Enzyme Activation in Peritoneal Cells from Mice Infected with Mycobacterium lepraemurium To THE EDITOR: In a previous study ( 5 ) we found that peri- toneal cells (PC) from NIH mice inoculated i.p. with 10 8 Mycobacterium lepraemurium (M1m) showed increased levels of several lysosomal hydrolases 4 months after inoc- ulation. Two months later, most of the en- zyme activities decreased to values equal to or lower than those found in the control group. This suggested a transient state of biochemical activation resulting, very likely ( 2 ), from the generation of an affective cell- mediated immune response (via lympho- kines) toward the mycobacterial antigens, and led us to study the kinetics of such bio- chemical activation in the PC population (mostly macrophages) during the entire pe- riod of infection. We inoculated 150 NIH female mice (8 weeks old, 20-24 g) i.p. with 10 8 Aihn bacilli freshly separated ( 4 ) from lepromas from previously infected animals. Similar, non-inoculated animals served as controls. Groups of 15 animals were sacri- ficed at 2-week intervals following inocu- lation to collect PC as described elsewhere ( 5 ). Four days before PC collection, the an- imals were injected i.p. with 2.0 ml of light mineral oil (Sigma). Cell suspensions were pooled, separated from the oil in a separa- tion funnel, washed, adjusted to 20 to 22 x
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476^ International Journal of Leprosy^ 1986
TABLE 2. Recovery of M. leprae isolated from skin biopsies taken from patients withdifferent types of disease.
Disease type"Biopsy pool Tissue
homogenatesTwo-phase separation
(x10' AFB
(
To Recover
- x 100%)ca
no. ofspecimens
(x10' AFB ml - ')(a)
Lower layer(b)
Upper layer(c)
LL old cases 5 8.9 0.0 0.0LL new cases 8 281.0 0.0 131.1 47LL with reactivation 4 131.4 3.1 87.6 67LL with ENL 4 8.1 0.0 7.0 86BL 7 71.0 0.0 31.4 44TT 4 0.0 0.0 0.0BT 3 0.0 0.0 0.0Mouse foot pad 6 3.1 0.0 0.8 26
from armadillo liver tissues (supplied byIMMLEP) in that the former did not reactwith any of the tuberculosis patients' seratested, while the latter reacted with 5%-9%of these sera ( 2 ).
Tropical DiseasesInstitute for Medical Research50588 Kuala LumpurMalaysia
REFERENCES1. DRAPER, P. Protocol 1/79. Sixth Programme Re-
port. WHO Special Programme for Research andTraining in Tropical Diseases. Geneva: WorldHealth Organization, 1983, chapter 8, p. 219. TDR/PR-6/83.8-LEP.
2. GAN, S. C. Antigen characterifation of Mycobac-terium leprae, thesis, University of Malaya, 1985.
3. KRONVALL, G., STANi -oRD, J. L. and WALSH, G. P.Studies of mycobacterial antigens, with special ref-erence to Mycobacterium leprae. Infect. Immun. 13(1976) 1132-1138.
Enzyme Activation in Peritoneal Cells from MiceInfected with Mycobacterium lepraemurium
To THE EDITOR:In a previous study ( 5 ) we found that peri-
toneal cells (PC) from NIH mice inoculatedi.p. with 10 8 Mycobacterium lepraemurium(M1m) showed increased levels of severallysosomal hydrolases 4 months after inoc-ulation. Two months later, most of the en-zyme activities decreased to values equal toor lower than those found in the controlgroup. This suggested a transient state ofbiochemical activation resulting, very likely(2 ), from the generation of an affective cell-mediated immune response (via lympho-kines) toward the mycobacterial antigens,and led us to study the kinetics of such bio-
chemical activation in the PC population(mostly macrophages) during the entire pe-riod of infection. We inoculated 150 NIHfemale mice (8 weeks old, 20-24 g) i.p. with10 8 Aihn bacilli freshly separated (4 ) fromlepromas from previously infected animals.Similar, non-inoculated animals served ascontrols. Groups of 15 animals were sacri-ficed at 2-week intervals following inocu-lation to collect PC as described elsewhere( 5 ). Four days before PC collection, the an-imals were injected i.p. with 2.0 ml of lightmineral oil (Sigma). Cell suspensions werepooled, separated from the oil in a separa-tion funnel, washed, adjusted to 20 to 22 x
FIG. I. Number of PC collected from normal (■) and Mini-infected (11) mice at several post-inoculationtimes. The percent values for infected animals (0) are also given in relation to the values found (here normalizedas 100%) in the control animals (•). Each point is the average from 3 determinations.
