Vol. 47, No. 1INFECTION AND IMMUNITY, Jan. 1985, p. 288-2930019-9567/85/010288-06$02.00/0Copyright © 1985, American Society for Microbiology

Genetic Resistance to Murine Cryptococcosis: the Beige Mutation(Chediak-Higashi Syndrome) in Mice

GABRIEL MARQUIS,1t SERGE MONTPLAISIR,l* MICHELINE PELLETIER,2 SERGE MOUSSEAU,1 ANDPIERRE AUGER1

Department of Microbiology and Immunology' and Department of Pathology,2 Universite de Montreal, Montreal,Quebec, Canada H3C 3J7

Received 31 May 1984/Accepted 25 September 1984

The influence of the bgl and bg2 mutations on the susceptibflity of mice to experimental cryptococcosis wasstudied in inbred mice of the C57BL/6J and C3H/HeJ strains. Although infected animals with the bglbggenotype had a significantly shorter lifespan than bgl+ or +/+ animals, C3H/He beige-2J mice were lesssusceptible than C57BL/6 beige-J mice when compared with nonbeige mice of similar background. On days 18and 19 after infection, quantitation of cryptococci in the brain, liver, and spleen revealed that the overallburden of organisms in infected C57BL/6 beige-J mice was in excess of one log unit above that found in thebrain, liver, and spleen of infected C57BL/6 +/+ mice. At that time, C57BL/6 beige-J mice showed a 53%increase in mean brain weight, a 67.8% decrease in mean liver weight, and a 58.6% decrease in mean spleenweight, when compared with uninfected animals of the same age and genetical lineage. The correspondingfigures for C57BL/6 +/+ mice were a 32% increase in mean brain weight, a 41.4% decrease in mean liverweight, and a 23.4% decrease in mean spleen weight. From these data, it is concluded that (i) the beigemutation in mice is associated with increased susceptibility to cryptococcosis, (ii) the accrued susceptibility ofthe beige mutant is related to more rapid changes in the weight profile of the target organs as well as to a higherrate of growth or decreased clearance of Cryptococcus neoformans or both, and (iii) other autosomal genes arelikely to be involved in the genetic control of susceptibility to murine cryptococcosis.

The beige mutation in mice, which is analogous to Chediak-Higashi syndrome in humans, leads to decreased neutralprotease activity in polymorphonuclear leukocytes (34),marked impairment in natural killer (NK) cell function (26),pigment dilution, large melanin granules, large lysosomalgranules (19), and increased susceptibility to infections (9).The Chediak-Higashi mutation has been mapped at thecentromeric region on chromosome 13, and four distinctallelic mutant genes, bgJ, bg2J, bg511, and bg, are recognized(4). After recent indications that NK cells may play a role innatural host resistance to Cryptococcus neoformans (23), weinvestigated the influence of the bgJ and bg2J alleles on thecourse of experimental cryptococcosis in strains of inbredmice.

MATERIALS AND METHODSAnimals. Inbred mice of the following strains were origi-

nally obtained from the Jackson Laboratory, Bar Harbor,Maine: C57BL/6J (+/+), C57BL/6J beige-J (bg'Ibg'),C3H/HeJ (bg21l+), and C3H/He beige-2J (bg2JIbg'j). Breed-ing colonies were established in our animal facilities andmaintained for a 12-month acclimation period before theanimals were used in this study. BALB/cN nulnu and nul+mice were obtained from breeding colonies maintained byMicheline Pelletier, Department of Pathology, Universite deMontreail. Animals were housed and handled in accordancewith National Institutes of Health guidelines.Organism. C. neoformans ATCC 13690 was obtained from

the American Type Culture Collection and was maintained

* Corresponding author.t Present address: Department of Physiology, McIntyre Medical

Center, McGill University, Montreal, Quebec, Canada H3G 1Y6.

by passage in BALB/c nude mice. Quantitative assessmentof growth was performed by the standard plate count tech-nique on serial dilutions of brain homogenates. Plates show-ing the highest log titer of growth were used for seeding onbrain-liver-heart agar semisolid medium (Difco Laborato-ries, Detroit, Mich.). The culture was harvested after incu-bation at 37°C for 48 h, dispensed in 1.0-ml aliquots intoscrew-capped vials, and stored at -85°C until needed. Thisfrozen stock was periodically renewed. India ink prepara-tions of the isolate revealed a rather thin capsule.

