INFECTION AND IMMUNITY, Aug. 1976, p. 509-521 Copyright © 1976 American Society for Microbiology
Vol. 14, No. 2 Printed in U.S.A.
Analysis of the Intradermal Response Against a Soluble Cercarial Antigenic Preparation from Schistosoma mansoni S. P. KATZ'* AND D. G. COLLEY Department of Microbiology, Vanderbilt University School of Medicine and the Veterans Administration Hospital, Nashville, Tennessee 37232
Received for publication 23 March 1976
Intradermal testing was performed with a soluble cercarial antigenic preparation (CAP) from Schistosoma mansoni cercariae in CBA/J mice multiply infected with S. mansoni or sensitized with CAP. Both an early (5-h) response and a late (24- to 48-h) reaction to CAP, as measured by increase in dermal thickness, was elicited after injection of antigen into the ears of either multiply infected (3X-75) or CAP-sensitized (CAP/complete Freund adjuvant [CFA]) mice. Histopathological examination showed that the early response was primarily vascular in nature and involved a polymorphonuclear cell infiltrate in and around dilated capillaries. The late reaction to CAP consisted of a perivascular cellular infiltrate of polymorphonuclear and mononuclear cell types. Passive transfer of 3X-75-infected or CAP/CFA-sensitized serum (0.4 ml) to normal mice conveyed the ability to mount an early (5-h) response to CAP which was marked histopathologically by a prominent polymorphonuclear cell infiltrate. The majority of the responsiveness in normal mice after administration of lymph node cells (40 x 106) from multiply infected or CAP/CFA-sensitized mice was observed 24 to 48 h after injection of CAP and was mononuclear in nature.
Immunodiagnosis of schistosomiasis by intradermal skin testing has been achieved by numerous investigators using a variety of antigenic preparations (8, 10, 17). Analysis of intradermal reactivity during the course of experimental infection has also been utilized to assess the development of immune capabilities to schistosomal antigens (2). In addition, dermal activity has long been associated with those events surrounding initiation of schistosomiasis by cercarial penetration (4, 15, 19), and the inflammatory response noted in the skin of some animals has been implicated as contributing to the mechanism of resistance to initial infection with the disease (13). Intensified tissue reactivity to invading cercariae has been described also in a number of experimental hosts either harboring a preceding infection (9, 12, 14) or previously challenged with irradiated cercariae (6, 7). Analysis of the immunological basis of the dermal reactivity against cercariae as well as cercarial antigens by mice with a mature schistosomal infection has been reported (3). The present study examines the immunological reactivity elicited by a soluble cercarial antigenic preparation (CAP) in mice either multiply infected with cercariae or sensitized with ' Present address: Department of Pathology (Immunology), Yale University School of Medicine, New Haven, CN 06510.
CAP. The induction and elicitation of immune capabilities by CAP in animals so infected or exposed has been previously demonstrated using in vitro methods of analysis (11). Anti-CAP intradermal reactivity was assessed by comparing the increase in ear thickness observed in either multiply infected or CAP-sensitized mice after injection of CAP. These tissues were also examined histopathologically. Further defmition of these observed immunological responses was made by passive transfer ofeither serum or lymphoid cells from infected or sensitized mice to normal mice, followed by skin testing with CAP. MATERUILS AND METHODS Infection and sensitization. All animals used were male, inbred mice of the strain CBA/J (Jackson Memorial Laboratories, Bar Harbor, Me.), 6 to 8 weeks of age. Infection of these animals was accomplished by subcutaneous inoculation with 75 cercariae of a Puerto Rican strain of S. mansoni, followed by two injections at weekly intervals of 75 cercariae (3X-75). Mice were sensitized by subcutaneous injection of 20 ,ug of protein of a soluble CAP emulsified in complete Freund adjuvant (CFA) and two sequential weekly injections of 20 gg of protein of CAP alone (CAP/CFA). CAP was prepared as previously described using lyophilized cercariae homogenized in Dulbecco balanced salt solution at a concentration of 20,000 cercariae per ml (11). Intradermal reactivity. Intradermal reactivity to
509
510
INFECT. IMMUN.
