Selected immune responses of adult cotton rats (Sigmodon hispidus) to dietary restriction

Cowp.

Biochem.

Vol. 104A, No.

Physiol.

0300-9629/93 $6.00 + 0.00

3. pp. 593-599, 1993

0 1993Pergamon Press Ltd

Printed in Great Britain

SELECTED IMMUNE RESPONSES OF ADULT COTTON RATS (SIGMODON HISPIDUS) TO DIETARY RESTRICTION ROBERT L. LOCHMILLER,MICHELLE R. VESTEYand SCOTT T. MCMURRY Department of Zoology, Oklahoma State University, Stillwater, OK 74078, U.S.A (Tel. 405-744-5555)

(Received 18 June 1992; accepted 16 July 1992) Abstract-l.

We examined the influence of moderate to severe dietary restriction on immune system function in adult cotton rats. Animals (N = 80) were fed ad lib. (controls) or restricted [moderate = 80% ad lib. for 1 or 2 weeks; severe = 80% ad lib. for 1 or 2 weeks followed by 40% ad lib. for one (week 3) or two (week 4) additional weeks] amounts of food for 1-t weeks. 2. Average body weight loss for severely restricted animals in week 4 was 17%; dietary treatments had no measurable effect on hematological parameters (hematocrit, white blood cell count), lymphoid organ weights (thymus gland, spleen, popliteal lymph nodes), and mononuclear cell yields from lymphoid organs. 3. Cell-mediated immune function was assessed in vitro by a lymphoproliferative response assay and in viuo by a delayed-type hypersensitivity response assay. 4. Proliferative responses of spleen cell cultures stimulated with concanavalin A (Con A, Conaualiu ensiformis) and pokeweed (PWM, Phytolucca americana) were normal or significantly greater among moderately restricted than control cotton rats during week 1 and week 2. 5. Lymphoproliferative responses of severely restricted animals were normal or reduced during week 3 and week 4. 6. Delayed-type hypersensitivity responses to the contact antigen oxazolone were significantly depressed among severely restricted animals in week 4 compared to controls. 7. In comparison with laboratory rodent strains, our initial results indicate that immune system function in adult cotton rats is not as sensitive to short term (14 weeks) periods of dietary restriction. 8. Immune system function was related to changes in body weight as a result of feed restriction.

specific mortality in fluctuating wild populations of small mammals have largely been unsuccessful (Mihok er al., 1988). Small mammals who develop a disease condition are probably eliminated from the population through predation well before causespecific mortality can be determined. We have been elucidating possible mechanisms by which nutrition influences mortality rates in cotton rat populations. In this study we examined the consequences of short-term, moderate feed restriction on measures of cell-mediated immunity in adult cotton rats. Recent studies with Microtus arvalis (Dobrowolska and Adamczewska-Andrzerjewska, 1991) and Antechinus stuartii (Barker et al., 1978; Bradley et al., 1980) have provided evidence of relationships between altered immunocompetence and population dynamics.

INTRODUCTION It has become clear that an intimate relationship exists between nutritional status, immunocompete Ice, and disease in humans (reviewed by Chandra ar d Newberne, 1977; Gershwin ef al., 1985). Investiga tors have demonstrated that severe malnutrition can result in significant atrophy of major lymphoid organs (Bell et al., 1976) and impairment of cell-mediited immunity (Chandra et al., 1982), humoral in munity (Filteau et al., 1987), macrophage phagocy tic activity (Keusch et al., 1978), and other nonspe-

ciic defense mechanisms (Michalek et nl., 1974). A’lditionally, even moderate malnutrition may alter inmunocompetence (McMurray et al., 1981). Impaired immunity can lead to decreased resistance to di (ease agents, including bacteria, viruses, protozoa, ard parasites (Keusch and Katz, 1979). Malnutrition is undoubtedly a common occurrence in wild herbivore populations (White, 1978; Hansson, 1979, Belovsky, 1986), but we know little about its re ationship to immunocompetence and consequent alterations in population dynamics. Many small m;ammal populations such as the cotton rat (Sigmodon hispidus) fluctuate widely both within and between years (Goertz, 1964; Fleharty et al., 1972), possibly in response to changing nutritional conditions in the habitat. Attempts to document cause-