Preparation of inocula. Brain-liver-heart agar medium (50ml) was seeded with the thawed contents of a culture vial.The culture was harvested after incubation at 37°C for 72 h.The organism was pelleted by centrifugation at 500 x g andrepeatedly washed with sterile 0.9% saline until residual agarfrom the medium had been flushed away. A sample contain-ing the resuspended organism was diluted 100-fold with 0.9%saline. The number of cells was counted in a hemacytometerchamber, followed by preparation of suspensions with finalconcentrations of 1 x 106 and 3 x 105 yeast cells per ml. Theviability of blastospores was greater than 99% as establishedby a dye-exclusion method (2).Animal inoculation. Unless otherwise stated, mice were

infected at 40 to 44 days of age by injecting 0.1 ml of thestandardized yeast cell suspension into the right or leftlateral tail vein. Pairs of animals of related genetic lineagewere infected simultaneously. Infected animals were housedin a physically separate facility. Mortalities were recorded atleast on a daily basis.

Monitoring the infection. Upon death, animals were au-topsied, and their brain, liver, and spleen were separatelydispensed into sterile, tared, 30-ml, capped polycarbonatetubes, each tube containing 10 stainless steel ball bearings(1/4 in. [ca. 0.64 cm]). Net organ weights were determined

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BEIGE MUTATION IN MURINE CRYPTOCOCCOSIS 289

TABLE 1. MST for C57BL/6 beige-J, C57BL/6 +/+, C3H/He beige-2J, and C3H/He bgl+ mice challenged with 105 C. neoformans cells

MstoGenotypeNo.°fSEM%Specific CHaplotypesMousen Genotype No.eof aMsT SEM lysis (YACL1) CoatH2Da,,Tastrain mice (days) ~~~~~~~~~~(E/T= 50:1)a color H2D Q23 Tl

C57BL/6J +/+ 16 22.2 1.174 23.8 aBCD b a bbglbg 17 16.7 0.391 9.9

C3H/HeJ bgl+ 29 20.4 0.414 28.9 ABCD k b bbglbg 42 19.0 0.324 1.5

aAverage values for baseline NK activity were obtained from the literature (7,22,32). The NK-mediated cytotoxicity was determined by incubating splenocytesfrom unsensitized normal animals with 51Cr-labeled YAC-1 tumor cells, an in vitro subline of the Moloney virus-induced T cell lymphoma of A/Sn origin. E/T,Effector to target ratio.

with an analytical balance. A volume of saline equal to 10times the recorded organ weight was then dispensed intoeach tube. Grinding was achieved by vigorous mechanicalshaking (S8220 mixer; American Scientific Products, McGawPark, Ill.). Serial 10-fold dilutions of homogenates wereprepared. After mixing, 10-,ul samples were removed fromselected dilutions and uniformly spread on brain heart infu-sion agar in 90-mm petri dishes. Bacterial contamination wasprevented by the addition of penicillin (20 U/ml) and genta-micin (10 ,ug/ml) to the culture plates. Counts were recordedafter incubation at 37°C for 72 h. When animals could not beprocessed at once, they were immediately frozen in sealedpackages at -20°C. Counts obtained on these frozen cadav-ers were similar to those obtained from animals which wereprocessed immediately.

Histopathological studies. Infected animals were sacrificedat various time intervals, and specimens were fixed in 10%buffered formaldehyde solution. Tissues were embedded inparaffin, sectioned (5 ,um thickness), and stained with hema-toxylin and eosin (H&E) or periodic acid-Schiff.

