Footpad Infected with Mycobacterium leprae

10
INFECTION AND IMMUNITY, Feb. 1972, p. 238-247 Copyright © 1972 American Society for Microbiology Vol. 5, No. 2 Prinited in U.S.A. Ultrastructural Changes in Cells of the Mouse Footpad Infected with Mycobacterium leprae MICHAEL J. EVANS AND LOUIS LEVY Life Scienices Divisio)1, Stan2ford Research Institute, Menlo Park, California 94025, and Leprosy Research Ullit, Public Health Service Hospital, Sani Francisco, Californlia 94118 Received for publication 22 September 1971 Ultrastructural changes in cells of the mouse footpad are described which oc- curred during the log phase of multiplication, the plateau, and the stationary phase of growth of Mycobacterium leprae. BALB/c mice were inoculated in the right hind footpad with 5 x 103 organisms and sacrificed in pairs at 86 to 173 days after inoculation. Tissue samples were prepared for electron microscopy by standard techniques. During the early growth phase of M. leprae in the mouse footpad, few organisms can be detected. Those present are in macrophages and are bound by a single membrane. The cytoplasm of the macrophage is less dense around the organism. There are few lysosomes, and the bacteria do not appear to be degenerat- ing. At the peak of the growth phase, the organisms within a macrophage are bound by either a single or double membrane. There is an increased number of vacuoles, which are also bound by a double membrane, and lysosomes. During the stationary phase, most of the macrophages have taken on a vacuolar appearance and contain lysosomes. The vacuoles are bound by a double membrane, as are most of the organisms within the macrophage. Many of these organisms appear to be degenerating. Occasionally, organisms are encountered in the sarcoplasm of striated muscle. They are usually bound by a single membrane and do not appear to be degenerating. The relationship between Mycobacterium leprae and the tissues they infect is of considerable interest. In tissue samples from human leprosy patients and from mice and rats infected with M. leprae, bacteria are found in macrophages of the loose connective tissues and in the cells mak- ing up nerve and muscle tissue (1, 8). Within tis- sue macrophages, the bacilli usually are sur- rounded by an electron-transparent area bounded by a membrane, and many appear to be in various stages of degeneration. In nerve and muscle cells, not as many bacilli appear to be degenerating (5, 6). The mouse footpad infected with M. leprae is a suitable system in which to study the host- parasite relationship in leprosy because it per- mits serial sampling of the tissue as the infection progresses. After the inoculation of a small amount of viable M. leprae into the footpad of an immunologically competent mouse, there is a lag phase lasting 30 to 60 days, during which period there is no detectable increase in the num- ber of organisms (9). Then, M. leprae enters a logarithmic phase of multiplication, during which time the number of organisms increases, with an average doubling time of 12.5 days. Toward the end of the logarithmic phase, numbers of acid- fast bacilli (AFB) may be readily seen within cells lying in the loose connective tissue just beneath the plantar skin and surrounding the nerve-and- vessel bundles. Despite the presence of the or- ganisms, there is a minimum of inflammatory response in the footpad tissues. Shortly after the number of organisms passes 106 per footpad (the "cplateau," 130 to 150 days after inoculation), multiplication ceases; at about this time, the foot- pad tissue displays evidence of a low-grade in- flammatory process characterized by the presence of a round-cell infiltrate. At no time is there gross disease of the footpad. After cessation of multi- plication, death of M. leprae begins. Although the number of AFB does not change very much dur- ing this stationary phase, death of organisms can be demonstrated by mouse passage (4, 10). The purpose of the study reported below was to describe ultrastructural changes in cells of the mouse footpad as the leprosy infection progressed through the logarithmic phase of multiplication, the plateau, and the beginning of the stationary phase. 238

Transcript of Footpad Infected with Mycobacterium leprae

Page 1: Footpad Infected with Mycobacterium leprae

INFECTION AND IMMUNITY, Feb. 1972, p. 238-247Copyright © 1972 American Society for Microbiology

Vol. 5, No. 2Prinited in U.S.A.

