INFECTION AND IMMUNITY, Aug. 1977, p. 439-446
Vol. 17, No. 2
Copyright © 1977 American Society for Microbiology
Printed in U.S.A.
Development of Delayed-Type Hypersensitivity During Mycobacterium lepraemurium Infection in Mice L. W. POULTER* AND M. J. LEFFORD Trudeau Institute, Inc., Saranac Lake, New York 12983
Received for publication 16 February 1977
Various preparations of Mycobacterium lepraemurium were used to elicit delayed-type hypersensitivity in the footpad of mice infected with this organism. With a sonicated preparation of the mycobacterium, a significant increase in footpad swelling was elicited in mice infected with M. lepraemurium 5 weeks previously, but not in BCG-infected animals or uninfected controls. This footpad reaction was shown to peak at 24 h and to be associated with an infiltration of mononuclear cells. The kinetics of footpad swelling, its association with lymphoproliferation, and its dependence on T lymphocytes were each examined. The results support the hypothesis that this is a delayed-type hypersensitivity reaction. The ability to transfer this reactivity to normal mice with cells but not serum offers further confirmation that this hypersensitivity is dependent on cellmediated immunological mechanisms rather than humoral antibody. The relevance of this to the study of the immunological response of mice to murine leprosy is discussed. In human leprosy, there is a close correlation between resistance to the mycobacterium and the development of cell-mediated immunity (CMI) (6, 15). After Mycobacterium lepraemurium infection in mice, it has been demonstrated that the host generates some degree of resistance to reinfection (1). As the inflammatory reaction that develops at the site of M. lepraemurium infection has been shown to be similar to a delayed-type hypersensitivity (DTH) reaction (3), there is circumstantial evidence of the induction of CMI. The relationship between CMI and resistance in other bacterial infections is well established (9, 10, 18), and there is some evidence that similar mechanisms are responsible for the resistance generated to M. lepraemurium (3). As yet, however, no definitive proof of specific CMI developing as a result of M. lepraemurium infection has been reported. Although the level of resistance to secondary challenge can be determined at varying times during the primary infection, the nature of such acquired immunity remains uncertain. In any case, the experimental protocol required to demonstrate resistance is such that the immunological status of the host may well have changed during this time span. Thus, methods of demonstrating resistance alone do not lend themselves well to critical analysis of the immunological mechanisms involved. Since there is some evidence to support the proposition that CMI develops during M. kepraemurium infec-
tion (3), it was thought that this might be detectable by the expression of a DTH response at the site of an intradermal challenge (8, 12). It must be emphasized that the term CMI is used in the present paper only to describe the presence of acquired cell-mediated immunological mechanisms and not protective immunity. Although no correlation with protective immunity was determined, the demonstration of DTH in an M. lepraemurium-infected animal illustrates the development of a cell-mediated immunological response that can be easily quantitated 24 to 48 h after challenge. Besides confirming the development of CMI, therefore, this reaction could be used to analyze certain changes in immunological status at precise time points during this chronic infection. This paper describes the successful demonstration of DTH after M. lepraemurium infection, as well as the kinetics and characteristics of this reaction. MATERIALS AND METHODS Animals. Inbred hybrid mice of the B6D2 F1 strain (C7BI/6 x DBA/2) (parental strains from Jackson Laboratories, Bar Harbor, Me.) of either sex, 6 to 8 weeks old, were used. These mice are relatively resistant to M. lepraemurium, having a mean survival time of 32 weeks after the intravenous injection of 108 viable organisms. For some experiments, mice were thymectomized within 24 h of birth and used at 8 weeks of age. Mycobacteria. M. lepraemurium (Hawaii strain) was maintained by repeated passage in susceptible 439
440
POULTER AND LEFFORD
