Rats

H. R. P. MILLERI AND Y. NAWA~ Department of Immunology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra City, Australian Capital Territory 2601, Australia (Accepted

for publication

14 November

1978)

MILLER, H. R. P., AND NAWA, Y. 1979. Nippostrongylus brasiliensis: Intestinal gobletcell response in adoptively immunized rats. Experimental Parasitology 47, 81-90. Goblet-cell differentiation was studied in the intestinal epithelium of rats infected with the nematode Nippostrongylus brasiliensis. An increase in the proportion of goblet cells occurred at the time of worm expulsion in rats infected with 1000 or 4000 third stage larvae. Adoptive immunization of infected rats with immune-thoracic duct lymphocytes (TDL) induced extensive goblet-cell differentiation whereas the transfer of immune-TDL into normal rats had no effect. The extent of goblet-cell differentiation in adoptively immunized infected rats was proportional to the number of cells transferred. A goblet-cell response also occurred in adoptively immunized rats harboring implanted “normal” and “damaged” worms but recipients of normal worms which were not given cells were unable either to expel their worm burden or to induce a goblet-cell response. Experiments in which the parasites were expelled with an anthelmintic drug suggested that the gobletcell increase was not simply a repair process associated with the expulsion of the parasites. In all situations where immune expulsion of the parasites occurred, there was a concomitant rise in the proportion of goblet cells. These experiments suggest that thoracic duct lymphocytes either directly or indirectly regulate the differentiation of intestinal goblet cells. INDEX DESCRIPTORS: Nippostrongylus bras&en&; Nematode, parasitic; Rat; Goblet-cell differentiation; Lymphocytes; Thoracic duct; Epithelium, intestinal; Immunity, mucosal; Immunization, adoptive.

of its natural host, the rat, are not fully elucidated. Most workers agree that antibody h as some deleterious effect on the adult parasite (Ogilvie and Love 1974) and it is thought that the final phase of expulsion is a nonspecific event mediated by lymphocytes (Ogilvie and Love 1974) although there is evidence that prostaglandin E1 also plays a role (Kelly and Dineen 1976). Since the parasites live

INTRODUCTION

The protective mechanisms responsible for the elimination of the nematode Nippostrongylus brasiliensis from the jejunum 1 Present address: Department of Pathology, Animal Diseases Research Association, Moredun Institute, 408 Gilmerton Road, Edinburgh EH17 7JH, Scotland, U.K.; and to whom all correspondence should be sent. 2 Present address: Department of Anatomy, Kumamoto University Medical School, 2-2-l Honjo, Kumamoto, Japan.

within

the

lumen

of

the

gut

and

do

not

penetrate the mucosa (Symons 1965) any 81 0014-4894/79/010081-10$02.00/O All

Copyright 0 1979 rights of reproduction

by Academic Press, Inc. in any form reserved.

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FIG. 1. Light micrographs of rat intestinal mucosa stained with PAS-alcian blue to demonstrate goblet cells ( X250). (a) The area of epithelium in which goblet cells are counted is illustrated in this section of normal intestine. (b) Fourteen days after infection with 4000 brasiZien.sis there is an increase in the proportion of goblet cells, third stage Nippostrongylus many of which appear to be discharging their contents. (c) Eight days after infection with

Nippostrongylus

bradiensis:

soluble mediator of worm epulsion would need to traverse a relatively intact epithelium and function in the highly reducing environment of the intestinal lumen. The role of the intestinal epithelium in the mucosal response has received relatively little attention, although villous atrophy and crypt hyperplasia are known to occur during infection (Ferguson and Jarrett 1975, Symons 1965). However, studies by Wells (1963) in rats infected with N. brasilimsis and by Dobson (1966) in parasitized sheep have established that increased numbers of goblet cells are present in parasitized epithelia. Since mucus is reported to have parasiticidal properties (Frick and Ackert 1948) and to inhibit oxygen consumption by nematode larvae (Dobson 1967), increased numbers of goblet cells could be of some significance in the protection of mucosal surfaces against parasitic infection. Immunity against N. brasiliensis is conferred by the adoptive transfer of immunethoracic duct lymphocytes (TDL) and cells drained from donor rats harboring a primary infection confer a high degree of protection (Nawa and Miller 1978, Ogilvie et al. 1977). In the present paper which is a sequel to an already published study of worm burden kinetics (Nawa and Miller 1978), we have examined the intestinal epithelium for changes in gobletcell differentiation which might be associated with immune expulsion of the parasite. The results confirm that elimination of the parasite is associated with an increase in the proportion of intestinal goblet cells (Wells 1963) and establish that this effect can be adoptively transferred by immune-TDL.