Statistical analysis. The data were fed to a computerdatabase. Uninfected animals (n = 27) were sacrificed at themedian survival time of corresponding experimental sub-groups to obtain reference values for organ weights. InfectedC57BL/6 (+/+) mice were sacrificed at the median survivaltime of C57BL/6 beige-J (bglbg) mice to compare tissuecounts in respective strains. Whenever appropriate, thevariations in organ weights were compared after adjustmentfor multiple comparison of all pairs of means includingexperimental and control populations (Bonferroni test). TheKaplan-Meier product-limit estimate was used to analyzesurvival data and plot the survival function. The Breslow(generalized Wilcoxon) and Mantel-Cox (generalized Sav-age) statistics were applied to compare the survival func-tions of genetically distinct subgroups; there were no cen-sored observations. The Student t test procedure for unpairedsamples was applied in comparing mean weight values,mean tissue counts, or mean survival between subgroups,the standard error of the mean being presented with thesevalues in the text and tables.

TABLE 2. MST for C57BL/6 beige-J and +/+ mice infected at 6and 8 weeks of age with 3 x 104 C. neoformans cells

Genotype No. of Age MST SEMmice (weeks) (days)

+/+ 27 6 24.2 0.612bglbg 26 6 19.0 0.309+/+ 22 8 25.7 0.975bglbg 24 8 17.5 0.587

RESULTS

Influence of the beige mutation on susceptibility. Table 1presents the mean survival time (MST) for each populationin the study (dose, 105 yeast cells per mouse) in relation topublished data on NK activity (specific cytotoxicity toYAC-1 tumor cells), coat color, and H-2-associated genes.When differences in MST for beige and nonbeige mice(AMST) were examined, the beige mutation had a greaterimpact on survival in the C57BL/6 strain (AMST, 5.5 days;P < 0.001) than in the C3H/He strain (AMST, 1.4 days; P =0.009). These findings could not be explained by a differencein the baseline NK activity of these two strains of mice; onthe basis of H-2 and non-H-2 regulatory influences, slightlyhigher responses in both NK and natural cytotoxicity werelikely to be seen in the C3H/He environment. In addition,the difference at the agouti coat color locus is not likely to bemeaningful, since both C57BL/6 +/+ and C3H/HeJ +/+mice express the resistance phenotype and have similartissue counts upon death (unpublished data).

Further studies were carried out only with the C57BL/6strain. The infecting dose of cryptococci was reduced to 3 x104 yeast cells per mouse in an attempt to better discriminatethe two genotypes. A group of animals was infected at a laterage to evaluate the impact of this factor on the duration ofsurvival. The results are summarized in Table 2.The reduction of the infecting dose had little influence on

AMST (from 5.5 to 5.2 days) but definitely reduced thescattering of survival observations previously noted in theC57BL/6 population. The effect of age was unequivocal as

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DAYS OF INFECTION

FIG. 1. Survival plots for 6-week-old (U, O) and 8-week-old(A, A) C57BL/6 beige-J and +/+ mice infected with 3 x 104 C.neoformans (ATCC 13690) cells.

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290 MARQUIS ET AL.

LU.625

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DAYS OF INFECTION

FIG. 2. Survival plots for 6-week-old C3H/He beige-2J (*) andbgl+ (*) mice infected with 105 C. neoformans (ATCC 13690) cells.

the AMST for the 54-day-old age group increased to 8.2days.

Survival analysis. Plots of survival functions for the 42-and 54-day-old age groups are presented in Fig. 1. Thedifference in AMST between the two age groups was mainlyascribable to an increase in early mortality for the bglbggenotype, as the 75th quantile of the distribution droppedfrom 18 days in the 42-day-old age group to 15 days in the54-day-old age group (P = 0.05). When the survival rates ofC57BL/6 beige-J mice were compared with those of theparent strain, P values were found to be less than 0.0001.Figure 2 presents the survival functions of C3H/He bglbgand bgl+ mice. The P value for these data was established asfollows: 0.007 < P < 0.018.Organ weight profile. Uninfected C57BL/6 beige-J and