mice. Purified suspensions were obtained from the livers and spleens by using the method of Draper (4) and stored at -70°C. Inocula of known size were prepared by appropriate dilution of the stock suspension after making counts of acid-fast bacilli in stained smears (14). Heat-killed (HK) M. lepraemurium were prepared by heating a suspension of live organisms at 100°C for 20 min. Sonicated (Son) M. lepraemurium was prepared by sonicating viable suspensions of cells for 15 min with a sonicator (Bronwill Biosonic IV, VWR Scientific) set at 80% of maximum. HK M. kepraemurium was also sonicated in an identical manner for use in some experiments. Frozen and thawed (FT) M. lepraemurium was prepared by freezing viable cells to -70°C for 2 h six times and thawing to room temperature for 30 min between each freeze. In some experiments, BCG Pasteur (TMC 1011) grown at this Institute was used. Primary infection. Normal mice were infected with an appropriate number of mycobacteria contained in 0.04 ml of sterile saline, injected subcutaneously (s.c.) into the left hind footpad (LHFP). Elicitation of DTH. At varying times after primary infection with M. lepraemurium or BCG, animals were injected s.c. in the right hind footpad (RHFP) with either living, HK, Son, or FT preparations of M. lepraemurium. These suspensions were injected in a volume of 0.04 ml of sterile saline. In all cases, the thickness of the RHFP was measured with dial-gauge calipers before injection and at varying times thereafter. Lymphocyte proliferation. Cellular proliferation in the draining popliteal lymph node (PLN) was measured weekly after M. lepraemurium infection. Twenty microcuries of tritiated thymidine (specific activity, 3 Ci/mmol; New England Nuclear Corp., Boston, Mass.) was injected intravenously (i.v.), and 30 min later the draining and contralateral PLN were removed, homogenized, and washed with cold trichloroacetic acid, and the deoxyribonucleic acid was extracted with hot trichloroacetic acid as described previously (17). Radioactivity was measured in a liquid scintillation counter and expressed as counts per minute. Cell transfer. Mice infected 5 weeks previously with 108 M. lepraemurium (s.c. in LHFP) were injected intraperitoneally with 2 ml of 5% casein. Three days later, the mice were killed and the peritoneal exudate cells (PECs) were washed out with phosphate-buffered saline (PBS) containing 1% fetal calf serum and heparin (10 U/ml). The cells from 50 donor mice were pooled, and adherent cells were removed by incubating the cell suspension twice in large plastic petri dishes for 1 h at 37°C in an atmosphere of 5% CO2. After each incubation, the nonadherent cells were removed by vigorous washing with fresh PBS solution. The remaining cells were spun at 200 x g for 15 min and suspended at a concentration of 2 x 108/ml. For controls, PECs were collected from normal mice in an identical manner. Viability was determined by trypan blue exclusion and was found to be consistently above 90%. Systemic cell transfer was performed by the injection of 108 PECs in 0.5 ml of PBS i.v. into normal
INFECT. IMMUN.
recipient mice. These mice were challenged with 108 equivalents of Son M. lepraemurium in the LHFP either immediately after cell transfer or 24 h later. Local cell transfer was performed by the method of Metaxas and Metaxas-Buehler (11). Nonadherent PECs, 107, from infected or normal animals were mixed with 108 equivalents of Son M. lepraemurium in 0.05 ml and injected into the LHFP of normal recipient mice. Any increase in footpad thickness was measued 6, 12, 18, 24, 48, and 72 h later. Control groups of mice were injected with antigen alone or cells alone. As a positive control, a group of animals infected at the same time as the donor mice was challenged with 108 equivalents of Son M. lepraemurium to confirm that the cell donors had developed the capacity to express DTH. Serum transfer. Blood was collected aseptically by cardiac puncture from normal or M. lepraemurium-infected mice (108 M. lepraemurium, 5 weeks previously). The serum was separated out, and groups of normal recipients were infected with 0.5 ml i.v. These animals were challenged in the LHFP with 108 equivalents of Son M. lepraemurium immediately, and footpad swelling was measured 24 h later. As a direct comparison to the local cell transfer (see above), local serum transfers were performed. Serum, 0.05 ml, from infected or normal animals was mixed with 108 equivalents of Son M. lepraemurium and injected s.c. into the LHFP of normal mice. Footpad thickness was measured 6, 12, 18, 24, 48, and 72 h later. Control groups of mice received serum alone. Histology. Specimens for histological examination were fixed in phosphate-buffered 10% Formalin for 24 h, dehydrated in alcohol, and embedded in hydroxyethyl methacrylate. Thin sections (1 tLm) were stained with hematoxylin and eosin. Statistical analysis. There were at least five animals in each experimental group. Results are expressed as the mean and standard deviation within these groups, and statistical significance was determined with Student's t test for nonpaired data.