Ultrastructural Changes in Cells of the MouseFootpad Infected with Mycobacterium leprae

MICHAEL J. EVANS AND LOUIS LEVYLife Scienices Divisio)1, Stan2ford Research Institute, Menlo Park, California 94025, and Leprosy Research Ullit,

Public Health Service Hospital, Sani Francisco, Californlia 94118

Received for publication 22 September 1971

Ultrastructural changes in cells of the mouse footpad are described which oc-curred during the log phase of multiplication, the plateau, and the stationaryphase of growth of Mycobacterium leprae. BALB/c mice were inoculated in the righthind footpad with 5 x 103 organisms and sacrificed in pairs at 86 to 173 days afterinoculation. Tissue samples were prepared for electron microscopy by standardtechniques. During the early growth phase of M. leprae in the mouse footpad, feworganisms can be detected. Those present are in macrophages and are bound by asingle membrane. The cytoplasm of the macrophage is less dense around theorganism. There are few lysosomes, and the bacteria do not appear to be degenerat-ing. At the peak of the growth phase, the organisms within a macrophage arebound by either a single or double membrane. There is an increased number ofvacuoles, which are also bound by a double membrane, and lysosomes. During thestationary phase, most of the macrophages have taken on a vacuolar appearance andcontain lysosomes. The vacuoles are bound by a double membrane, as are mostof the organisms within the macrophage. Many of these organisms appear to bedegenerating. Occasionally, organisms are encountered in the sarcoplasm ofstriated muscle. They are usually bound by a single membrane and do not appear tobe degenerating.

The relationship between Mycobacteriumleprae and the tissues they infect is of considerableinterest. In tissue samples from human leprosypatients and from mice and rats infected withM. leprae, bacteria are found in macrophages ofthe loose connective tissues and in the cells mak-ing up nerve and muscle tissue (1, 8). Within tis-sue macrophages, the bacilli usually are sur-rounded by an electron-transparent area boundedby a membrane, and many appear to be in variousstages of degeneration. In nerve and muscle cells,not as many bacilli appear to be degenerating(5, 6).The mouse footpad infected with M. leprae is

a suitable system in which to study the host-parasite relationship in leprosy because it per-mits serial sampling of the tissue as the infectionprogresses. After the inoculation of a smallamount of viable M. leprae into the footpad ofan immunologically competent mouse, there is alag phase lasting 30 to 60 days, during whichperiod there is no detectable increase in the num-ber of organisms (9). Then, M. leprae enters alogarithmic phase of multiplication, during whichtime the number of organisms increases, with an

average doubling time of 12.5 days. Toward theend of the logarithmic phase, numbers of acid-fast bacilli (AFB) may be readily seen within cellslying in the loose connective tissue just beneaththe plantar skin and surrounding the nerve-and-vessel bundles. Despite the presence of the or-ganisms, there is a minimum of inflammatoryresponse in the footpad tissues. Shortly after thenumber of organisms passes 106 per footpad (the"cplateau," 130 to 150 days after inoculation),multiplication ceases; at about this time, the foot-pad tissue displays evidence of a low-grade in-flammatory process characterized by the presenceof a round-cell infiltrate. At no time is there grossdisease of the footpad. After cessation of multi-plication, death of M. leprae begins. Although thenumber of AFB does not change very much dur-ing this stationary phase, death of organisms canbe demonstrated by mouse passage (4, 10).The purpose of the study reported below was to

describe ultrastructural changes in cells of themouse footpad as the leprosy infection progressedthrough the logarithmic phase of multiplication,the plateau, and the beginning of the stationaryphase.

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MATERIALS AND METHODS

Shepard's mouse footpad technique for the cultiva-tion of M. leprae was used in these experiments (9,12). Inoculation of the mice and the harvesting of M.leprce were performed in San Francisco. The micestudied were taken from groups of locally bred

BALB/c mice inoculated with M. leprae in other ex-periments. The M. leprae were all from the same strainof organisms originally isolated from a patient byShepard and carried since then in mouse passage bothin Shepard's laboratory and in San Francisco. Fromeach group of mice from which individuals were ta-

TABLE 1. Sunmmary of experimenits from which mice were takeni for electronz microscopy (EM)