MATERIALS

AND METHODS

Animals and experimental design

Adult cotton rats used in this study were 4-6 months-old and born to parents maintained in our outbred laboratory colony. Animals were maintained on a commercial laboratory ration for rodents (Purina Mills, St Louis, MO) under a 15L:9D light-dark cycle at room temperature. A total of 80 593

594

ROBERT L. LOCHMILLER

offspring from 20 litters were weighed and randomly assigned to a control (laboratory chow fed ad lib.) or restricted (moderate and severe) dietary group for l-4 weeks duration. Animals within each treatment group (N = 10) were blocked according to litter and housed in pairs in hanging wire-bottom cages (24 x 18 x 18 cm). Moderately restricted animals were fed 80% ad lib. (22 g chow/cage) for one or two weeks; severely restricted animals were fed 80% ad lib. during the first 2 weeks and 40% ad lib. (11 g chow/cage) for 1 (week 3) or 2 (week 4) additional weeks. Ad lib. feed intake was measured prior to the start of the trial. Average body weight loss for animals in the 4 week, severely restricted group was about 17% of their initial weight. One cotton rat (severely restricted, week 3) died of unknown causes during the trial. Cotton rats terminated on weeks l-4 were anesthetized with an intramuscular injection of ketamine hydrochloride (Aveco Co., Inc., Fort Dodge, IA 50501) at 50 mg/kg body wt. Terminal body weights were recorded and percentage change in body weight determined as 100 x (initial - terminal)/initial. Blood for hematologies was collected via the retro-orbital sinus plexus into tubes containing EDTA-K,. Animals were killed by cervical dislocation while under ketamine anesthesia. Lymphoid organs and hematology Weights of thymus gland, spleen, and paired popliteal lymph nodes were determined to the nearest 0.1 mg. All tissues were freed of adhering fat before weighing. Mononuclear cell yields were determined for thymus gland and lymph nodes (see below for spleen) by gently dissociating cells using a glass-onglass tissue homogenizer (0.15 mm clearance) in phosphate buffered saline (PBS). Viable cell counts were performed with a hemacytometer after lysing erythrocytes in T&buffered 0.83% ammonium chloride and staining with trypan blue. Hematocrits were determined by the microcapillary tube-centrifuge method. White blood cell (WBC) counts were determined manually using a hemacytometer. Lymphoproliferative response assay Cell-mediated immunity was assessed in vitro using lymphoproliferation assay. Spleens were aseptically removed, placed in preweighed 15 x 60 mm sterile petri dishes containing RPM1 1640 culture medium (Sigma Chemical Co., St Louis, MO 63178) and weighed. Spleens were cut into 3-4 pieces and gently dissociated in a sterile tissue homogenizer containing 5 ml ice-cold supplemented RPM1 medium (RPMI-S) consisting of 1.025% L-glutamine (200 mM, Sigma Chemical Co.), 1.0% Na pyruvate (100 mM Sigma Chemical Co.), 1.0% non-essential amino acids (10 mM, 100 x , Sigma Chemical Co.), 1.O% penicillin (10,000 U/ml)-streptomycin (10 mg/ml) solution

et al.