C57BL/6 mice were sacrificed at the median survival time ofeach genotype. For infected C57BL/6 beige-J and C57BL/6mice, changes in the weight of the brain, liver, and spleen,expressed as percentage values of appropriate uninfectedcontrol groups, were in concordance at the time of death.Representative figures for the 42-day-old age group chal-lenged with 3 x 104 cryptococci are: a 59.3 to 61.4% increasein the brain weight, a 63.8 to 65.8% decrease in the liverweight, and a 56.5 to 58.9% decrease in the spleen weight.With the 105 dose, the average increase in brain weightvalues for C57BL/6 beige-J and C57BL/6 mice exceededcontrol values by only 25.8 to 26.7% (P = 0.001 for allcomparisons with both control and low-dose experimentalpopulations). Clear differences in the organ weight profilesof the two genotypes were noted when infected C57BL/6animals (dose, 3 x 104; age, 42 days) were sacrificed at themedian survival time of the beige-J mutant; on days 18 and19, when experimental groups were compared with theirrespective control population, there was a 32 versus 53%increase in the brain weight (P = 0.001), a 41.4 versus 67.8%decrease in liver weight (P = 0.001), and 23.4 versus 58.6%decrease in spleen weight (P = 0.001) for +/+ and bglbgmice, respectively. All recorded variations in organ weightwere significant at the P = 0.001 level relative to appropriateuninfected control groups. Normal C57BL/6 mice werefound to have significantly higher mean organ weight valuesthan normal C57BL/6 beige-J animals (brain, 433.6 ± 5 mgversus 375 ± 6 mg, P < 0.001; liver, 1,386.4 ± 58 mg versus

1,203.1 + 51 mg, P = 0.027; spleen, 90 + 4 mg versus 66.2± 4 mg, P < 0.001).

Tissue counts. Figure 3 presents cumulative results ofquantitation of cryptococci in tissues of C57BL/6 andC57BL/6 beige-J mice on days 18 and 19 after infection with3 x 104 yeast cells per mouse. At that time C57BL/6 micehad lower counts than C57BL/6 beige-J mice in the brain(mean, 1.38 log units; P < 0.0001), liver (mean, 1.31 logunits; P < 0.0001), and spleen (mean, 1.98 log units; P <0.0001). For the two different infecting doses used in thisstudy, quantitation of cryptococci was systematically per-formed upon death in the C57BL/6 strain. For the twodifferent infecting doses, mean liver counts were found to beconsistently lower in C57BL/6 +/+ mice. The averagegrowth in the liver, in log units, at the time of death for +/+and bglbg mice was 6.206 ± 0.251 versus 7.188 ± 0.088when 105 cryptococci were injected (P = 0.002) and 6.785 ±0.114 versus 7.130 ± 0.064 when 3 x 104 cryptococci wereinjected (P = 0.012). No significant. difference in colonycounts in the brain or spleen was seen at the time of death,although with the 105 dose there was a definite trend towardlower mean counts of cryptococci in C57BL/6 mice relativeto C57BL/6 beige-J mice (brain, 7.678 ± 0.090 versus 7.929± 0.048, P = 0.022; spleen, 6.842 ± 0.178 versus 7.486 ±0.225, P = 0.034).

Histopathology. Well-defined, cystic collections of fungiwere scattered in the cerebrum and cerebellum of bothC57BL/6 and C57BL/6 beige-J mice. No inflammatory cellswere demonstrated in or around the cystic cavities exceptfor some necrotic macrophages in the center of the cysts. Inthe leptomeninges, there were isolated or focally clusteredcryptococci with a few polymorphonuclear leukocytes, lym-phocytes, and macrophages.

Histological studies of the liver revealed that both C57BL/6and C57BL/6 beige-J mice developed a granulomatous hep-atitis, although the architecture of the granulomas varied. Inthe C57BL/6 +/+ mice, accumulations of macrophagescontaining only few cryptococci were surrounded by a largenumber of lymphocytes and a few polymorphonuclear leuko-

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BRAIN LIVER SPLEENFIG. 3. Mean tissue colony counts of cryptococci on days 18 and

19 of infection for C57BL/6 beige-J and +/+ mice. Standarddeviations are illustrated.