Expt no.Time ofharvesta(days)

m 6-8-70 126

m 9-3-69

m 10-8-69

m 5-19-70

m 5-13-70

m 8-5-69

m 4-9-70

m 4-30-70

m 3-17-70

m 3-11-70

147

148

117

166167

168175

117124132138139141145152166180194208

139

139154

162

160

139152

146

152

Yield ofM;1. lepraeb(X 105)

7.64.15.413.012.65.95.4

0.40.40.21.45.93.2

11.311.25.0

2.24.219.96.511.119.014.419.430.430.121.317.8

11.5

21.011.5

18.4

37.5

24.521.0

11.9

25.9

Mean time toplateau', c

(days)

145

176

138

145

125

151

135

117

143

134

Time ofsacrifice forEMa (days)

86

120

90

106

112

150

142

125

168

173

Time before (+) Designation ofor after (-) IDsgainoplateau (days) EM prepn

+59

+56

+48

+39

+13

+1

-7

-8

-25

-39

A

B

C

D

E

F

G

H

I

J

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a Days since inoculation.' Number of organisms per footpad, based on a harvest from the pooled tissue of four footpads.c The time to plateau is calculated assuming a constant doubling time of 13 days for this strain of

M. leprae in these BALB/c mice.

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ken for histologic study, at least one harvest of M.leprae was performed. Harvests were carried out onthe pooled footpad tissue of at least four mice. Thetime to plateau was calculated from the number oforganisms first inoculated, the number harvested, andthe time from inoculation to harvest, assuming a con-stant doubling time during logarithmic multiplication.

Because killing of M. leprae in the mice beginsshortly after the plateau of multiplication is achieved,the proportion of viable organisms will vary amongharvests and among the inocula prepared from theharvests. Therefore, the time course of multiplicationof M. leprae will vary from experiment to experiment.To be able to superimpose growth curves from morethan one experiment, it is necessary to use a timescale related to the time to plateau rather than to thetime of inoculation. This method was used here.

Electron microscopy studies were carried out inMenlo Park, Calif. The footpad was dissected freeand fixed in 2%7 gluteraldehyde buffered with caco-dylate to pH 7.2 for up to 24 hr. It was then sliced,washed in buffer, postfixed in 1% Os04 containing7.5% sucrose buffered with Veronal acetate to pH 7.2,dehydrated in alcohol, and embedded flat in Beemcapsules with Araldite. Samples for light microscopywere cut with a glass knife at 1.0 ,um and stained withToluidine Blue. Samples for electron microscopy werecut with a diamond knife on a Porter Blum MT2 ultra-microtome, placed on nickel grids, stained with uranylacetate and lead citrate, and viewed with a Philips200 electron microscope.

RESUTLTSThe data describing the experiments from which

mice were taken for electron microscopic studyof the infected footpad tissue are presented inTable 1. The results of harvests for each experi-ment are shown, together with the calculatedtime to plateau, the time mice were taken for elec-tron microscopic study, and the time of study rela-tive to the time of plateau. Figure 1, in which thelog number of M. leprae recovered in each of theharvests listed in Table I has been plotted as a

function of the time before or after plateau,demonstrates that the timing of the electron mi-croscopic studies in the manner used in this ex-

periment is valid. The results of the harvests maybe seen to fall along two straight lines, the re-

gression equations for which are:

log no. of M. leprae = 5.83 + 0.022(7.74 - no. of days before plateau) (1)

log no. of M. leprae = 6.32 + 0.0006(no. of days after plateau 43.33) (2)

Line 1, which describes the logarithmic phaseof multiplication, has a correlation coefficient of0.93; the logarithmic increase in bacterial num-

bers of 0.022 per day represents a doubling time of13.7 days. The logarithmic phase of multiplica-tion may be seen to extend from 59 days beforeuntil 15 days after the plateau value of 106 M.leprae per footpad has been achieved.

7.0

a

m11

0

0

cc

6.0

5.0

o'-0

0

3.0100 80 60 40 20 0 -20 -40 -60 -80 -100

TIME BEFORE (+) OR AFTER (-) PLATEAU -- days

FiG. 1. Composite growth curve of Mycobacteriumleprae inz the 10 groups of mice from which individualswere taken for histologic study. The log number ofM. leprae recovered per footpad in each harvest isplotted as a function of the time before or after theplateau of 106 organisms/footpad was achieved ineach group of mice. Curve may be resolved into twocomponents: line 1, logarithmic phase; and line 2,stationary phase of multiplication. The letters Athrough J indicate the time with respect to plateauwhen mice were sacrificed for electron microscopy.