RESULTS Response to parasite and elicitation of DTH. Mice were infected with 108 M. lepraemurium into the LHFP, and both hind feet were measured at weekly intervals. After 3 weeks a detectable increase in the thickness of the LHFP was seen, which then enlarged relatively rapidly between weeks 3 and 6 (Fig. 1). As this increase is thought to be associated with an immunological response (3), 5 weeks after infection was selected as an appropriate time for the demonstration of DTH. Mice were infected with either 108 M. lepraemurium or 106 BCG in the LHFP, and 5 weeks later groups of these mice and normal controls were challenged in the RHFP with either 108 live M. lepraemurium or equivalent concentrations of HK, Son, or FT M. lepraemurium. The results (Fig. 2) showed that challenge with live, Son, or FT M.
VOL. 17, 1977
E
DTH DURING MURINE LEPROSY
Son M. lepraemurium. The RHFP were measured before testing and 6, 12, 24, 48, and 72 h later. In both infected and control mice, there was a rapid swelling of the RHFP, which subsided by 12 h (Fig. 3). This was followed, however, by a delayed reaction, which peaked 24 h after challenge. At this time, the feet of the infected mice were significantly thicker than those of the controls (P < 0.001). At 48 h, the reaction had subsided and, although still greater than in the control mice, the difference was not statistically significant. The reaction had almost totally disappeared by 72 h. The use of a concentration of 108 Son M.
10
z u v
0
a
.....
8 0 61
w
.
2 Z
i va,
2
4
6
10 11
8 I
Weeks
FIG. 1. Development of increas ed footpad thckness in B6D2F1 mice after the s.c. injection ofl1o8 M. lepraemurium at this site. Ten a nimals were measured before infection and each weeek thereafter. The mean increase in thickness is reco rded.
-
0*8
- 07
04
LIVE
Son
HK
MLM
FT
CHALLENGE
FIG. 2. Comparison of the abili .onm.ur,l rn,tinnluecllul-tull t(RAT LY.LiLIJ WU CsIC-ss Ur Z- -a Mp footpad reaction in the RHFP of mice infected 5 weeks previously in the LHFP with 108 M. lepraemurium (open bars). The eliciting dose was either 108 live M. lepraemurium or 108 equivalents ofHK, Son, or FT M. lepraemurium. The solid bars represent the reaction in normal mice, and the striped bars represent the reaction in animals infected with 10 BCG in the LHFP 5 weeks previously. Means of five mice ± n 1LMf . Ur UL-CUIsa U/
441
tyM) ofariout p4rh-
standard deviation.
lepraemurium was justified by a dose-response experiment, the results of which appear in Fig. 4. Mice infected with M. lepraemurium 5 weeks previously were tested with graded concentrations of M. lepraemurium. A dose equivalent to 10W M. lepraemurium elicited the largest (P < 0.001) increase in the footpad thickness in the infected mice without producing a nonspecific reaction in normal mice. When the dose was raised to 109 M. lepraemurium equivalents, there was substantial nonspecific swelling in the control animals. It should be noted here, however, that different batches of the Son preparation caused peak reactions at different concentrations. This result probably derives from the fact that neither the absolute number nor the viability of M. lepraemurium in a given preparation can be accurately determined. It is, therefore, necessary to assay each new preparation of sonic extract before experimental use. Histology. Footpad tissue of M. lepraemuE
09
0-8 z
07
v
0
0-6
x
0*5
0 0
0-4
lepraemurium preparations caused a signifi- z 03 cant 24-h increase in footpad thickness in the 0-2 infected animals but not in the normal controls; however, the HK M. kepraemurium failed to 0-1 elicit such a response. The use of live M. leprae72 48 24 6 12 murium for challenge, however, also caused a HOURS positive response in BCG-infected animals. The Son M. lepraemurium preparation was thereFIG. 3. Kinetics of footpad swelling in the RHFP fore chosen for use in subsequent experiments, after s.c. injection of 108 equivalents of Son M. since it produced the largest specific response. lepraemurium in animals infected with 108 M. leKinetics of the footpad response. Mice that praemurium in the LHFP 5 weeks previously (solid S c
v
had been infected in the LHFP with 108 M.
lepraemurium 5 weeks previously and normal controls were challenged in the RHFP with 108
line). The broken line represents the response in uninfected animals. Means of five mice + standard deviation.
442
INFECT. IMMUN.
POULTER AND LEFFORD
E-
E E
z
0-8
I
o 0 at
0-6
/
0L O 0-4 0
cm
[3H]thymidine. It was found (Fig. 8) that a significant increase in deoxyribonucleic acid synthesis could be detected 1 week after infection (P
Vol. 17, No. 2
Copyright © 1977 American Society for Microbiology
Printed in U.S.A.