KATZ AND COLLEY
CAP was assayed 3 weeks after initial infection with cercarial extracts have been demonstrated to S. mansoni cercariae or sensitization with CAP. generate (5). Normal mice, neither infected with cercariae nor Histological examination of mouse ears sensitized with CAP, were used as controls. The after injection of CAP. Histopathological analantigen preparation, 0.03 ml (15 ug total of protein) yses indicated that the responses generated of CAP, was administered intradermally into the pinna of mouse ears using glass disposable syringes within 3X-75 and CAP/CFA ears challenged (Becton, Dickson and Co., Rutherford, N.J.) and 27- with CAP over the course of the study were gauge needles (1, 2). The thickness of the individual comparable. Early (5-h) biopsies from these anears was determined before injection of CAP and 5, imals were edematous and contained polymor14, 24, 48, 72, and 96 h later with an engineer's phonuclear (PMN) leukocyte infiltrates which micrometer. The reactivity is expressed as the mean were seen in and around dilated capillaries increase in thickness in 10-3 cm at these times in (Fig. 2). Normal mice likewise exhibited edema relationship to time zero ± the standard error of the and PMN cell infiltration 5 h after intradermal mean. Passive transfer of lymph node cells and serum. exposure to CAP (Fig. 3). However, the intenThe ability of lymph node (LN) cells and serum from sity of the reaction in tissue from these animals 3X-75 and CAP/CFA-sensitized mice to passively was not as pronounced as that in 3X-75- and transfer anti-CAP skin reactivity to normal mice CAP/CFA-challenged mice and, furthermore, was investigated. LN cells were obtained from donor did not progress with time. However, 14 h after mice by gentle teasing in RPMI-1640 medium. The injection of CAP, microscopic examination of cells were counted and adjusted to a concentration of ears from 3X-75- and CAP/CFA-infected mice 100 x 106 per ml. Either LN cells or pooled serum revealed perivascular cuffing of cells predomifrom donor mice were injected intravenously into nantly mononuclear (MN) in nature (Fig. 4). normal mice via the lateral tail vein in 0.4-ml vol- This response was elevated by 24 h and conumes. Eighteen hours after passive transfer, recipient mice were analyzed for intradermal reactivity to CAP. Tissue histology. At various times after skin testing with CAP, ears were surgically removed and immediately fixed in 10% neutral buffered formalin. 14 Tissues were processed in an Autotechnicon (Tech74) nicon Instruments, Tarrytown, N.Y.) before embed74 (64 ding in Paraplast (Sherwood Medical Industries, St. 12Louis, Mo.) for sectioning. Sections 5 ,um thick were (4 cut, stained with hematoxylin and eosin, and examined microscopically. (20) Statistical analysis. Student's t test was used Z 101 I to determine the significance of the differences be~~~~~~~~~(68(' tween the mean values obtained from experimen- z tally treated groups of mice and those results obw N (~~~~~~~~~ tained from normal mice challenged with CAP. Val- ~~~~~~~~~~~~ ~~~~~~~~~~(24) ues of P < 0.05 were considered significant. ~6 \ Z _92 -
(74)
RESULTS Development of ear thickening in response to CAP. Analysis of anti-CAP intradermal reactivity was done by measuring the increase in ear thickness at various times after injection of CAP into the ears of normal, multiply infected (3X-75) and CAP-sensitized (CAP/CFA) mice. The mean increase in thickness + standard error of the mean at these times is presented in Fig. 1. Both 3X-75-infected and CAP/ CFA-sensitized animals exhibited early as well as late responsiveness to CAP. All ofthe experimental points were highly significant (P < 0.0001) when compared with the corresponding responses of normal animals to CAP injection. The early (5-h) increase in ear thickness of normal mice in response to CAP may be attributed to an anaphylatoxin activity that some
5
14
24
48
72
96
HOURS AFTER CHALLENGE INJECTION
FIG. 1. Intradermal responsiveness to CAP. The increase in ear thickness of normal mice (0), multiply infected (3X-75) mice (A), and CAP-sensitized (CAPICFA) mice (-) at 5,14, 24, 48, 72, and 96 h after intradermal challenge with CAP. Intradermal skin testing was done 3 weeks after initial infection or sensitization. The results are expressed as the mean increase in 10-3 cm + standard error of the mean. The number of determinations which contribute to each mean is indicated in parentheses.