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FIG. 2. Low-power view of a mouse footpad. Sec-tioned at I ,um and stained with Toluidine Blue. X 125.

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X WXgE F LINE 2

A LINEl -B

240 INFECT. IMUNITY

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M. LEPRAE IN THE MOUSE FOOTPAD

Line 2 describes a stationary phase of bacterialmultiplication. Although bacterial numbers arenot decreasing at this time, the proportion ofviable organisms is shown to decrease after thepeak of multiplication has been reached (4, 10).

Preparations A through D represent early

logarithmic muitiplication, whereas E was madelate in the logarithmic phase; F, G, and H repre-sent the plateau, and preparations I and J weremade during the end of logarithmic multiplica-tion and the early portion of the stationary phase.An example of the area of mouse footpad tis-

5FIG. 3. Light micrograph of organisms (arrow) in a macrophage of the loose connective tissue, 56 days beJore

plateau was reached. X 1,250.FIG. 4. Organismis in macrophages 13 days before plateau. They are surrounded by a single membrane. Note

lack of lysosomes, vacuoles, anid residual bodies. X 17,500.FIG. 5. An organiism in a macrophage 13 days before plateau. Note the cytological detail in the organism, the

single membrane surrounding it (arrow), and the less dense area of cytoplasm that surrounds it (halo). X 67,250.

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b.A

7'*.

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sue that was studied is illustrated in Fig. 2. Crosssections of the footpad like this allowed us tostudy the epidermis, hypodermis, muscles, nerves,tendons, and the loose connective tissue aroundthese structures. These sections were scanned withthe light microscope, and an area containingorganisms was selected. This area was thentrimmed and sectioned for electron microscopy.

The footpads from 10 different mice were usedto study the logarithmic phase of M. leprae.Eight animals were sacrificed at times from 59 to39 days before plateau was reached (preparationsA through D). In these tissue sections, very feworganisms were observed. Two mice were sacri-ficed 13 days before plateau was reached (prep-aration E). They were similar to the other ani-

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FIG. 6. Light micrcogrcpli of organisms in mnacr-ophages 1 clay beJore platealu. X 1,250.FIG. 7. Organisms in macrophages 8 days after plateaul. Notice the membrane thlat has formed around the

periphery of the halo anid entcloses thle organisms (arrow); also membrane-boutid residual bodies (R). X 17,500.FIG. 8. Exan-mples of organiisms in the cytoplasm of a macrophage 8 days after plateaui: (i) bound by a single

membrane and surrounded by a h/alo; (ii) boinid by a single membrane, surroiiiided by a halo, with another mem-brane at the periphery of the halo; (iii) suirroinided by a double membrane. X 67,250.

242 INFECT. IMMUNITY

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M. LEPRAE IN THE MOUSE FOOTPAD

mals except that there were more organisms. Theorganisms were usually within fibroblast-like cellsof the loose connective tissue (Fig. 3). Becausethese cells contain organisms, we have referredto them as macrophages; however, they containvery few lysosomes, vacuoles, or residual bodies(Fig. 4). The organisms in the macrophagesusually have good cytological detail and are sur-rounded by a single membrane. Outside thismembrane, the cytoplasm of the cell is usually less

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dense, forming a halo around the organisms (Fig.5). There are no other membranes around theorganisms at this time. The macrophages con-taining organisms tend to be in groups in theloose connective tissue. No organisms are seenin the extracellular space or in striated musclefibers.The footpads from six mice were used to study

the plateau of M. leprae inefection (preparationsF through H). The animals were sacrificed at in-

K .

FIG. 9. Organiisms in foamy macrophages I day before plateau . In conitrast to other macrophages at this timeanid earlier times (Fig. 4), there are opaquie droplets (OD), foamy vacuioles (FV), anid 1ysosomes (L). X 22,750.