Development of Delayed-Type Hypersensitivity During Mycobacterium lepraemurium Infection in Mice L. W. POULTER* AND M. J. LEFFORD Trudeau Institute, Inc., Saranac Lake, New York 12983
Received for publication 16 February 1977
Various preparations of Mycobacterium lepraemurium were used to elicit delayed-type hypersensitivity in the footpad of mice infected with this organism. With a sonicated preparation of the mycobacterium, a significant increase in footpad swelling was elicited in mice infected with M. lepraemurium 5 weeks previously, but not in BCG-infected animals or uninfected controls. This footpad reaction was shown to peak at 24 h and to be associated with an infiltration of mononuclear cells. The kinetics of footpad swelling, its association with lymphoproliferation, and its dependence on T lymphocytes were each examined. The results support the hypothesis that this is a delayed-type hypersensitivity reaction. The ability to transfer this reactivity to normal mice with cells but not serum offers further confirmation that this hypersensitivity is dependent on cellmediated immunological mechanisms rather than humoral antibody. The relevance of this to the study of the immunological response of mice to murine leprosy is discussed. In human leprosy, there is a close correlation between resistance to the mycobacterium and the development of cell-mediated immunity (CMI) (6, 15). After Mycobacterium lepraemurium infection in mice, it has been demonstrated that the host generates some degree of resistance to reinfection (1). As the inflammatory reaction that develops at the site of M. lepraemurium infection has been shown to be similar to a delayed-type hypersensitivity (DTH) reaction (3), there is circumstantial evidence of the induction of CMI. The relationship between CMI and resistance in other bacterial infections is well established (9, 10, 18), and there is some evidence that similar mechanisms are responsible for the resistance generated to M. lepraemurium (3). As yet, however, no definitive proof of specific CMI developing as a result of M. lepraemurium infection has been reported. Although the level of resistance to secondary challenge can be determined at varying times during the primary infection, the nature of such acquired immunity remains uncertain. In any case, the experimental protocol required to demonstrate resistance is such that the immunological status of the host may well have changed during this time span. Thus, methods of demonstrating resistance alone do not lend themselves well to critical analysis of the immunological mechanisms involved. Since there is some evidence to support the proposition that CMI develops during M. kepraemurium infec-
tion (3), it was thought that this might be detectable by the expression of a DTH response at the site of an intradermal challenge (8, 12). It must be emphasized that the term CMI is used in the present paper only to describe the presence of acquired cell-mediated immunological mechanisms and not protective immunity. Although no correlation with protective immunity was determined, the demonstration of DTH in an M. lepraemurium-infected animal illustrates the development of a cell-mediated immunological response that can be easily quantitated 24 to 48 h after challenge. Besides confirming the development of CMI, therefore, this reaction could be used to analyze certain changes in immunological status at precise time points during this chronic infection. This paper describes the successful demonstration of DTH after M. lepraemurium infection, as well as the kinetics and characteristics of this reaction. MATERIALS AND METHODS Animals. Inbred hybrid mice of the B6D2 F1 strain (C7BI/6 x DBA/2) (parental strains from Jackson Laboratories, Bar Harbor, Me.) of either sex, 6 to 8 weeks old, were used. These mice are relatively resistant to M. lepraemurium, having a mean survival time of 32 weeks after the intravenous injection of 108 viable organisms. For some experiments, mice were thymectomized within 24 h of birth and used at 8 weeks of age. Mycobacteria. M. lepraemurium (Hawaii strain) was maintained by repeated passage in susceptible 439
440
POULTER AND LEFFORD