VOL. 14, 1976
INTRADERMAL REACTIVITY TO S. MANSONI CAP
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FIG. 2. Dermal reactions in the ear of a multiply infected (3X-75) mouse 5 h after challenge injection of CAP. Dilated dermal capillaries containing many PMN cells (hematoxylin and eosin, magnification x960).
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FIG. 3. Normal mouse ear 5 h after injection of CAP. Slight edema with dilated capillaries containing a few PMN cells (hematoxylin and eosin, magnification x384).
sisted of a mixed PMN and MN cellular infiltrate which was found throughout the dermis (Fig. 5). At this time, sections of tissue from normal CAP-injected mice lacked any evidence of responsiveness to CAP (Fig. 6). The mixed cellular responsiveness exhibited by 3X-75-infected and CAP/CFA-sensitized animals to CAP persisted, although with less intensity, until termination of the experiment at 96 h. Passive transfer of intradermal reactivity to CAP. Separation of the early and late responses to CAP noted in multiply infected and CAP-sensitized mice was accomplished by the passive transfer of either LN or sera from these animals to normal recipients before skin testing with CAP. These experiments revealed that the passive transfer of serum conveyed to normal mice a nominal ability to mount an early 5h anti-CAP response, whereas the late developing capacity of reactivity was transferred by the LN cell population of previously infected or sensitized mice (Fig. 7 and 8). Although the degree of reactivity passively transferred was not great, statistical comparison of the experimental data with the normal reactivity revealed quantitative and kinetic differences between
recipients of serum and those receiving LN cells. The activity exhibited by mice receiving 3X-75 serum was significant only at 5 and 14 h (P 0.05), whereas that of LN cell recipients was not significant until 14 h and remained significant thereafter (P . 0.05). Transfer of CAP/CFA LN cells resulted in anti-CAP activity in normal recipients, which was significant when analyzed at 14, 24, 48, and 72 h after injection of CAP (P 0.05), but not at 5 h postchallenge. CAP/CFA serum recipients displayed significant anti-CAP reactivity at 5, 14, and 24 h. Histological analysis of anti-CAP activity after passive transfer. Examination of tissue from mice passively administered serum obtained from 3X-75-infected or CAP/CFA-sensitized animals revealed that challenge with CAP induced edema and a PMN infiltrate which was seen in and around dilated capillaries by 5 h after challenge (Fig. 9). At 14 h such ears were still edematous and had dilated capillaries containing a few PMN cells, as well as an increased mixed dermal infiltrate consisting of MN and predominantly PMN cell types (Fig. 10). By 24 h the reaction was not as intense, -
-
VOL. 14, 1976
INTRADERMAL REACTIVITY TO S. MANSONI CAP
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FIG. 4. Multiply infected (3X-75) mouse ear 14 h after CAP injection. Perivascular cuffing of a mixed cellular infiltrate (hematoxylin and eosin, magnification x384).
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although PMN cells were still evident in the capillaries ofthe ear (Fig. 11). Prior injection of 3X-75 or CAP/CFA LN cells conveyed to normal mice the ability to respond to an intradermal challenge with CAP with a late (24- to 48-h), predominantly MN response (Fig. 12). Although a moderate early (5-h) response to CAP marked by edema and the presence of PMN leucocytes was noted in these animals, this reactivity was analogous to that seen in normal mice challenged with CAP.
DISCUSSION Previous experiments by several investigators have suggested that dermal reactivity against cercariae, those organisms that initiate infection with schistosomiasis, may be of importance in host resistance to reinfection with
'N~ ~ ~ ~ ~ ~ ~T
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the disease (7, 9, 12, 14). Such responsiveness contribute also to the ability of some experimental hosts to resist a primary schistosomal infection. This is suggested by the work of von Lichtenberg et al. (13), which has shown that some animals which are naturally resistant to schistosomal infection exhibit vigorous dermal reactivity in response to initial cercarial exposure. Analysis of the immunological nature of secondary dermal responsiveness directed against cercariae or a cercarial extract has been done previously in mice possessing a sexually mature infection of S. mansoni (3) and in monkeys previously challenged with irradiated S. japonicum cercariae (6). The present studies have examined the immunological nature of the intradermal skin reactivity elicited in inbred mice by a challenge may
INTRADERMAL REACTIVITY TO S. MANSONI CAP
VOL. 14, 1976
-
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e~~~-
--
0,
Vol. 14, No. 2 Printed in U.S.A.