(Sigma Chemical Co.), 0.1% 2-mercaptoethanol (2 x lo-’ M, 1: 1000 dilution in sterile PBS, Sigma Chemical Co.), and 10.0% normal horse serum (Sigma Chemical Co). Cells were allowed to settle for 1Omin and supernatant decanted into sterile 16 x 125 mm screw-cap culture tubes. Cells were centrifuged for 7 min at 10°C and 275 g, the supernatant was decanted, and pellet resuspended in 5 ml RPMI-S (this wash step was repeated twice). Viable cell counts were performed with a hemacytometer after lysing erythrocytes from a small aliquot with Tris-buffered 0.83% ammonium chloride and staining with trypan blue; mononuclear cell viability was consistently > 90%. Lymphocyte proliferation, or blast transformation, of T and B cells in response to polyclonal mitogenic stimulation was assessed in vitro using the plant lectins concanavahn A (Con A) and pokeweed mitogen (PWM), respectively, obtained from Sigma Chemical Co. The spleen cell suspension was adjusted to a final concentration of 500,000 cells/90 ~1 in RPMI-S. Four concentrations of each mitogen (Con A at 2.5, 5, 10, and 20pg/ml of culture; PWM at 0.156, 0.313, 0.625, and 1.25 pg/ml of culture) were added (10 ~1 volumes) to duplicate 90 ~1 of aliquots of the final cell suspension in 96-well, flat-bottom microtiter plates (Flow Laboratories, McLean, VA 22102). Unstimulated control cultures were used as blanks. Cell cultures were incubated for 72 hr at 37°C in a humidified incubator with 5% CO,-95% air. Lymphocyte proliferation was assessed by cellular reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, Sigma Chemical Co.] using the procedure described by Mosmann (1983). Briefly, after 69 hr of incubation, 10 nl of MTT (5 mg/ml in sterile PBS) were added to each well, incubated the remaining 3 hr, and cultures terminated with the addition of 160 ~1 of acid-isopropanol (0.04N HCl in isopropanol). End-points were recorded as absorbances at 570/630 nm on a Titertek Multiskan Plus MK II spectrophotometer (Flow Laboratories) against unstimulated controls as blanks. Delayed- type hypersens. tivity response assay Cell-mediated immunity was assessed in vivo by measuring delayed contact hypersensitivity to oxazalone (4-ethoxy-methylene-2-phenyl-oxazol-5-one; Sigma Chemical Co.) as described by Jones (1984). Briefly, animals were sensitized with a percutaneous application of 100 ~1 of a 3% solution of oxazalone in absolute alcohol to their shaved abdomen. Seven days later animals were challenged with 25 ~1 of oxazalone solution to each surface of the left ear and 25 ~1 of vehicle to each surface of the right ear. Ear thickness was measured to the nearest 0.005 mm using a constant tension digital micrometer. End point responses were expressed as the difference in thickness between the challenged (left) and control (right) ear.

595

Immune responses of diet restricted Sigmodon Statistical analyses

I, Means

Table

bematological

morphology and immune response assays between control and moderately restricted dietary groups within each week were tested by analysis of variance with litter treated as a bhck effect (PROC GLM; SAS Institute Inc., 1982). Prl jbably as a result of both genetic differences and co~npetition between individuals within cages, weight ch;tnges fluctuated widely within both dietary groups. Su~tantial weight loss was observed among a few an mals in the control group and several animals in the restricted group maintained initial body weight. As a result, Pearson correlation coefficients (SAS In: titute Inc., 1982) were also calculated to evaluate the relationships between selected immune response pa.ameters and percentage change in body weight. Differences

in immune

organ

RESULTS

nitial body weights averaged 199.9 + 3.1 g (SE) an1 did not differ between diet treatment groups. W :ight loss among moderately restricted cotton rats during the 1st two weeks of the trial was not substantial (Fig. l), but increased for severely restricted co ton rats in week 3 and week 4 compared to co rtrols (P < 0.05). Average weight loss of severely re: tricted cotton rats after 4 weeks was 17.6 f 2.4%. HCmatology

white blood cell counts ranged from 1.68 to 21 04 x 103/mm3 blood and showed no significant (P > 0.05) difference between diet treatment groups wihin any week (Table 1). Hematocrits were not intluenced significantly (P > 0.05) by moderate or se1‘ere dietary restriction (Table 1). LJ mphoid organ responses