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tervals from 1 day before to 8 days after plateauhad been reached. Many more organisms werefound at this time (Fig. 6). Almost all are locatedin macrophages of the hypodermis and the looseconnective tissue around muscle fibers. Uponcloser analysis, it was found that the organismswere in three different conditions and that therewas a strong tendency for all of them to be in the

same condition in any given cell. In the first con-dition, the organisms are surrounded by a singlemembrane, and few lsyosomes or residual bodiesare present in the macrophage. These cells aresimilar to macrophages reported in the earlygrowth phase (Fig. 4 and 5). Secondly, the or-ganisms are surrounded by two membranes (Fig.7 and 8). In these cells, there may or may not be a

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FIG. 10 and 11. Organisms in a macroplhage I day before plateau, showing the relationship of opaque droplets(OD), foamy vacuoles (FV), and a lysosome-like combination (C) of these to the organisms. Note the doublemembranes around these structures (arrows). X 39,000.

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significant number of lysosomes and residualbodies. Figure 8 shows an organism surroundedby a double membrane. Notice that the secondmembrane forms at the periphery of the haloaround the organism. The two membranes appearto come together so that the organisms are sur-rounded tightly. Finally, organisms are observedin "foamy" macrophages and appear to have de-.K. .

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- 3 ik' ;"r

generated (Fig. 9, 10, 11). There are numerousvacuolated structures, some containing organisms,together with occasional lysosomes and opaquedroplets (Fig. 9). In foamy macrophages, bacteriamay be found inside vacuoles that contain (i)opaque droplets, (ii) dense granules similar tothose in the foamy structures, or (iii) a combina-tion of the two (Fig. 10 and 11). Macrophages in

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FIG. 12. Light micrograph of organiisms in a striated muscle fiber 25 days after plateau. X 1,250.FIG. 13. Electron micrograph of same tissue as in Fig. 11. The organisms lie in the sarcoplasm and are not

associated with double membranes (as are organisms in the macrophages. X 39,000.

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the second and third condition are characteristicof immunologically activated cells (3, 15, 16).The footpads from four mice were used to study

the stationary phase (25 to 39 days after plateau)of M. leprae infection (preparations I and J).There is an increase in the number of macro-phages, forming a diffuse granuloma in the looseconnective tissue. Most of the macrophages thatcontain organisms are vacuolated and look likethose in Fig. 9. However, an occasional macro-phage was observed that contained bacteria sur-rounded by a single or double membrane similarto those in Fig. 4 and 7. At this time, organismswere also found in the striated muscle fibers oftwo animals (Fig. 12). The organisms are in thesarcoplasm and are not surrounded by a doublemembrane; they did not appear to be degenerating(Fig. 13).

DISCUSSION

These cellular changes are consistent with tis-sue changes reported during the development ofdelayed hypersensitivity and cellular immunityduring the pathogenesis of tuberculosis (3). Wedid not directly measure delayed hypersensitivity;however, we detected ultrastructural changes inmacrophages that are associated with the onset ofcellular immunity. Development of cellular im-munity requires activation of macrophages toincrease their digestive and microbicidal capa-bilities, and proliferation to increase their number(2, 3). During the logarithmic growth phase, therewere very few ultrastructural changes in tissuemacrophages that would signify their activation.However, when the number of organisms reachedabout 106, a point after which immunologicalfactors have been shown to check the growth ofM. leprae in the footpads of mice (8, 11), therewere changes in macrophages that are associatedwith the onset of cellular immunity (1, 13, 14).These macrophages contained lysosomes, re-sidual bodies, and organisms in vacuoles, andthere was an increase in the number of macro-phages. These observations suggest that themacrophage population had been immunologi-cally activated.