Analysis of the Intradermal Response Against a Soluble Cercarial Antigenic Preparation from Schistosoma mansoni S. P. KATZ'* AND D. G. COLLEY Department of Microbiology, Vanderbilt University School of Medicine and the Veterans Administration Hospital, Nashville, Tennessee 37232
Received for publication 23 March 1976
Intradermal testing was performed with a soluble cercarial antigenic preparation (CAP) from Schistosoma mansoni cercariae in CBA/J mice multiply infected with S. mansoni or sensitized with CAP. Both an early (5-h) response and a late (24- to 48-h) reaction to CAP, as measured by increase in dermal thickness, was elicited after injection of antigen into the ears of either multiply infected (3X-75) or CAP-sensitized (CAP/complete Freund adjuvant [CFA]) mice. Histopathological examination showed that the early response was primarily vascular in nature and involved a polymorphonuclear cell infiltrate in and around dilated capillaries. The late reaction to CAP consisted of a perivascular cellular infiltrate of polymorphonuclear and mononuclear cell types. Passive transfer of 3X-75-infected or CAP/CFA-sensitized serum (0.4 ml) to normal mice conveyed the ability to mount an early (5-h) response to CAP which was marked histopathologically by a prominent polymorphonuclear cell infiltrate. The majority of the responsiveness in normal mice after administration of lymph node cells (40 x 106) from multiply infected or CAP/CFA-sensitized mice was observed 24 to 48 h after injection of CAP and was mononuclear in nature.
Immunodiagnosis of schistosomiasis by intradermal skin testing has been achieved by numerous investigators using a variety of antigenic preparations (8, 10, 17). Analysis of intradermal reactivity during the course of experimental infection has also been utilized to assess the development of immune capabilities to schistosomal antigens (2). In addition, dermal activity has long been associated with those events surrounding initiation of schistosomiasis by cercarial penetration (4, 15, 19), and the inflammatory response noted in the skin of some animals has been implicated as contributing to the mechanism of resistance to initial infection with the disease (13). Intensified tissue reactivity to invading cercariae has been described also in a number of experimental hosts either harboring a preceding infection (9, 12, 14) or previously challenged with irradiated cercariae (6, 7). Analysis of the immunological basis of the dermal reactivity against cercariae as well as cercarial antigens by mice with a mature schistosomal infection has been reported (3). The present study examines the immunological reactivity elicited by a soluble cercarial antigenic preparation (CAP) in mice either multiply infected with cercariae or sensitized with ' Present address: Department of Pathology (Immunology), Yale University School of Medicine, New Haven, CN 06510.
CAP. The induction and elicitation of immune capabilities by CAP in animals so infected or exposed has been previously demonstrated using in vitro methods of analysis (11). Anti-CAP intradermal reactivity was assessed by comparing the increase in ear thickness observed in either multiply infected or CAP-sensitized mice after injection of CAP. These tissues were also examined histopathologically. Further defmition of these observed immunological responses was made by passive transfer ofeither serum or lymphoid cells from infected or sensitized mice to normal mice, followed by skin testing with CAP. MATERUILS AND METHODS Infection and sensitization. All animals used were male, inbred mice of the strain CBA/J (Jackson Memorial Laboratories, Bar Harbor, Me.), 6 to 8 weeks of age. Infection of these animals was accomplished by subcutaneous inoculation with 75 cercariae of a Puerto Rican strain of S. mansoni, followed by two injections at weekly intervals of 75 cercariae (3X-75). Mice were sensitized by subcutaneous injection of 20 ,ug of protein of a soluble CAP emulsified in complete Freund adjuvant (CFA) and two sequential weekly injections of 20 gg of protein of CAP alone (CAP/CFA). CAP was prepared as previously described using lyophilized cercariae homogenized in Dulbecco balanced salt solution at a concentration of 20,000 cercariae per ml (11). Intradermal reactivity. Intradermal reactivity to
509
510
INFECT. IMMUN.