Moderate and severe dietary restriction had no significant (P > 0.05) influence on weights of thymus

20 c g

Control 0 Restricted

and

corresponding standard

and immunological

Parameter White

blood

Hematocrit Liver,

SE

7.24

0.37

Mononuclear

cell yield

0.21

weight

(cells x IO’)

9.56

0.55

(cells x IO’/mg)

7.85

0.37

34.3

(mg)

cell yield

(cells x IO’)

Mononuclear

cell yield

(cells x 106/mg)

node,

4.4

122.1

Mononuclear lymph

0.4

5.64

(mg) cell yield

weight

(mg,

I.4

4.82

paired)

0.34

I .34

0.06

4.7

0.,2

Mononuclear

cell yield

(cells x 106)

I .93

0.02

Mononuclear

cell yield

(cells x lO’/mg)

3.85

0.39

(N = 79); values did not differ trol,

moderately

observations

restricted,

were

significantly

(P > 0.05)

and severely

restricted

among groups

conso all

pooled

gland, spleen, paired popliteal lymph nodes, and liver compared to controls within each week (Table 1). The thymus gland in 4-6 month-old cotton rats is relatively small compared with juveniles due to an obligatory age-related reduction in size. Mononuclear cell yields from thymus gland, spleen, and lymph nodes, expressed both on an absolute and relative (per mg tissue) basis, showed no significant (P > 0.05) difference between diet treatment groups during any week. Cell yields were greatest in the spleen, averaging 9.56 f 0.55 x 10’ cells (Table 1); paired popliteal lymph nodes yielded about a tenth as many mononuclear cells. Pearson correlation .analysis indicated a significant depression in mononuclear cell yield in the thymus gland with increased loss of body weight (Table 2). In vitro cell-mediated immune responses All cultures of spleen cells were responsive to the T-cell mitogen Con A and the T-B-cell mitogen PWM (Table 3). Optimum concentrations for mitogenie stimulation of our cell culture system was predetermined to be 5.0 pg/ml for Con A and b.625pg/ml for PWM. -

B8

Week

rats

Mean

(g)

gland,

of selected

42.2

Spleen, weight Mononuclear

Popliteal

(SE)

of adult cotton

cells (cells x IO’jmm’) (%)

weight

Thymus

errors

characteristics

of Trial

Fig. 1. Mean (standard error bars) change in body weight [initial - final)/initial) x 1001of adult cotton rats fed ad lib. (control), moderately restricted (80% ad lib. for 1-2 weeks), or severely restricted (80% ad lib. for 2 weeks and 40% ad lib. for l-2 weeks) diets.

596

ROBERT L. LBCHMILLER et al.

Table 2. Pearson correlation coefficients for the relationship between selected measures of immune system function and percentage change in body weight of adult cotton rats fed ad lib. or restricted diets for 2, 3, or 4 weeks Immune parameter Thymus mononuclear cell yield Delayed-type hypersensitivity response Lymphoproliferation Con A, 5.0 @g/ml 10.0 pg/ml 20.0 pg/ml PWM, 0.156 fig/ml 0.313pg/ml 0.625 ueiml I.250 b$ml

Week 2 Week 3 Week 4 Overall (N = 20) (N = 19) (N = 20) (N = 79)

0.46;

0.57”

0.24’

0.60”

0.2a** 0.24’ 0.30” 0.29**

0.51*

0.53” 0.49’ 0.46’

0.26. 0.28” 0.29’. 0.28**

Overall correlation coefficients were determined by pooling all 79 animals from weeks l-4. fP < 0.05. **p < 0.01.