RAT GOBLET CELL RESPONSE

83

MATERIALS AND METHODS

Host animals. Female (PvG/c x DA)Fi rats 11 to 13 weeks old were used in all experiments. They were fed a standard diet and were allowed tap water ad libiturn. Females from a colony of randomly bred Wistar rats were used to maintain the parasites. Nippostrongylus brasiliensis. Standard methods were used for the culture and maintenance of the parasite (Jennings et al. 1963). Infective third-stage larvae (L,), adjusted to the required concentration, were injected subcutaneously in 0.5 ml saline. Adult worms were harvested on the sixth (“normal” worms) or eleventh day (“damaged” worms) of infection from (PvG/c x DA)F 1 rats infected with 4000 L3 (Nawa and Miller 1978). Cell transfer. Previous experiments had established that TDL harvested on the tenth day (Day 10 TDL) of infection with 4000 L3 conferred a high degree of protection in infected recipient rats (Nawa and Miller 1978). Thoracic duct cannulation was carried out as described previously (Nawa and Miller 1978). Five units of heparin in 1 ml of saline were injected subcutaneously into cell donors just after cannulation. Thoracic duct lymph was collected overnight in sterile flasks containing 10 ml of medium 199 with 20 units of heparin. TDL were harvested and washed three times with Hanks’ balanced salt solution containing 1% bovine serum albumin and were finally adjusted to the required concentration of viable cells with this medium. The viability of the cells was more than 95% as judged by phasecontrast microscopy. Recipient rats were infected on the day of cell transfer either

1000 L, there is moderately severe atrophy and edema of the villus. Goblet cells are reduced in size and the epithelium has a flattened appearance. (d) Eight days after infection with 1000 L, and adoptive immunization with 1 X 10” Day 10 thoracic duct lymphocytes goblet cells are abundant. Note also that the villous architecture is fully restored when compared with lc.

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subcutaneously with 1000 L3 or by the intraduodenal implantation of adult worms. Histological techniques. Since the parasites infest the anterior third of the intestine, a segment of jejunum 1.5 cm in length and 20 cm from the duodenum was ligated and injected with 0.1 ml freshly prepared 4% formaldehyde in phosphate-buffered saline. The segments were fixed by immersion for 24 hr before they were split longitudinally and their content of worms were counted. One half of each segment was gently flattened, without damaging the mucosa, between two glass slides separated from each other by a distance of 1 mm and was dehydrated in an asending ethanol series. The flattened segments were then trimmed along their longitudinal axes and were cleared in chloroform. They were embedded in paraffin wax and oriented so that 4- to 6-pm sections were cut at right angles to the mucosal surface. This orientation gave the sectioned septate villi a finger-like appearance. Sections were stained with periodic acid/Schiff (PAS) and alcian blue (Mowry 1963) and were counterstained with hematoxylin. Method of counting goblet cells. Preliminary studies confirmed previous observations that during the early stages of infection (Days 6 to S) there is partial villous atrophy together with a decrease in villus height : crypt depth ratio (v/c ratio) (Ferguson and Jarret 1975) and that just before worm expulsion (Days 10 to 14) there is extensive regeneration of the mucosa with a concomitant increase in villous size (Miller and Jarrett 1971). Each section was assessed to determine the extent of villous atrophy or regeneration before goblet and absorptive cells were counted. The counts were done in such a way as to include representative areas of villous atrophy and regeneration. At least 10 villi were counted in each of two sections from a minimum of five rats/ group. In those sections with variable

NAWA

villus morphology 15-20 representative villi were counted. Only villous epithelium was counted, beginning at the base of each villus but excluding the tip (Fig. la). A minimum of 1000 absorptive cells was counted in each section and the proportion of goblet cells present in the villi was expressed as the number of goblet cells per 1000 absorptive cells. RESULTS

Since the experimental protocol for this study has already been published in detail (Nawa and Miller 1978), the numbers of rats in each group and the Nippostrongylus brasiliensis worm-burden kinetics will only be briefly described at the beginning of each section of the results. Goblet Cells during