10 6 PC per ml, divided into 2 ml aliquots,and kept frozen until used.
Enzyme determinations have been pre-viously detailed ( 5 ) and include: deoxyri-bonuclease (DNase), ribonuclease (RNase),/3-glucuronidase ((3-Glu), acid phosphatase(AcPh), lipase (Lip), acid proteinase (Pro),and lysozyme (Lys). An additional enzymeactivity, that of peroxidase (PO), was as-sayed in conical, 5 ml glass tubes to whichthe following were added: 0.1 M phosphatebuffer, pH 6.0 (0.3 ml); 0.02 M o-dianisi-dine hydrochloride in water (0.05 ml); theappropriately diluted enzyme preparation(0.3 ml) (After thawing, the cell suspensionwas sonicated for 10 sec at a low intensityjust to disperse clumps.); distilled water tomake 2.4 ml; and 0.03 M H2O, (0.1 ml).After shaking and incubation of the tubesat 25°C for 20 min, the reaction was stoppedby adding 0.5 ml of 40% trichloroacetic acid(TCA), the tubes centrifuged (3000 x g x10 min), the supernatant discarded, the re-sulting brownish precipitate dissolved in 3.0ml dioxan, and the optical density (OD) ofthe colored supernatant from a final centri-fugation was read at 460 nm against a diox-an blank. PO activity is given in referenceto authentic PO samples (P8375, Sigma) asunits per 1 x 10° cells (1 unit is the amount
of enzyme needed to produce an OD changeof 0.1 under the assay conditions).
The progress of the infection was period-ically monitored by measuring the spleenweights in relation to the body weights. (Thespleen is the organ most affected followingi.p. inoculation of mice with Mini.)
We found a progressive loss of total PCin the infected group with a proportionalincrease in the macrophage population (Fig.1). Shortly after the inoculation, the numberof PC per infected mouse increased non-specifically in response to the i.p. depositionof Allm but decreased steadily thereafter.By day 132 post-inoculation, this value wasnearly 60% below the one found in the con-trol group with almost 100% of the cellsbeing morphologically and functionallymacrophages.
In spite of these facts, most of the assayedenzyme activities in the PC from the Allm-infected animals showed a progressive ele-vation that peaked between 40 and 75 dayspost-inoculation, with a tendency to de-crease thereafter until the end of the exper-iment (Fig. 2). The infected animals thatwere not further manipulated, and whichsurvived the infection for over 6 to 8 months,showed increasing signs of the disease thateventually killed them. RNase and PO ac-
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478^ International Journal of Leprosy^ 1986
14 28 42 56^70 81. 98 112
DAYS OF INFECTION
Fin. 2. Enzyme activities in PC from normal (N)
and Mbn-infected mice at several post-inoculation
times. The enzyme activities (and amounts of protein)tested were: 1 = DNase (240 pg); 2 = RNase (1O pg);
pg); 6 = Lys (112 pg); 7 = Pro (329 pg); 8 = PO (400pg). Each point is the average of 3 determinations.
Results are shown normalized as percent values. Shad-owed areas show the period of peak activation.
tivities did not increase in response to theinfection. On the contrary, PO activity grad-ually fell and completely disappeared by day110 following inoculation. This behavior ofPO was related to the diminution of cellsother than macrophages, including PO-richneutrophils.
The progress of the infection was neverarrested (Fig. 3). Spleen enlargement in-creased until day 135 (4.5 months) post-inoculation, after which it did not changesignificantly.
From these data, several conclusions can
>cw0z- 3Uz
- 2c)_
0^1^2^3^4^5^6
TIME OF INFECTION (MONTHS)
FR;. 3. Progress of the .111ir infection in terms ofspleen enlargement. At each post-infection time shown,
3 1n7n-inoculated (*) and 3 normal controls (^) wererandomly selected to calculate the splenic indexes ac-cording to the formula:
spleen weight/body weight in ,tilin-infected mice
^
spleen weight/body weight in control mice^•
Points are averages of 3 determinations.
be drawn: a) infection of mice with Mb)/apparently induces biochemical activationin their PC population; b) such PC activa-tion is not permanent but transitory, andpeaks between 40 and 75 days post-infec-tion, depending on the enzyme; c) some en-zyme activities increase more than the oth-ers, with somewhat different kinetics (In thisstudy, RNase did not show evidence of ac-tivation.); d) the total number of PC andthe proportion of cells other than macro-phages fall in proportion to the duration ofinfection; e) PO activity does not increasebut falls steadily and completely disappearsby the end of the fourth month of infection;f) despite these changes, the murine diseaseprogresses in an apparently unlimited man-ner.