Lymphoproliferative responses of cotton rats were variable, but appeared to be influenced by moderate dietary restriction. Proliferative responses of spleen cell cultures stimulated with 10.0 pg Con A/ml were significantly greater (P < 0.05) among moderately restricted than control cotton rats during week 1 and week 2. A similar trend was apparent for cultures stimulated with 5.0 pg Con A/ml. Spleen cell cultures stimulated with PWM from moderately restricted cotton rats during week 1, and to some degree week 2, appeared to show an increased ability to undergo blastogenesis as well. Mean absorbency value for cultures of moderately restricted animals stimulated with 0.313 pg PWM/ml was 34% higher (P < 0.05) than the mean absorbance value for cells of control animals in week 1. In general, lymphoproliferative responses of severely restricted animals were comparable with controls or were slightly reduced after week 3 or week 4. The mean absorbance value for cell cultures of restricted animals stimulated with 0.625 pg PWM/ml was 39% lower (P < 0.05) than controls in week 4. A similar trend was suggested by absorbances for cultures stimulated with 0.313 pg PWM/ml. Unstimulated cultures from severely restricted cotton rats in week 3 showed a tendency for lower spontaneous proliferation compared to control animals. Significant correlations were evident between lymphoproliferative responsiveness and change in body weight (Table 2). Opposite cell-mediated immune responses of severely restricted animals in week 3 and week 4, compared to those in the first 2 weeks, contributed to low overall correlation coefficients for these relationships. In vivo cell-mediated immune responses No significant differences (P > 0.05) among responses in the delayed oxazolone contact hypersensitivity test were observed between dietary groups

Immune

responses

30T

c

251 I

E

20

x .?f

.$ Lo

Control

s

10

m

a I

a a

15

0

Restricted

; = E x

591

Sigmodon

of diet restricted

a a

a

a

x rJ n iI

Week

i

i

of Trial

Fig. 2. Delayed-type hypersensitivity responsiveness to a percutaneous application of oxazolone in sensitized adult cotton rats as influenced by l-4 weeks of ad lib. (control) or restricted (moderately restricted received 80% ad lib. for 1-2 weeks; severely restricted received 80% ad lib. for 2 weeks and 40% ad. lib. for l-2 weeks) diets. Responsiveness was expressed as the mean increase in ear thickness in units of IO-*mm (standard error bars). Means within each week followed by different superscripts are significantly different (P < 0.05; ANOVA).

weeks l-3 (Fig. 2). Delayed-type hypersensidepressed tiv ity responses were significantly (P < 0.05) among severely restricted cotton rats in week 4 compared to controls. Only l/l0 cotton rats in :he restricted group had a response greater than the mc an response of controls (S/l0 > Mean) in week 4. Dc layed-type hypersensitivity responses were significa itly correlated with percentage change in body weight for week 2, week 3, and overall (Table 2). during

DISCUSSION

fhere were considerable individual differences in re! ponse for all measured parameters in this study. A m;.jor part of this variability can probably be attri>uted to genetic differences, both in terms of immune system physiology (Gasser and Silvers, 1974; SCmltz and Bailey, 1975) and nutritional requiremr.nts (Nesheim, 1972). Additionally, competition for food within a cage may have contributed to the va-iability in weight loss observed. Litter effects (tr:ated as a block effect in ANOVA) were highly significant (statistics not reported) for several immunological parameters in this study, supporting a genetic basis to the observed variability. Despite this inherent variability, results of this study suggest that cell-mediated immune function in adult cotton rats is alt:red in response to moderate weight loss from dietary restriction and that duration and severity of dir tary restriction will influence the nature of the immunological response. Lymphocyte function is routinely assessed quantitatively in vitro by the selective stimulation of T- and B- cells by mitogenic agents such as plant lectins (Stites et al., 1987). Lymphocyte activation in culture with mitogens represents a complex array of biochemical events, including cell-cell interactions,