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BEIGE MUTATION IN MURINE CRYPTOCOCCOSIS 291

cytes (Fig. 4a). Conversely, in the C57BL/6 beige-J mice,macrophages engorged with multiple blastospores were sur-rounded by a few lymphocytes and a large number ofpolymorphonuclear leukocytes (Fig. 4b). Well-demarcatedlymphoid follicles and few cystic collections of fungi wereobserved in the spleen of C57BL/6 mice. In the spleen ofbeige mice, cryptococcal cysts were more numerous andlymphoid follicles were more ill defined.

DISCUSSION

We have shown that the bg locus on chromosome 13influences natural host resistance to cryptococcosis. In-creased susceptibility was documented in two different in-bred strains, although the large difference in AMST betweenthe two genetic backgrounds indicates that other autosomalgenes probably contribute to prolonged survival and height-ened resistance. Evidence for a complex interplay of multi-genic influences is provided by the failure to demonstrateany accrued susceptibility of C57BL/6 beige-J mice, relativeto bgl+ or +/+ littermates, in experimental infections withListeria monocytogenes (6), Leishmania tropica (16),

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FIG. 4. (a) Photomicrograph of a granulomatous formation in theliver of a C57BL/6 +/+ mouse. Macrophages containing isolatedcryptococci are surrounded by a large number of lymphocytes and a

few polymorphonuclear leukocytes (x 1,200; H&E). (b) Photomicro-graph of a granulomatous formation in the liver of a C57BL/6 beige-Jmouse. Clusters of cryptococci and macrophages engorged withnumerous yeast cells are seen to be surrounded by a few lympho-cytes and a large number of polymorphonuclear leukocytes (x 1,200;H&E).

Plasmodium chabaudi (31), Trypanosoma rhodesiense (14),and Toxoplasma gondii (15). The difference in AMST in ourstudy is not ascribable to a difference at the Hc locus forhemolytic complement since both strains of mice carry theHc' allele which was shown to segregate with the resistancephenotype in a murine cryptococcosis model (25). By com-parative studies performed at the median survival time of thebeige mutant group, we were able to demonstrate that thebiological response to the infectious agent was different inbglbg and +/+ mice. Although both the changes in organweights and the growth of cryptococci in tissues tended to afinal common point, the time sequence of these events wasby no means comparable in the two genotypes. The liver wasthe single site where colony counts of the two genotypeswere most clearly different, at least during the late phase ofthe infection which was our concern in this study.A difference was noted in the relative survival time of our

two beige populations after challenge with C. neoformans.Although both had a shorter lifespan than the parent strain,C3H/He beige-2J mice were less susceptible than C57BL/6beige-J mice when compared with nonbeige mice of similarbackground. The reasons for such a difference are unclear.On the one hand, the genes bgJ and bg2J, although theproduct of two independent mutations, are believed to betrue alleles at the same locus with similar penetrance andbiological effects (4). On the other hand, no meaningfuldifference has so far been documented between the +/+ andbgl+ genotypes in the C57BL/strain, either through in vitroassays (26, 27) or in in vivo resistance to Candida albicans(9), L. monocytogenes (6), or P. chabaudi (31). An addi-tional factor was the presence of the Lpsd allele at the Lpslocus on chromosome 4 in the C3H/HeJ strain (35) which isknown to determine some impairment in the activation,bactericidal, and tumoricidal activity of macrophages (24,36); this mutant gene was seen as a significant factor whichwould further compromise the ability of C3H/HeJ beige-2Jmice to cope with C. neoformans. The kinetics of macro-phage-mediated cytolysis of tumor cells has already beenshown to be abnormal in mice with the bglbg genotype (20).Our results indicate that, despite their state of immunodefi-ciency, C3H/HeJ beige-2J mice displayed considerable re-sistance to C. neoformans. More work will be required torule out a difference in the biological effect of the bgJ andbg2J mutations or uncover genetic or immunological factorswhich may partially offset the negative influence of the Lpsdand bg2J genes on host resistance to infectious agents.Another question which must be addressed is the useful-