These observations suggest that a) :11/minfection of mice induces an early state ofcell-mediated immunity (CMI) ( 3 ) which in-fluences macrophage metabolism ('), in-cluding their microbicidal capabilities ( 2 );b) shortly after (or simultaneously with) thedevelopment of CMI, suppressor mecha-nisms are induced which turn off the "pro-tective effects" of CMI ( 6 ) including mac-rophage activation, allowing the "few"surviving bacilli to take over and assuring
54, 3^ Correspondence^ 479
the progress of the infection; c) the paralleland progressive decreases in PO activity andneutrophils from the PC of "11/m-infectedanimals suggest an effect of the infection onthis cell population. Further work is in pro-gress.
Departments of Immunologyand Biochemical Engineering
Escucla Nacional de CienciasBiologicas
I.P.N. Carpi() y Plan de AyalaCol. Santo Tomas11340 .1Iexico, D.F., Mexico
Acknowledgments. 0. Rojas-Espinosa holds a fel-lowship from COFAA, IPN. The study was financiallysupported by the CONACYT and the DG1,113N.
REFERENCES1. HA, D. K. K., LAWTON, J. W. M. and GARDNER, I.
D. Experimental murine leprosy: a biochemical study
emphasizing lysosomal enzyme changes in vivo andenzyme secretion by macrophages in vitro. Exp.Molec. Pathol. 40 (1984) 177-194.MAckANEss, G. B. and BLANDEN, R. V. Cellularimmunity. Progr. Allergy 11 (1967) 89-140.
3. PotriA ER, L. W. and LEErrouD, M. J. Relationshipbetween delayed type hypersensitivity and the pro-gression of Afycobacterium lepracmurium infec-tion. Infect. Immun. 20 (1978) 530-540.
4. PRABILAKARAN, K., HARRIS, E. B. and Kuo - DDEI-MLR, W. F. Binding of ' 4 C-labeled DOPA by My-cobacterium leprac in vivo. Int. J. Lepr. 44 (1976)58-64.
5. Roms-EspiNosA, 0., RoDR uitu.z-PAt.z, L.,GoNzALEz-CRuz, 0. and EsTRADA-PARRA, S.Phagocytosis in leprosy. 5. The effect of the infec-tion with .14a/bacterium lepramurizon on the levelof diverse hydrolytic lysosomal enzymes of murineperitoneal macrophages. Int. J. Lepr. 50 (1982) 306-315.
6. Rook, G. A. W. Suppressor cells of mouse and man.What is the evidence that they contribute to theaetiology of the mycobacterioses? Lepr. Rev. 53(1982) 306-312.
Lepromin Skin Test in Normal People inSingapore—A One-year Follow Up
To THE EDITOR:The lepromin test is an intradermal skin
test used to classify a case of Hansen's dis-ease into the tuberculoid or lepromatous va-riety ( 5 ). This survey was prompted by thefact that the incidence of lepromin positiv-ity in Singapore is unknown. Singapore is ahighly urbanized island in South East Asiawhich is endemic for Hansen's disease.
We prepared our human lepromin solu-tion in the manner recommended by WHO( 5 ). The solution contained 160 x 10' bacilliper ml prepared from a nodule of a lepro-matous leprosy patient, and 0.1 nil was in-jected in the usual manner intradermally inthe volar forearms of 120 normal volun-teers. The Mitsuda reaction was read at 21days by two people. Simultaneously, a tu-berculin test was done on the other forearm.One year later, 30 of the subjects were re-called and re-tested with armadillo-derived
lepromin containing 160 X 10' bacilli per
A positive result was defined as per WHOcriteria as being any induration 3 mm orgreater in diameter ('). Table 1 gives thenumber of cases according to age. There wasan overall positivity rate of 70.3% whichcorresponds to the rate found in most en-demic countries where similar studies have
TABLE 1. LCprOMin test (human) in 120normal subjects.