which are thought to correlate with the in uivo processes that occur during antigen-specific stimulation. Cells are routinely cultured with a range of concentrations of stimulant since altered lymphocyte function may result in shifts in dose-response curves. The in viuo delayed hypersensitivity test provides a quantitative means of assessing cell-mediated immune function in a host (Buckley, 1986). The delayed hypersensitivity reaction is a measurable result of a number of events including antigen recognition, lymphocyte and macrophage interaction, cellular infiltration, release of soluble lymphocyte mediators, local edema, and changes in vascularity. A quantitatively normal level of delayed cutaneous hypersensitivity often suggests that alterations in the mechanisms responsible for cellular immunity are of little pathophysiologic significance (Buckley, 1986). Lymphoproliferation assays suggested that early moderate dietary restriction can lead to a heightened responsiveness to Con A and PWM stimulation. In contrast, cotton rats subjected to prolonged (4 weeks) dietary restriction showed normal responsiveness to Con A and reduced responsiveness to PWM stimulation. The mechanism for the heightened lymphocyte responsiveness to mitogens during early dietary restriction is not known. Several investigators have documented similar responses to moderate protein-calorie restriction in laboratory animals (Malave et al., 1980; Weindruch et al., 1982; Hoffman-Goetz et al., 1985). Alteration in suppressor T-cell activity of moderately restricted laboratory mice has been demonstrated (Malave and Pocino, 1981). However, this may not be an adequate explanation for our observations, as delayed-type hypersensitivity responses, which involve the activation of suppressor T-cells (Liew, 1977), were not enhanced during weeks l-3.

598

ROBERT L. LOCHMILLER et al

Hoffman-Goetz et al. (1985) reported that enhanced monocyte-macrophage production of interleukin- 1 helped explain the observed heightened responses to Con A stimulation in calorie restricted laboratory rabbits. Changes in splenic subpopulations of lymphocytes (for example, proportion of Con A or PWM responsive lymphocytes) could also account for the observed changes in mitogenic responsiveness of moderately restricted cotton rats, as suggested by Weindruch et al. (1982). It is also possible that subclinical infection in restricted animals may have occurred, resulting in an artificial elevation in mitogenie responsiveness in weeks 1 and 2. Severe dietary restriction can often result in a significant suppression of mitogenic responsiveness of lymphocytes (Keusch and Katz, 1979); similar responses were only evident in week 4 in this study. Alterations in response as measured by this in vitro assay of cell-mediated immunity were supported by an observed depression in delayed cutaneous hypersensitivity (in vivo) of cotton rats to oxazolone. The depressed proliferative response of spleen cells from severely restricted cotton rats to PWM (compared to Con A) suggests an impairment of B-cell function. There is evidence that oxazolone contact sensitivity in mice, in addition to involving T-cells, also involves B-cells and antibody to some degree (Phanuphak et al., 1974). Alternatively, T suppressor cells of cotton rats may be less sensitive to caloric restriction than other subpopulations, and after 4 weeks may represent a much larger proportion of T-cells. This could account for the suppressed delayed-type hypersensitivity response, and what appears to be a B-cell impairment in the lymphoproliferative response, could reflect a suppression of B-cell responses to PWM by T suppressor cells. Malnutrition, both severe and moderate, in humans has been associated with defective sensitization and recognition processes in delayed type hypersensitivity reactions to contact antigens (Smythe et al., 1971; Edelman et al., 1973; Chandra and Newberne, 1977). However, several laboratory animal studies have indicated that protein-calorie malnutrition can often enhance (Fernandes et al., 1976; Malave and Pocino, 1981) or have no affect on (Williams et al., 1979) cell-mediated immunity as measured by delayed hypersensitivity responses. Many of these apparent contradictions can probably be attributed to differences in animal models (species and strains), nutritional histories of subjects, ages, and experimental designs. Obviously, a considerable amount of caution must be exercised when extrapolating results derived from one animal model to another (Fernandes et al., 1976). Our initial results indicate that immune system function in adult cotton rats is responsive to changes in nutritional status. Short-term nutritional restriction may actually lead to a heightened level of immunocompetence, but prolonged dietary restriction appears to result in a gradual suppression of