ness of Chediak-Higashi mice as a model for NK celldeficiency. Recent reports show only a reduction in thebaseline NK activity measured in beige mice (5, 22). Inaddition, these NK effector cells are by no means unrespon-sive to environmental stimuli as a two- to threefold increasein NK activity has been documented after infection withvesicular stomatitis virus (22) or in vivo stimulation with aninterferon inducer (15). Significant in vitro NK cell activa-tion by Corynebacterium parvum has also been reported forthe bglbg genotype (5), although in both instances theoverall activity of the beige mutant is about one half of thatobserved in similarly treated +/+ or bgl+ mice. Anotherunexplored area is the ability of these effector cells to copewith various numbers of microorganisms: is this mechanismpotent or easily overwhelmed? An interesting hypothesis isthat there is a difference in the two beige populations in thisstudy in their respective ability to activate NK effector cellsafter challenge with C. neoformans. A differential responsein NK activation could be supported by the genetic environ-

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292 MARQUIS ET AL.

ment, such as the presence in the C3H/HeJ strain of theH-2Dk and Qa2,3b haplotypes, which are known to posi-tively influence NK and natural cytotoxic cell reactivity (17,33). Host- or organism-derived inhibitory substances couldalso modulate NK cell reactivity (1). More work is needed todefine more clearly the role of NK cells in host resistance tocryptococcosis and other infectious agents.

Careful consideration must also be given to the fact thatother important effector mechanisms of host resistance maybe altered in Chediak-Higashi mice. These mice have beenused for years as a model for abnormal granulocytes. Thenumber of circulating polymorphonuclear leukocytes is notreduced (3), but they display abnormal bactericidal kinetics(12, 13) as a result of a delay in forming phagolysosomes(29). The metabolic burst of Chediak-Higashi granulocytes isnormal, whereas iodination and oxygen consumption areincreased above normal levels (28, 29). Their chemotacticactivity is abnormal in vitro (12, 13), but cellular exudativeresponses were not shown to be diminished in mice with thebglbg genotype (21). Gadebusch has shown that normalmouse granulocytes can actively ingest C. neoformans (11).Others have demonstrated that this organism is very sus-ceptible to multiple components of the human neutrophil:myeloperoxidase-dependent oxidative mechanisms, lowconcentrations of hydrogen peroxide, as well as cationicproteins (8, 18). The ability of beige mutant mice to mount astrong inflammatory response to C. neoformans was ascer-tained by the histopathological findings in our study (Fig.4b). Therefore, the granulocyte abnormality is not likely tohave a primary role in explaining how C57BL/6 beige-J micecould develop tissue burdens of crytococci in excess of a fulllog unit over C57BL/6 +/+ mice on days 18 and 19 of theinfection.Another probable mechanism operating against C. neofor-

mans involves cytotoxic T lymphocytes. Specifically sensi-tized lymphoid cells, which were shown to be capable ofinhibiting the growth of the yeast, should be present in thespleen of infected animals at the end of week 1 (10). Theability of beige mice to develop normal levels of cytotoxic Tlymphocytes is in doubt. Although some investigators didnot find any evidence of impairment (22, 27), others havereported a reduction in inducible T lymphocyte cytotoxicityto tumor and virus-infected cells ranging from one-half toone-third of that of nonbeige control mice (30, 37). Indeed,our histopathological results indicate that fully developedgranulomas with a predominance of lymphocytes are infre-quently encountered in mice with the bglbg genotype. Fur-ther study of the growth-inhibiting activity of specific cyto-toxic T lymphocytes against C. neoformans in the beigemodel will be helpful in gaining a better understanding ofhost defenses in cryptococcosis.

ACKNOWLEDGMENTSWe thank Juneann W. Murphy and Tim Yoshino for reviewing this

manuscript.This work was supported in part by the Fondation Justine-Lacoste-

Beaubien, the Medical Research Council of Canada, and theUniversitd de Montreal, G.M. is grateful to the Medical ResearchCouncil of Canada for the current postdoctoral fellowship award.

LITERATURE CITED1. Albright, J. W., K.-Y. Huang, and J. F. Albright. 1983. Natural

killer activity in mice infected with Trypanosoma musculi.Infect. Immun. 40:869-875.