Activation of the macrophages was also associ-ated with the appearance of a second membranearound the organism. During the logarithmicgrowth phase, when the macrophages were notactivated, most organisms were surrounded by asingle membrane. However, as the plateau wasreached and the macrophages were activated, mostorganisms were now surrounded by two mem-branes. The second membrane may be associatedwith lysosome-bacillus interaction since bacteria

bounded by double membranes are seen withlysosomes and vacuoles, whereas organismsbound by single membranes are not. We have nodirect evidence from which to determine whetherthe second membrane is of cytoplasmic or bac-terial origin. However, it appears first at somedistance from the organism, at the outer limit ofthe electron-transparent area, suggesting that it isproduced by the cytoplasm.M. leprae has been reported in striated muscle

fibers, and it was suggested that this may be animportant site for their multiplication (5, 6, 7).In the present study, the majority of the organismswe observed were in macrophages. They were notfound in muscle cells until after plateau wasreached. Because most multiplication of or-ganisms takes place during the growth phase be-fore plateau is reached, it suggests that the or-ganisms multiply in macrophages (1, 2). The mainargument against the macrophage as a site formultiplication of M. leprae is the unfavorableenvironment produced by an activated macro-phage (6). We have shown that during the loga-rithmic phase the macrophages do not appear tobe activated. If this is the case, then the macro-phages would not inhibit multiplication of theorganisms. However, after plateau, when themacrophages are activated, they may not be asfavorable a site for bacterial multiplication. Atthis time multiplication in muscle cells might bethe preferred site.

ACKNOWLEDGMENT

This work was supported in part by the U.S. Leprosy Panel ofthe U.S.-Japan Cooperative Medical Science Program adminis-tered by the Geographic Medicine Branch, National Institute ofAllergy and Infectious Diseases (grants Al 10110 and Al 07801).It was presented in part at the Sixth Annual Leprosy Conference,Atlanta, Georgia, 10-12 March 1971.

LITERATURE CITED

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2. Berthrong, M. 1968. The macrophage-tubercle bacillus rela-tionship and resistance to tuberculosis. Ann. N.Y. Acad.Sci. 154:157-166.

3. Dannenberg, A. M., Jr. 1968. Cellular hypersensitivity andcellular immunity in the pathogenesis of tuberculosis:specificity, systemic and local nature, and associated macro-phage enzymes. Bacteriol. Rev. 32:85-102.

4. Levy, L. 1970. Death of Mycobacteriu,n leprae in mice andthe additional effect of dapsone administration. Proc. Soc.Exp. Biol. Med. 135:745-749.

5. Palmer, E., R. J. W. Rees, and A. G. M. Weddell. 1965. Siteof imiultiplication of hum-an leprosy bacilli inoculated intothe footpads of mice. Nature (London) 206:521-523.

6. Pearson, J. M. H. 1970. Mjcobacterium leprae in the striatedmuscle of patients with leprosy. Leprosy Rev. 41:155-166.

7. Rees, R. J. W., and A. G. M. Weddell. 1968. Experimentalmodels for studying leprosy. Ann. N.Y. Acad. Sci. 154:214-236.

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8. Rees, R. J. W., and A. G. M. Weddell. 1970. Transmissionof human leprosy to the mouse and its clinical implications.Trans. Roy. Soc. Trop. Med. Hyg. 64:31-42.

9. Shepard, C. 1960. The experimental disease that follows theinjection of human leprosy bacilli into foot-pads of mice. J.Exp. Med. 112:445-454.

10. Shepard, C. C., and Y. T. Chang. 1967. Effect of DDS on

established infections with Mycobacterium leprae in mice.Int. J. Leprosy 35:52-57.

11. Shepard, C. C., and C. C. Congdon. 1968. Increased growthof Mycobacterium leprae in thymectomized-irradiated miceafter foot pad inoculation. Int. J. Leprosy 36:224-227.

12. Shepard, C. C., and D. H. McRae. 1968. A method for count-ing acid-fast bacteria. Int. J. Leprosy 35:78-82.

13. Skinsnes, 0. K. 1968. Comparative pathogenesis of myco-

bacterioses. Ann. N.Y. Acad. Sci. 154:19-31.14. Spector, W. G. 1969. The granulomatous inflammatory

exudate. Int. Rev. Exp. Pathol. 8:1-55.15. Weissmann, G., and P. Dukor. 1967. The role of lysosomes in

immune responses. Advan. Immunol. 12:283-331.16. Yang, H. Y., and 0. K. Skinsnes. 1969. Intracellular modula-

tion in cellular immunity. I. Morphologic studies of macro-phages in murine leprosy under conditions of immunityenhancement and suppression. Int. J. Leprosy 37:111-129.

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