Primary

Infection

Goblet cell kinetics were studied in rats harboring a 4000 L, infection. Groups of five rats were killed at 48-hr intervals beginning 6 days after infection. The worm-burden kinetics were similar to those described by Jarrett et al. (1968) with parasite expulsion completed by Day 14. Six and eight days after infection with 4000 L.?, during the stable phase of infestation, the proportion of goblet cells was depressed when compared with normal rats (Fig. 2). The few goblet cells that were visible were smaller than normal and in some instances appeared to be discharged. The mucin within these cells and within goblet cells in normal rats was acidic and stained blue in the PAS-alcian blue sequence. Ten days after infection there was extensive villous regeneration and in such areas the proportion of goblet cells was increased. However, goblet cells were rare in the regions of villous atrophy and overall the proportion of goblet cells was only slightly greater than the levels in normal rats (Fig. 2). As the mucosa continued to regenerate, a further increase in the proportion of goblet cells occurred on Day 12, reaching a peak on

Nippostrongylus

I...... 6

DAYS

8

10

12

brasiliensis:

14

16

AFTER INFECTION

FIG. 2. The change in the proportion of goblet cells in rats harboring a 4000 third stage Nippostrongylus brasiliensis infection. ( l ) Infected rats; ( A )uninfected control. Vertical bars indicate 2 SE.

Day 14 (Figs. lb and 2). At this time 20 to 30% of the goblet cells in the villi stained pink, suggesting that they contained a neutral mucin. Worm expulsion was complete by Day 14 and the proportion of goblet cells had declined slightly by Day 16 (Fig. 2). Goblet Rats

Cells in Adaptively

Immunized

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RAT GOBLET CELL RESPONSE

Day 10 to reach a peak on Day 12 when there were 357 + 10 goblet cells/1000 absorptive cells (Fig. 3). Goblet cells in the adoptively immunized animals were increased above control levels on Day 6 reaching a peak 8 to 10 days (Fig. 3) after infection and declining on Day 12. A further difference between the two groups on Day 8 was that the villi were fully regenerated in the recipients of Day 10 TDL (Fig. Id) whereas villous atrophy was moderately severe in the controls (Fig. lc). At the time of worm expulsion in both groups, many of the goblet cells apparently contained neutral mucin since they stained pink in the PAS-alcian blue sequence. Dose-Response Infection

Experiment

after

Larval

Because the previous experiment suggested that TDL influence the differentiation of goblet cells, this relationship was tsted further by examining the epithelium of rats infected with 1000 Ls and given different doses of Day 10 TDL. Worm counts in this experiment have already been described and worm expulsion was found to be a function of the number of cells transferred (Nawa and Miller 1978). The effects of transferring 1 x 107, 5 X

The kinetics of goblet-cell differentiation were studied in rats harboring 1000 La which had been adoptively immunized on the day of infection with Day 10 TDL. Infected rats were allocated to two groups of 20 and were either injected intravenously with 1 x lo8 Day 10 TDL in 1 ml Hanks’ solution containing bovine serum or were given 1 ml albumin (HBSA) ? HBSA alone (Nawa and Miller 1978). / Five rats from each group were killed at if 2-day intervals between Days 6 and 12 : 2 200 postinfection for worm burden and goblet" d' cell counts. The worm-burden kinetics in ; ,opr./;b i2 i4 this experiment have already been de0 scribed in full (Nawa and Miller 1978). DAYS AFTER INFECTION The goblet-cell response in the two FIG. 3. Kinetics of goblet-cell differentiation in groups reflected the worm-burden kinetics. normal ( 0) and adoptively immunized rats ( l ) The proportions of goblet cells were de- infected with 1000 third stage Nippostrongylus brapressed 6 and 8 (Figs. lc and 3) days after siliensis. ( A) Uninfected control. Vertical bars infection in the controls but increased 011 depict f SE.

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/t

5 5?r%TFG LOG ,,, NOTDL

TRANSFERRED

FIG. 4. A logarithmic plot of the relationship between the number of Day 10 TDL transferred and the increase in the proportion of goblet cells ( O-O) in rats infected with 1000 third stage Nippostrongylus brasiliensis. Also depicted are the infected controls ( e ), normal (A ), and adoptively immunized uninfected rats (a). 2 SE is shown by vertical bars.