2. Auger, P., G. Marquis, and L. Dallaire. 1979. Viability assess-ment by dye exclusion. A fluorescent method for fungal cells.Arch. Dermatol. 115:1195-1196.

3. Bennett, J. M., R. S. Blume, and S. M. Wolff. 1969. Character-ization and significance of abnormal leukocyte granules in thebeige mouse: a possible homologue for Chediak-Higashi Aleu-tian trait. J. Lab. Clin. Med. 73:235-243.

4. Brandt, E. J., R. T. Swank, and E. K. Novak. 1981. The murineChediak-Higashi mutation and other murine pigmentation mu-tations, p. 99-117. In M. E. Gershwin and B. Merchant (ed.),Immunologic defects in laboratory animals, vol. 1. PlenumPublishing Corp., New York.

5. Brunda, M. J., H. T. Holden, and R. B. Heberman. 1980.Augmentation of natural killer cell activity of beige mice byinterferon and interferon inducers, p. 411-415. In R. B. Herber-man (ed.), Natural cell-mediated immunity against tumors.Academic Press, Inc., New York.

6. Cheers, C., and J. Macgeorge. 1982. Genetic and cellularmechanisms of natural resistance to intracellular bacteria, p.1521-1526. In R. B. Herberman (ed.), NK cells and othernatural effector cells. Academic Press, Inc., New York.

7. Clark, E. A., L. D. Schultz, and S. B. Pollack. 1981. Mutationsin mice that influence natural killer (NK) cell activity. Immuno-genetics 12:601-613.

8. Diamond, R. D., R. K. Root, and J. E. Bennett. 1972. Factorsinfluencing killing of Cryptococcus neoformans by human leuko-cytes in vitro. J. Infect. Dis. 125:367-376.

9. Elin, R. J., J. B. Edelin, and S. M. Wolff. 1974. Infection andimmunoglobulin concentrations in Chediak-Higashi mice. In-fect. Immun. 10:88-91.

10. Fung, P. Y. S., and J. W. Murphy. 1982. In vitro interactions ofimmune lymphocytes and Cryptococcus neoformans. Infect.Immun. 36:1128-1138.

11. Gadebusch, H. H. 1959. Phagocytosis of Cryptococcus neofor-mans in anemic mice. J. Bacteriol. 78:259-262.

12. Gallin, J. I., J. S. Bujak, E. Patten, and S. M. Wolff. 1974.Granulocyte function in the Chediak-Higashi syndrome of mice.Blood 43:201-206.

13. Gallin, J. I., R. J. Elin, R. T. Hubert, A. S. Fauci, M. A.Kaliner, and S. M. Wolff. 1979. Efficacy of ascorbic acid inChediak-Higashi syndrome (CHS): studies in humans and mice.Blood 53:226-234.

14. Jones, J. F., and G. E. Hancock. 1983. Trypanosomiasis in micewith naturally occurring immunodeficiencies. Infect. Immun.42:848-851.

15. Kamiyama, T. 1984. Toxoplasma-induced activities of perito-neal and spleen natural killer cells from beige mice againstthymocytes and YAC-1 lymphoma targets. Infect. Immun.43:973-980.

16. Kirkpatrick, C. E., and J. P. Farrell. 1982. Leishmaniasis inbeige mice. Infect. Immun. 38:1208-1216.

17. Klein, G. 0. 1982. NK-activity against YAC-1 is regulated bytwo H-2 associated genes, p. 275-280. In R. B. Herberman(ed.), NK cells and other natural effector cells. Academic Press,Inc., New York.

18. Lehrer, R. I., and K. M. Ladra. 1977. Fungicidal components ofmammalian granulocytes active against Cryptococcus neofor-mans. J. Infect. Dis. 136:96-99.

19. Lutzner, M. A., C. T. Lowrie, and H. W. Jordan. 1967. Giantgranules in leukocytes of the beige mouse. J. Hered. 58:299-300.