cell-mediated immunity. It is important to note that animals used in this study were adults and that other age classes may have responded differently to similar experimental treatments. Cotton rats were also heavier at the start of this trial than most adults collected in the wild; largely a reflection of differences in lipid reserves. Consequences of such disparity in body composition on metabolic responses to feed restriction and immune system function are unknown. It is probably unwise to state that immune system function of moderately or severely restricted cotton rats in this study was impaired. Considerably more work will be necessary before we can correlate observed changes in immune function tests with alterations in immunocompetence, and thus increased susceptibility to infection. Acknowledgements-We acknowledge the financial support provided by the Kerr Foundation, National Science Foundation (BSR-8657043), and the Department of Zoology, Oklahoma State University. The technical assistance provided by D. Nash and B. C. Fields in animal husbandry was greatly appreciated.

REFERENCES Barker I. K., Beveridge I., Bradley A. J. and Lee A. K. (1978) Observations on spontaneous stress-related mortality among males of the dasyurid marsupial Antechinus stuartii Macleay. Aust. J. Zool. 26, 435-447. Bell R. G., Hazel1 L. A. and Price P. (1976) Influence of dietary protein restriction on immune competence. II. Effect on lymphoid tissue. Clin. exp. Immunol. 26, 314-326. Belvosky G. E. (1986) Generalist herbivore foraging and its role in competitive interactions. Am. Zool. 26, 51-69. Bradley A. J., McDonald I. R. and Lee A. K. (1980) Stress and mortality in a small marsupial (Antechinus stuarfii Mcleay). Gen. camp. Endocr. 40, 188-200. Buckley C. E. (1986) Delayed hypersensitivity skin testing. In Manual of Clinical Laboratory Immunology (Edited H. and Fahey J. L.), by Rose N. R., Freidman pp. 260-273. American Society for Microbiology, Washington, DC. Chandra R. K. and Newberne P. M. (1977) Nufrition, Immunity, and Infection. Plenum Press, New York. Chandra R. K.. Joshi P., Au A., Woodford G. and Chandra S. (1982) Nutrition and immunocompetence of the elderly: effect of short-term nutritional supplementation on cell-mediated immunity and lymphocyte subsets. Nufr. Res. 2, 223-232. Dobrowolska A. and Adamczewska-Andrzejewska K. A. (1991) Seasonal and longterm changes in serum gammaglobulin levels in comparng the physiology and population densitv of the common vole, Microtus arvatis Pall, 1779. J. Intkrdiscipl. Cyc. Res. 22, I-19. Edelman R.. Suskind R.. Olson R. E. and Sirisinha S. (1973) Mechanisms of defective delayed cutaneous hypersensitiv: ity in children with protein-calorie malnutrition. Lancer 1, 506-508. Femandes G., Yunis E. J. and Good R. A. (1976) Influence of protein restriction on immune functions in NZB mice. J. Immunol. 116, 782-790. Filteau S. M.. Perrv K. J. and Woodward B. (1987) Triiodothyronine -improves the primary antibody response to sheep red blood cells in severely undernourished weanling mice. Proc. Sot. exp. biol. Med. 185, 427-433.

Immune responses of diet restricted ~jg~o~o~ Fleharty E. D., Choate J. R. and Mares M. A. (1972) Fluctuations in population density of the hispid cotton rat: factors influencing a “crash”. Bull. Southern Calif: Acad. Sci. 71, 132-138.

Gasser D. L. and Silvers W. K. (1974) Genetic determinants ofimmunological responsiveness. Adv. Zmmunol. 18, I-66. Gershwin M. E., Beach R. S. and Hurley L. S. (1985) Nutrition und Zmmunify. Academic .Press, Orlando, FL. Goertz J. W. (1964) The influence of habitat quality upon density of cotton rat populations. Ecol. Monogr. 34, 359-381.