lo’, 1 x 108, and 4 x 10S Day 10 TDL into groups of five infected rats were examined 8 days after infection and cell transfer. A group of five infected rats not given cells and a group of five worm-free rats given 1 x lo8 Day 10 TDL were killed at the same time and served as controls for goblet-cell counts. When the proportions of goblet cells in the infected recipients of Day 10 TDL were plotted on a logarithmic scale against the number of cells transferred, a linear relationship was observed (Fig. 4). However, the proportion of goblet cells in the uninfected recipients of 1 x lo8 Day 10 TDL was similar to that of normal controls (Fig. 4). These results establish that Day 10 TDL in some way influence gobletcell differentiation and that the recipients must harbor parasites for this to occur. Implantation

of Adult

NAWA

degree of protection against both “normal” and “damaged” worms (Nawa and Miller 1978). An additional unusual feature of infection with implanted adult worms was the tendency of the parasites to aggregate into several large clumps. Clumping of this nature was not observed after larval infections. Groups of five infected control rats and of five recipients of 1 x lo8 Day 10 TDL were killed on Days 5 and 7 after “normal” worm transfer and on Days 3 and 5 after “damaged” worm transfer and goblet cells were counted. No increase in the proportion of goblet cells occurred in the normal recipients of “normal” worms but in the adoptively immunized rats the proportion of goblet cells was greater than in normal uninfected rats or in infected controls 5 and 7 days after implantation (Fig. 5). A rapid increase in the proportion of goblet cells was seen by Day 5 in the normal recipients of “damaged” worms and an increase was apparent on Day 3 in the adoptively immunized rats infected with “damaged” worms (Fig. 6). These experiments establish that adoptive immmlization with Day 10 TDL induces goblet-cell differentiation in the recipients of both “normal” and “damaged” worms. They also suggest that normal re-

Worms

The kinetics of expulsion of 600 implanted “normal” and “damaged” worms have been reported in full, together with the effect of adoptive immunization and, contrary to previous data (Ogilvie and Love 1974), Day 10 TDL conferred a high

TATION

F’Ic. 5. Kinetics of the goblet-cell response in normal ( 0) and adoptively immunized ( l ) recipients of 600 “normal” adult Nipportronfi~jlus bradiensis. ( n ) Uninfected control. Vertical bars indicate k SE.

Nippostrongyhs

bra&e&s:

RAT

GOBLET

CELL

87

RESPONSE

Goblet-Cell Response after Nonimmunological Expulsion of the Parasites

5 ,001 2

s

DAYS

4

AFTER

6

8

IMPLANTATION

FIG. 6. Kinetics

of goblet-cell differentiation in ( 0) and adoptively immunized ( l ) reof 600 “damaged” adult Nippostrongylus brdiensis. (A ) Uninfected control. * SE is depicted by vertical bars. normal cipients

cipients of “normal” worms are less able to mount a goblet-cell response than are normal recipients of “damaged” worms. Dose-Response Experiment tation of Adult Worms

after Implan-

The relationships between the adoptive response, worm expulsion, and gobletcell differentiation were explored further by transferring doses of Day 10 TDL into rats infected by intraduodenal implantation of normal or damaged worms. Groups of five recipients of normal worms were killed on Day 5 after infection and groups of five recipients of damaged worms were killed on Day 3. The doses of transferred cells and the worm burdens have been described in full (Nawa and Miller 1978). The proportions of goblet cells in the recipients of “normal” and “damaged worms were plotted logarithmically against the number of TDL transferred (Fig. 7). Goblet-cell differentiation was increased in recipients of 1, 2, and 4 x lo* Day 10 TDL when compared with normal controls or infected controls not given cells (Fig. 7), but a relationship between the increase in the proportion of goblet cells and the number of TDL transferred was not established.

Because the proportion of goblet cells increased at the time of worm expulsion, the possibility that this was a repair phenomenon associated with epithelial regeneration was investigated. This was done by treating rats with an anthelmintic drug at various stages of a primary infection and examining the goblet-cell response in the mucosa. (PvG/c x DA) F1 rats were infected with 1000 L3 and groups of five rats were given 44 mg/kg of thiabendazole (Thibenzole, Merck, Sharpe and Dohme) (Murray et al. 1971) in 0.5 ml water by stomach tube and were killed 48 to 72 hr later. Worm expulsion was complete within 48 hr after treatment with this drug and, apart from the group treated on Day 4 where a mean of 5 * 1 worms were present on Day 7, all groups were free of parasites. Five infected control rats given 0.5 ml water by stomach tube harbored 650 to 800 worms at the time of killing. An additional five rats infected with 4000 Ls were treated on Day 6 and were free of

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LOG ,. NO.TDL

8.0

8.5

TRANSFERRED

FIG. 7. A logarithmic plot of the relationship between the number of Day 10 TDL transferred and the increase in the proportion of goblet cells in recipients of 600 “normal” ( l ) or “damaged” Nippostronglus brdiensis (0). The infected control recipients of normal or damaged worms, and normal (A) and adoptively immunized uninfected (a) rats are also shown. Vertical bars record I+ SE.