mice. Purified suspensions were obtained from the livers and spleens by using the method of Draper (4) and stored at -70°C. Inocula of known size were prepared by appropriate dilution of the stock suspension after making counts of acid-fast bacilli in stained smears (14). Heat-killed (HK) M. lepraemurium were prepared by heating a suspension of live organisms at 100°C for 20 min. Sonicated (Son) M. lepraemurium was prepared by sonicating viable suspensions of cells for 15 min with a sonicator (Bronwill Biosonic IV, VWR Scientific) set at 80% of maximum. HK M. kepraemurium was also sonicated in an identical manner for use in some experiments. Frozen and thawed (FT) M. lepraemurium was prepared by freezing viable cells to -70°C for 2 h six times and thawing to room temperature for 30 min between each freeze. In some experiments, BCG Pasteur (TMC 1011) grown at this Institute was used. Primary infection. Normal mice were infected with an appropriate number of mycobacteria contained in 0.04 ml of sterile saline, injected subcutaneously (s.c.) into the left hind footpad (LHFP). Elicitation of DTH. At varying times after primary infection with M. lepraemurium or BCG, animals were injected s.c. in the right hind footpad (RHFP) with either living, HK, Son, or FT preparations of M. lepraemurium. These suspensions were injected in a volume of 0.04 ml of sterile saline. In all cases, the thickness of the RHFP was measured with dial-gauge calipers before injection and at varying times thereafter. Lymphocyte proliferation. Cellular proliferation in the draining popliteal lymph node (PLN) was measured weekly after M. lepraemurium infection. Twenty microcuries of tritiated thymidine (specific activity, 3 Ci/mmol; New England Nuclear Corp., Boston, Mass.) was injected intravenously (i.v.), and 30 min later the draining and contralateral PLN were removed, homogenized, and washed with cold trichloroacetic acid, and the deoxyribonucleic acid was extracted with hot trichloroacetic acid as described previously (17). Radioactivity was measured in a liquid scintillation counter and expressed as counts per minute. Cell transfer. Mice infected 5 weeks previously with 108 M. lepraemurium (s.c. in LHFP) were injected intraperitoneally with 2 ml of 5% casein. Three days later, the mice were killed and the peritoneal exudate cells (PECs) were washed out with phosphate-buffered saline (PBS) containing 1% fetal calf serum and heparin (10 U/ml). The cells from 50 donor mice were pooled, and adherent cells were removed by incubating the cell suspension twice in large plastic petri dishes for 1 h at 37°C in an atmosphere of 5% CO2. After each incubation, the nonadherent cells were removed by vigorous washing with fresh PBS solution. The remaining cells were spun at 200 x g for 15 min and suspended at a concentration of 2 x 108/ml. For controls, PECs were collected from normal mice in an identical manner. Viability was determined by trypan blue exclusion and was found to be consistently above 90%. Systemic cell transfer was performed by the injection of 108 PECs in 0.5 ml of PBS i.v. into normal
INFECT. IMMUN.
recipient mice. These mice were challenged with 108 equivalents of Son M. lepraemurium in the LHFP either immediately after cell transfer or 24 h later. Local cell transfer was performed by the method of Metaxas and Metaxas-Buehler (11). Nonadherent PECs, 107, from infected or normal animals were mixed with 108 equivalents of Son M. lepraemurium in 0.05 ml and injected into the LHFP of normal recipient mice. Any increase in footpad thickness was measued 6, 12, 18, 24, 48, and 72 h later. Control groups of mice were injected with antigen alone or cells alone. As a positive control, a group of animals infected at the same time as the donor mice was challenged with 108 equivalents of Son M. lepraemurium to confirm that the cell donors had developed the capacity to express DTH. Serum transfer. Blood was collected aseptically by cardiac puncture from normal or M. lepraemurium-infected mice (108 M. lepraemurium, 5 weeks previously). The serum was separated out, and groups of normal recipients were infected with 0.5 ml i.v. These animals were challenged in the LHFP with 108 equivalents of Son M. lepraemurium immediately, and footpad swelling was measured 24 h later. As a direct comparison to the local cell transfer (see above), local serum transfers were performed. Serum, 0.05 ml, from infected or normal animals was mixed with 108 equivalents of Son M. lepraemurium and injected s.c. into the LHFP of normal mice. Footpad thickness was measured 6, 12, 18, 24, 48, and 72 h later. Control groups of mice received serum alone. Histology. Specimens for histological examination were fixed in phosphate-buffered 10% Formalin for 24 h, dehydrated in alcohol, and embedded in hydroxyethyl methacrylate. Thin sections (1 tLm) were stained with hematoxylin and eosin. Statistical analysis. There were at least five animals in each experimental group. Results are expressed as the mean and standard deviation within these groups, and statistical significance was determined with Student's t test for nonpaired data.