KATZ AND COLLEY
CAP was assayed 3 weeks after initial infection with cercarial extracts have been demonstrated to S. mansoni cercariae or sensitization with CAP. generate (5). Normal mice, neither infected with cercariae nor Histological examination of mouse ears sensitized with CAP, were used as controls. The after injection of CAP. Histopathological analantigen preparation, 0.03 ml (15 ug total of protein) yses indicated that the responses generated of CAP, was administered intradermally into the pinna of mouse ears using glass disposable syringes within 3X-75 and CAP/CFA ears challenged (Becton, Dickson and Co., Rutherford, N.J.) and 27- with CAP over the course of the study were gauge needles (1, 2). The thickness of the individual comparable. Early (5-h) biopsies from these anears was determined before injection of CAP and 5, imals were edematous and contained polymor14, 24, 48, 72, and 96 h later with an engineer's phonuclear (PMN) leukocyte infiltrates which micrometer. The reactivity is expressed as the mean were seen in and around dilated capillaries increase in thickness in 10-3 cm at these times in (Fig. 2). Normal mice likewise exhibited edema relationship to time zero ± the standard error of the and PMN cell infiltration 5 h after intradermal mean. Passive transfer of lymph node cells and serum. exposure to CAP (Fig. 3). However, the intenThe ability of lymph node (LN) cells and serum from sity of the reaction in tissue from these animals 3X-75 and CAP/CFA-sensitized mice to passively was not as pronounced as that in 3X-75- and transfer anti-CAP skin reactivity to normal mice CAP/CFA-challenged mice and, furthermore, was investigated. LN cells were obtained from donor did not progress with time. However, 14 h after mice by gentle teasing in RPMI-1640 medium. The injection of CAP, microscopic examination of cells were counted and adjusted to a concentration of ears from 3X-75- and CAP/CFA-infected mice 100 x 106 per ml. Either LN cells or pooled serum revealed perivascular cuffing of cells predomifrom donor mice were injected intravenously into nantly mononuclear (MN) in nature (Fig. 4). normal mice via the lateral tail vein in 0.4-ml vol- This response was elevated by 24 h and conumes. Eighteen hours after passive transfer, recipient mice were analyzed for intradermal reactivity to CAP. Tissue histology. At various times after skin testing with CAP, ears were surgically removed and immediately fixed in 10% neutral buffered formalin. 14 Tissues were processed in an Autotechnicon (Tech74) nicon Instruments, Tarrytown, N.Y.) before embed74 (64 ding in Paraplast (Sherwood Medical Industries, St. 12Louis, Mo.) for sectioning. Sections 5 ,um thick were (4 cut, stained with hematoxylin and eosin, and examined microscopically. (20) Statistical analysis. Student's t test was used Z 101 I to determine the significance of the differences be~~~~~~~~~(68(' tween the mean values obtained from experimen- z tally treated groups of mice and those results obw N (~~~~~~~~~ tained from normal mice challenged with CAP. Val- ~~~~~~~~~~~~ ~~~~~~~~~~(24) ues of P < 0.05 were considered significant. ~6 \ Z _92 -
(74)
RESULTS Development of ear thickening in response to CAP. Analysis of anti-CAP intradermal reactivity was done by measuring the increase in ear thickness at various times after injection of CAP into the ears of normal, multiply infected (3X-75) and CAP-sensitized (CAP/CFA) mice. The mean increase in thickness + standard error of the mean at these times is presented in Fig. 1. Both 3X-75-infected and CAP/ CFA-sensitized animals exhibited early as well as late responsiveness to CAP. All ofthe experimental points were highly significant (P < 0.0001) when compared with the corresponding responses of normal animals to CAP injection. The early (5-h) increase in ear thickness of normal mice in response to CAP may be attributed to an anaphylatoxin activity that some
5
14
24
48
72
96
HOURS AFTER CHALLENGE INJECTION
FIG. 1. Intradermal responsiveness to CAP. The increase in ear thickness of normal mice (0), multiply infected (3X-75) mice (A), and CAP-sensitized (CAPICFA) mice (-) at 5,14, 24, 48, 72, and 96 h after intradermal challenge with CAP. Intradermal skin testing was done 3 weeks after initial infection or sensitization. The results are expressed as the mean increase in 10-3 cm + standard error of the mean. The number of determinations which contribute to each mean is indicated in parentheses.
VOL. 14, 1976
INTRADERMAL REACTIVITY TO S. MANSONI CAP
"I*
4 44
!
-.
511
a.