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MILLER AND NAWA

parasites by Day 8 whereas the controls harbored approximately 2000 adult N. brasiliensis. Table I summarizes the changes in the proportion of goblet cells in the drug-treated rats. In all experiments the proportion of goblet cells returned to the levels seen in normal rats (Table I) or remained slightly depressed. An additional experiment in which normal rats were treated with the same dose of the anthelmintic drug established that the drug did not suppress goblet-cell differentiation (Table I). These results do not support the hypothesis that increased goblet-cell differentation is solely a repair phenomenon associated with worm expulsion. DISCUSSION

An increase in the proportion of goblet cells occurred in the villous epithelium during the immune expulsion of Nippostrongylus lwasiliensis from the rat intestine. This was true for several levels of infection and confirms a previous report where increased numbers of goblet cells were observed in the intestines of rats infected with N. hasiliensis (Wells 1963). The present experiments establish that adoptive immunization of infected rats with immune-TDL is also associated with an increase in the proportion of goblet cells. Furthermore, the extent of gobletcell differentiation in rats harbor parasites

Goblet Cell Itespollse in R&s Ilrfected with Sipp&ro?LgylUs brasilimsis alld Trcatrd with Thi:tbcrldazole ~--- - Goblet CdlS

Day treated ~4 6 8 6 0

Day killed

Infective dose of LI

Control -

7 8 10 8 2

1000 1 eon 1000 -1000

cells/lOUOabsorptive (mean + SE) Thiabendaeole treated

..~ 110f 8 206 i 26 217 f21 103 * 18 168f 8

l92i 216 i 178 * 117zt 173 i

4 14 15 8 12

established by larval infection was found Zo be a function of the number of TDL transferred. Such results suggest that intestinal immune responses which can be passively transferred with immune-TDL may, either directly or indirectly, exert some regulatory influence on mucosal epithelia. It was important to establish that increased goblet-cell differentiation was not simply a repair process associated with the loss of the parasites or with the release of stimulatory factors from the damaged worms. This appeared unlikely when, after clearing the parasites from the intestine with an anthehnintic drug, the proportion of goblet cells returned to the levels found in normal control rats. Nor was there any evidence that the increased proportions of goblet cells resulted from a decrease in the number of absorptive cells since, at the time of worm expulsion, villous architecture was fully restored. This was true in both normal and adoptively immunized rats and is in agreement with another report where an increase in villous size OCcurred at the time of worm expulsion (Miller and Jarrett 1971). Intestinal goblet-cell differentiation has been studied in a variety of experimental situations but the present results are, as far as we are aware, the first indication that this process may be regulated by circulating lymphocytes. However, it is not known if the lymphocytes themselves exert this effect or whether their products, such as antibody complex with antigen, or lymphokines are responsible for goblet-cell differentiation.

Nonspecific

stimuli

such as

whole-body irradiation alter the proportion of goblet cells in the gland cqpts and villi in normal rats (Van Dongen et al. 1976) and recent experiments in which chick embryonic duodenum was cultured in oitro have suggested that goblet-cell differentiation may be suppressed in viva by an unknown inhibitory factor. The latter study also demonstrated that thyroxine

Nippostrongyh

brasiliensis:

enhanced and hydrocortisone inhibited the in titro differentiation of goblet cells (Black and Moog 1977). There may, therefore, be several mechanisms by which mucosal immune responses affect epithelial differentiation. As yet there is no evidence that gobletcell differentiation is an essential step in the expulsion of Nippostrongylus bradiensi.s although the proportion of goblet cells invariably increased at the time of worm expulsion. This was true for larval infections and for the implantation of “damaged” worms. Conversely, after the implantation of “normal” worms no expulsion of the parasites occurred and there was no increase in the number of goblet cells. This would suggest that “normal” adult worms can suppress the rats mucosal response in normal recipients but that this suppressive effect is abolished in adoptively immunized recipients. The change in the staining properties of the goblet cells indicates that their accelerated differentiation is associated with a qualitative change in their content of mucin. Whether this is the result of an increased rate of production of mucin or whether it is a function of the increased rate of differentiation of the goblet cells remains to be determined. ACKNOWLEDGMENTS We thank Wendy Hughes and Judy Harriden for technical assistance and Anne Atkins for typing the manuscript.