H ansson L. (1979) Food as a limiting factor for small rodent numbers: tests of two hypotheses. Oecologia 37,297-3 14. Hoffmann-Goetz L., Beil R. C. and Keir R. (1985) Effect of protein malnutrition and interieukin I on in vitro rabbit lymphocyte mitogenesis. Nutr. Z7es.5, 769-780. Keusch G. T., Douglas S. D., Hammer G. and Braden K. (1978) Antibacterial functions of macrophages in experimental proteincalorie malnutrition. II. Cellular and humoral factors for chemotaxis, phagocytosis, and intracellular bactericidal activitv. J. Znfecf. Dis. 138. 134-142. K eusch G. T. and Katz M. (1979) .Mal~utrition and infection. In Nutrition: Pre- and Postnatal D~eio~ment (Edited by Winick M.), pp. 307-332. Plenum press, New York. L ew F. Y. (1977) Regulation of delayed-type hypersensitivity: I. T-suppressor cells for delayed-type hypersensitivity to sheep erythrocyte in mice. Eur. J. Zmmunol. 7,714-718. hi alave I., Nemeth A. and Pocino M. (1980) Changes in lymphocyte populations in protein-calorie deficient mice. Cell. Zmm~ol. 49, 2355249. halave I. and Pocino M. (1981) Nutrition and the regulation of the immune response. In Nufrifional Factors: Modulating EfSects and Metabolic Processes (Edited by Beers R. F. and Bassett. E. G.), pp. 383-404. Raven Press, New York. hi cMurray D. N., Loomis S. A., Casazza L. J., Rey H. and Miranda R. (1981) Development of impaired cell-mediated immunity in mild and moderate malnutrition. Am. J. clin. Nufr. 34, 68-77.

599

Michalek S. M., McGhee J. R. and Ghanta V. K. (1974) Complement levels in malnourished animals: measurement of complement in rat milk during the period of lactation. J. Reficuloendofhel. Sot. 16, 213-219. Mihok S., Lawton T. and Schwartz B. (1988) Fates and movements of meadow voles (Microms ~nnsyivanjcus) following a ~pulation decline. Can. J. Zok 66,323-328. Mosmann T. (1983) Rapid calorimetric assay for cellular growth and‘ survival:* application to proliferation and cytotoxicity assays. J. Zmmunol. Mefh. 65, 55-63. Nesheim M. C. (1972) Genetic variations in nicotinic acid requirements of chicks. J. Hered. 63, 347-350. Phanunhak P.. Moorhead J. W. and Claman H. N. (1974) Tolerance and contact sensitivity to DNFB in mice. L in viva detection by ear swelling and correlation with in vifro cell stimulation. J. Zmmunol. 112, 115-123. SAS Institute Inc. (1982) SAS User’s Guide: Sratistics, 1982 edn. SAS Institute Inc., Cary, NC. Shultz L. D. and Bailey D. W. (1975) Genetic control of contact sensitivity in mice: effect of H-2 and non H-2 loci. Zmmunogenefics 1, 570-583. Smythe P. M., Schonland M., Breeton-Stiles G. G., Coovadia H. M., Grace H. J., Leoning W. E. K., Mafoyane A., Parent M. A. and Vos G. H. (1971) Thymolymphatic deficiency and depression of cell-mediated immunity in protein-calorie malnutrition. Lancer 4, 9399943. Stites D, P., Stobo J. D. and Wells J. V. (1987) Basic and Clinical Immunology. Appleton and Lange, Los Altos. Weindruch R. H., Gottesman S. R. S. and Walford R. L. (1982) Modification of age-related immune decline in mice dietarily restricted from or after midadulthood. Proc. nafn. Acad. Sri. U.S.A. 79, 8988905.

White T. C. R. (1978) The importance of a relative shortage of food in animal ecology. Oecologia 33, 71-86. Williams E. A. J., Gebhardt B. M., Morton B. and Newberne P. M. (1979) Effects of early marginal methionine~holine deprivation on the development of the immune system in the rat. Am. J. chn. Nun. 32, 1214-1223.