'
FIG. 2. Dermal reactions in the ear of a multiply infected (3X-75) mouse 5 h after challenge injection of CAP. Dilated dermal capillaries containing many PMN cells (hematoxylin and eosin, magnification x960).
512
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FIG. 3. Normal mouse ear 5 h after injection of CAP. Slight edema with dilated capillaries containing a few PMN cells (hematoxylin and eosin, magnification x384).
sisted of a mixed PMN and MN cellular infiltrate which was found throughout the dermis (Fig. 5). At this time, sections of tissue from normal CAP-injected mice lacked any evidence of responsiveness to CAP (Fig. 6). The mixed cellular responsiveness exhibited by 3X-75-infected and CAP/CFA-sensitized animals to CAP persisted, although with less intensity, until termination of the experiment at 96 h. Passive transfer of intradermal reactivity to CAP. Separation of the early and late responses to CAP noted in multiply infected and CAP-sensitized mice was accomplished by the passive transfer of either LN or sera from these animals to normal recipients before skin testing with CAP. These experiments revealed that the passive transfer of serum conveyed to normal mice a nominal ability to mount an early 5h anti-CAP response, whereas the late developing capacity of reactivity was transferred by the LN cell population of previously infected or sensitized mice (Fig. 7 and 8). Although the degree of reactivity passively transferred was not great, statistical comparison of the experimental data with the normal reactivity revealed quantitative and kinetic differences between
recipients of serum and those receiving LN cells. The activity exhibited by mice receiving 3X-75 serum was significant only at 5 and 14 h (P 0.05), whereas that of LN cell recipients was not significant until 14 h and remained significant thereafter (P . 0.05). Transfer of CAP/CFA LN cells resulted in anti-CAP activity in normal recipients, which was significant when analyzed at 14, 24, 48, and 72 h after injection of CAP (P 0.05), but not at 5 h postchallenge. CAP/CFA serum recipients displayed significant anti-CAP reactivity at 5, 14, and 24 h. Histological analysis of anti-CAP activity after passive transfer. Examination of tissue from mice passively administered serum obtained from 3X-75-infected or CAP/CFA-sensitized animals revealed that challenge with CAP induced edema and a PMN infiltrate which was seen in and around dilated capillaries by 5 h after challenge (Fig. 9). At 14 h such ears were still edematous and had dilated capillaries containing a few PMN cells, as well as an increased mixed dermal infiltrate consisting of MN and predominantly PMN cell types (Fig. 10). By 24 h the reaction was not as intense, -
-
VOL. 14, 1976
INTRADERMAL REACTIVITY TO S. MANSONI CAP
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FIG. 4. Multiply infected (3X-75) mouse ear 14 h after CAP injection. Perivascular cuffing of a mixed cellular infiltrate (hematoxylin and eosin, magnification x384).
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although PMN cells were still evident in the capillaries ofthe ear (Fig. 11). Prior injection of 3X-75 or CAP/CFA LN cells conveyed to normal mice the ability to respond to an intradermal challenge with CAP with a late (24- to 48-h), predominantly MN response (Fig. 12). Although a moderate early (5-h) response to CAP marked by edema and the presence of PMN leucocytes was noted in these animals, this reactivity was analogous to that seen in normal mice challenged with CAP.
DISCUSSION Previous experiments by several investigators have suggested that dermal reactivity against cercariae, those organisms that initiate infection with schistosomiasis, may be of importance in host resistance to reinfection with
'N~ ~ ~ ~ ~ ~ ~T
oue2t haternecinofCP
n
osn
agiicto x8)
the disease (7, 9, 12, 14). Such responsiveness contribute also to the ability of some experimental hosts to resist a primary schistosomal infection. This is suggested by the work of von Lichtenberg et al. (13), which has shown that some animals which are naturally resistant to schistosomal infection exhibit vigorous dermal reactivity in response to initial cercarial exposure. Analysis of the immunological nature of secondary dermal responsiveness directed against cercariae or a cercarial extract has been done previously in mice possessing a sexually mature infection of S. mansoni (3) and in monkeys previously challenged with irradiated S. japonicum cercariae (6). The present studies have examined the immunological nature of the intradermal skin reactivity elicited in inbred mice by a challenge may
INTRADERMAL REACTIVITY TO S. MANSONI CAP
VOL. 14, 1976
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