REFERENCES BLACK, B. L., AND MOOG, F. 1977. Goblet cells in embryonic intestine: Accelerated differentiation in culture. Science 197, 368-370. DOBSON, C. 1966. Globule leucocytes, mucin and mucin cells in relation to Oesophagostomum columbianum infection in sheep. Austrnlian Journal of Science 28, 434. DOBSON, C. 1967. Changes in the protein content of the serum and intestinal mucus of sheep with reference to the histology of the gut and immunological response to Oesophago-

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stomum columbianum infections. Parasitology 57, 201-219. FERGUSON, A., AND JARRETT, E. E. E. 1975. Hypersensitivity reactions in the small intestine. 1. Thymus dependence of experimental “partial villous atrophy.” Gut 16, 114-117. FRICK, L. P., AND ACKERT, J. E. 1948. Further studies on duodenal mucus as a factor in age resistance of chickens to parasitism. Journal of Parasitology 34, 192-206. JARRETT, E. E. E., JARRETT, W. F. H., AND URQUHART, G. M. 1968. Quantitative studies on the kinetics of establishment and expulsion of intestinal nematode populations in susceptible and immune hosts. Parasitology 58, 625639. JENNINGS, F. W., MULLIGAN, W., AND URQUHART, G. M. 1963. Variables in x-ray “inactivation” of Nippostrongylus brasiliensis larvae. Experimental Parasitology 13, 367-373. KELLY, J. D., AND DINEEN, J. K. 1976. Prostaglandins in the gastrointestinal tract: Evidence for a role in worm expulsion. Australian Veterinary Journul 52, 391-397. MILLER, H. R. P., AND JARRETT, W. F. H. 1971. Immune reactions in mucous membranes. I. Intestinal mast cell response during helminth expulsion in the rat. Immunology 20, 277-288. MOWRY, R. W. 1963. The special value of methods that color both acidic and vicinal hydroxyl groups in the histochemical study of mucins. With revised directions for the colloidal iron stain, the use of alcian blue SGS, and their combinations with the periodic acid-Schiff reaction. Annals of the New York Academy of Sciences 106, 402-423. MURRAY, M., MILLER, H. R. P., SANFORD, J., AND JARFIETT, W. F. H. 1971. 5Hydroxytryptamine in intestinal immunological reactions. Its relationship to mast cell activity and worm expulsion in rats infected with Nippostrongylus brasiliensis. International Archives of Allergy and Applied Immunology 40, 236-247. NAWA, Y., AND MILLER, H. R. P. 1978. Protection against Nippostrongylus brasiliensis by adoptive immunization with immune thoracic duct lymphocytes. Cellular Immunology 37, 5160. OGILVIE, B. M., AND LOVE, R. J. 1974. COoperation between antibodies and cells in immunity to a nematode parasite. Transplantation Reviews 19, 147-168. OGILVIE, B. M., LOVE, R. J., JARRA, W., AND BROTNN, K. N. 1977. Nippostrongylus brasiliensis infection in rats. The celldar requirement for worm expulsion. Immunology 32, 521-528. SYMONS, L. E. A. 1965. Kinetics of the epithelial cells, and morphology of villi and crypts in

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the jejunum of the rat infected by the nematode Nippostrongylus brasiliensis. GastToenterology 49, 158-168. SYMONS, L. E. A. 1976. Scanning electron microscopy of the jejunum of the rat infected by the nematode Nippostrongylus brdiensis. lnternational Journal for Parasitology 6, 107-111. VAN DONGEN, J. M., KOOYP~IAN, J., AND VISSER,

NAWA

W. J. 1976. The influence of 400R x-irradiation on the number and the localization of mature and immature goblet cells and paneth cells in intestinal crypt and villus. Cell and Tissue Kinetics 9, 65-75. WELLS, P. D. 1963. Mucin-secreting cells in rats infected with Nippostrongylus brasiliensis. Experimental Parasitology 14, 15-22.

Nippostrongylus brasiliensis: intestinal goblet-cell response in adoptively immunized rats.

EXPERIMENTAL PARASITOLOGY 47, 81-90 (1979) Nipposfrongyhs brasiliensis: Response in Adoptively Intestinal Goblet-Cell Immunized Rats H. R. P. MILL...
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