20. Mahoney, K. H., S. S. Morse, and P. S. Morahan. 1980.Macrophage functions in beige (Chediak-Higashi syndrome)mice. Cancer Res. 40:3934-3939.

21. McGarry, M. P., E. J. Brandt, and R.T. Swank. 1976. Eosino-phil and neutrophil granulocyte exudation in the Chediak-Higashi (beige) mouse. Am. J. Pathol. 85:685-692.

22. McKinnon, K. P., A. H. Hale, and M. J. Ruebush. 1981.Elicitation of natural killer cells in beige mice by infection withvesicular stomatitis virus. Infect. Immun. 32:204-210.

23. Murphy, J. W., and D. 0. McDaniel. 1982. In vitro reactivity ofnatural killer (NK) cells against Cryptococcus neoformans. J.Immunol. 128:1577-1583.

24. O'Brien, A. D., E. S. Metcalf, and D. L. Rosenstreich. 1982.Defect in macrophage effector function confers Salmonellatyphimurium susceptibility on C3H/HeJ mice. Cell. Immunol.67:325-333.

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BEIGE MUTATION IN MURINE CRYPTOCOCCOSIS 293

25. Rhodes, J. C., L. S. Wicker, and W. J. Urba. 1980. Geneticcontrol of susceptibility to Cryptococcus neoformans in mice.Infect. Immun. 29:494-499.

26. Roder, J. C. 1979. The beige mutation in the mouse. I. A stemcell predetermined impairment in natural killer cell function. J.Immunol. 123:2168-2173.

27. Roder, J. C., M. L. Lohmann-Matthes, W. Domzig, and H.Wigzell. 1979. The beige mutation in the mouse. II. Selectivityof the natural killer (NK) cell defect. J. Immunol. 123:2174-2181.

28. Root, R. K. 1975. Comparison of other defects of granulocyteoxidative killing mechanisms with chronic granulomatous dis-ease, p. 201-226. In J. A. Bellanti and D. H. Dayton (ed.), Thephagocytic cell in host resistance. Raven Press, New York.

29. Root, R. K., A. S. Rosenthal, and D. J. Balestra. 1972. Abnormalbactericidal, metabolic, and lysosomal functions of Chediak-Higashi syndrome leukocytes. J. Clin. Invest. 51:649-665.

30. Saxena, R. K., Q. B. Saxena, and W. H. Alder. 1982. DefectiveT-cell response in beige mutant mice. Nature (London)295:240-241.

31. Skamene, E., M. M. Stevenson, and S. Lemieux. 1983. Murinemalaria: dissociation of natural killer (NK) cell activity andresistance to Plasmodium chabaudi. Parasite Immunol.

5:557-565.32. Stutman, O., and M. J. Cuttito. 1981. Normal levels of natural

cytotoxic cells against solid tumors in NK-deficient beige mice.Nature (London) 290:254-257.

33. Stutman, O., and M. J. Cuttito. 1982. Genetic control of NCactivity in the mouse: three genes located in chromosome 17, p.281-289. In R. B. Herberman (ed.), NK cells and other naturaleffector cells. Academic Press, Inc., New York.

34. Vassalli, J.-D., A. Granelli-Piperno, C. Griscelli, and E. Reich.1978. Specific protease deficiency in polymorphonuclear leuko-cytes of Chediak-Higashi syndrome and beige mice. J. Exp.Med. 147:1285-1290.

35. Vogel, S. N., A. C. Weinblatt, and D. L. Rosenstreich. 1981.Inherent macrophage defects in mice, p. 327-357. In M. E.Gershwin and B. Merchant (ed.), Immunologic defects in labo-ratory animals, vol. 1. Plenum Publishing Corp., New York.

36. Watson, J., R. Riblet, and B. J. Taylor. 1977. The response ofrecombinant inbred strains of mice to bacterial lipopolysaccha-rides. J. Immunol. 118:2088-2093.

37. Welsh, R. M., Jr., and R. W. Kiessling. 1980. Natural killer cellresponse to lymphocytic choriomeningitis virus in beige mice.Scand. J. Immunol. 11:363-367.

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