RESULTS Response to parasite and elicitation of DTH. Mice were infected with 108 M. lepraemurium into the LHFP, and both hind feet were measured at weekly intervals. After 3 weeks a detectable increase in the thickness of the LHFP was seen, which then enlarged relatively rapidly between weeks 3 and 6 (Fig. 1). As this increase is thought to be associated with an immunological response (3), 5 weeks after infection was selected as an appropriate time for the demonstration of DTH. Mice were infected with either 108 M. lepraemurium or 106 BCG in the LHFP, and 5 weeks later groups of these mice and normal controls were challenged in the RHFP with either 108 live M. lepraemurium or equivalent concentrations of HK, Son, or FT M. lepraemurium. The results (Fig. 2) showed that challenge with live, Son, or FT M.
VOL. 17, 1977
E
DTH DURING MURINE LEPROSY
Son M. lepraemurium. The RHFP were measured before testing and 6, 12, 24, 48, and 72 h later. In both infected and control mice, there was a rapid swelling of the RHFP, which subsided by 12 h (Fig. 3). This was followed, however, by a delayed reaction, which peaked 24 h after challenge. At this time, the feet of the infected mice were significantly thicker than those of the controls (P < 0.001). At 48 h, the reaction had subsided and, although still greater than in the control mice, the difference was not statistically significant. The reaction had almost totally disappeared by 72 h. The use of a concentration of 108 Son M.
10
z u v
0
a
.....
8 0 61
w
.
2 Z
i va,
2
4
6
10 11
8 I
Weeks
FIG. 1. Development of increas ed footpad thckness in B6D2F1 mice after the s.c. injection ofl1o8 M. lepraemurium at this site. Ten a nimals were measured before infection and each weeek thereafter. The mean increase in thickness is reco rded.
-
0*8
- 07
04
LIVE
Son
HK
MLM
FT
CHALLENGE
FIG. 2. Comparison of the abili .onm.ur,l rn,tinnluecllul-tull t(RAT LY.LiLIJ WU CsIC-ss Ur Z- -a Mp footpad reaction in the RHFP of mice infected 5 weeks previously in the LHFP with 108 M. lepraemurium (open bars). The eliciting dose was either 108 live M. lepraemurium or 108 equivalents ofHK, Son, or FT M. lepraemurium. The solid bars represent the reaction in normal mice, and the striped bars represent the reaction in animals infected with 10 BCG in the LHFP 5 weeks previously. Means of five mice ± n 1LMf . Ur UL-CUIsa U/
441
tyM) ofariout p4rh-
standard deviation.
lepraemurium was justified by a dose-response experiment, the results of which appear in Fig. 4. Mice infected with M. lepraemurium 5 weeks previously were tested with graded concentrations of M. lepraemurium. A dose equivalent to 10W M. lepraemurium elicited the largest (P < 0.001) increase in the footpad thickness in the infected mice without producing a nonspecific reaction in normal mice. When the dose was raised to 109 M. lepraemurium equivalents, there was substantial nonspecific swelling in the control animals. It should be noted here, however, that different batches of the Son preparation caused peak reactions at different concentrations. This result probably derives from the fact that neither the absolute number nor the viability of M. lepraemurium in a given preparation can be accurately determined. It is, therefore, necessary to assay each new preparation of sonic extract before experimental use. Histology. Footpad tissue of M. lepraemuE
09
0-8 z
07
v
0
0-6
x
0*5
0 0
0-4
lepraemurium preparations caused a signifi- z 03 cant 24-h increase in footpad thickness in the 0-2 infected animals but not in the normal controls; however, the HK M. kepraemurium failed to 0-1 elicit such a response. The use of live M. leprae72 48 24 6 12 murium for challenge, however, also caused a HOURS positive response in BCG-infected animals. The Son M. lepraemurium preparation was thereFIG. 3. Kinetics of footpad swelling in the RHFP fore chosen for use in subsequent experiments, after s.c. injection of 108 equivalents of Son M. since it produced the largest specific response. lepraemurium in animals infected with 108 M. leKinetics of the footpad response. Mice that praemurium in the LHFP 5 weeks previously (solid S c
v
had been infected in the LHFP with 108 M.
lepraemurium 5 weeks previously and normal controls were challenged in the RHFP with 108
line). The broken line represents the response in uninfected animals. Means of five mice + standard deviation.
442
INFECT. IMMUN.
POULTER AND LEFFORD
E-
E E
z
0-8
I
o 0 at
0-6
/
0L O 0-4 0
cm
[3H]thymidine. It was found (Fig. 8) that a significant increase in deoxyribonucleic acid synthesis could be detected 1 week after infection (P
