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Effect of two feeding formulas on immune responses and mortality in mice challenged with Listeria monocytogenes
Immunology Letters, 27 (1991) 45-48 Elsevier IMLET 01514
Effect of two feeding formulas on immune responses and mortality in mice challenged with Listeria monocytogenes R. K. C h a n d r a , M a r y a n n e Baker, Shengli W h a n g a n d Bing Au Departments of Pediatrics, Medicine and Immunology, Memorial University of Newfoundland, St. John's, Newfoundland, Canada (Received 15 June 1990; revision received 28 July 1990; accepted 26 September 1990)
1. Summary
2. Introduction
Cell-mediated immunity and natural killer cells play an important role against facultative intracellular organisms. The effect of two commercially available tube feeding formulas used for patients with acute or chronic debilitating and life-threatening illnesses was studied in mice challenged with Listeria monocytogenes. C57BL/6 x DBA/2 F t hybrid mice were given ad libitum access to one of two formulas or to chow. Sixty mice in each of the feeding groups were challenged with 4.8 × 103 organisms intraperitoneally. Mortality was significantly less in animals fed Impact ® , a formula enriched with arginine, R N A and selected fatty acids. This was associated with reduced number of viable organisms in the spleen on day 7 after challenge. There was no difference in the spleen/body weight index between the different groups. Delayed cutaneous hypersensitivity was slightly higher in the Impact ® group but this was not statistically significant. Natural killer cell activity was significantly higher in the Impact ® group compared with the other two feeding regimens. These observations suggest that selective manipulation of the composition of tube feeding formulas may have a significant impact on immune responses and on morbidity and mortality following infectious challenge.
Malnutrition is the commonest cause of immunodeficiency worldwide. The frequent presence of nutritional deficiencies in developing countries is well known. It has now been revealed that mild to moderate undernutrition is not uncommon, even in industrialized countries. For example, iron deficiency is seen in at least 9°7o of the North American population. In hospitalized patients, obvious protein-energy malnutrition is frequent. The combination of malnutrition and infection is responsible for morbidity and mortality in all age groups. In patients with a variety of primary diseases, such as cancer and Crohn's inflammatory bowel disease, nutritional deficiencies further worsen the picture and increase the risk of infectious complications. Although much of the initial work on nutrition and immunity was done on young children in developing countries, the general principle that nutrition is a critical determinant of immunocompetence is applicable universally. The enormous cost of health care required by patients with trauma, burns, cancer and a variety of other surgical and medical conditions has led to attempts at prevention of infectious and other complications. One such approach is the design and use of feeding formulas that may be expected to enhance immunocompetence and reduce morbidity and mortality due to infection. We have studied the impact of feeding two different formulas in mice challenged with an intracellular organism, Listeria mono-
Key words." Listeria; Enteral feeding; Immune response Correspondence to: Professor R. K. Chandra, Health Sciences
cytogenes.
Centre, Memorial University of Newfoundland, St. John's, Newfoundland AIB 3V6, Canada. 0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
45
3. Materials and Methods Six- to eight-week-old C 5 7 B L / 6 x D B A / 2 F 1 hybrid male mice were housed singly u n d e r controlled e n v i r o n m e n t a l c o n d i t i o n s ( 2 2 - 2 4 ° C temperature, 4 4 - 5 5 % relative humidity, 0 8 : 0 0 - 2 0 : 0 0 lighting hours). Three groups were studied: O n e was given a n o n - p u r i f i e d diet (Rodent Chow No. 5012, TABLE 1 Composition of feeding formulasa. Nutrient
IMPACT~
OSMOLITE®
Energy Proteins Arginine From intact protein Added free L-arginine Carbohydrate Fat MCT Linoleic acid EPA and DHA Total ~-3 fatty acids Yeast RNA Vitamin A Vitamin D Vitamin E Vitamin C Niacin Pantothenic acid Vitamin B6 Riboflavin Thiamine Folic Acid Biotin Vitamin KI Vitamin B12 Sodium Potassium Chloride Calcium Phosphorus Magnesium Zinc Iron Manganese Copper Iodine Selenium Chromium Molybdenum
420 kJ 5.6 g 1358 mg 158 mg 1200 mg 13.2 g 2.8 g 0.7 g 0.22 g 0.17 g 0.22 g 135 mg 670 IU 27.0 1U 6.0 IU 8.0 mg 2.0 mg 0.67 mg 0.15 mg 0.17 mg 0.20 mg 0.04 mg 0.020 mg 0.0067 mg 0.0008 mg 110 mg 130 mg 130 mg 80 mg 80 mg 27.0 mg 1.50 mg 1.20 mg 0.20 mg 0.17 mg 0.010 mg 0.001 mg 0.001 mg 0.002 mg
445 kJ 4.5 g 164 mg 164 mg 0 14.2 g 3.7 g 1.8 g 1.2 g 0 0.03 g 0 380 IU 30.6 IU 3.5 IU 22.8 mg 2.28 mg 1.14 mg 0.23 mg 0.2 mg 0.17 mg 0.046 mg 0.035 mg 0.0055 mg 0.0007 mg 94 mg 157 mg 145 mg 76 mg 76 mg 30.4 mg 1.71 mg 1.37 mg 0.38 mg 0.15 mg 0.012 mg * * *
a Amounts are shown per 100 ml. * Concentration not provided in available literature. EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid. 46
Ralston P u r i n a , St. Louis, MO). The second group was fed a n experimental f o r m u l a enriched with arginine, R N A a n d n-3 fatty acids, a n d recently marketed as I M P A C T ® (Sandoz N u t r i t i o n , M i n n e a p o l i s , MN). The third group received O S M O L I T E H N ® ( A b b o t t Laboratories, Montreal, PQ). The composition o f the two formulas is given in Table 1. Sixty mice in each o f the three feeding groups were challenged with 4.8 × 103 L. monocytogenes given intraperitoneally. The onset o f feeding the two form u l a s a n d the infectious challenge occurred simultaneously o n the same m o r n i n g . Virulence of the m i c r o o r g a n i s m was m a i n t a i n e d by c o n t i n u o u s passage t h r o u g h m o u s e spleens. The a n i m a l s were exa m i n e d a n d weighed daily. T h e spleen of those a n i m a l s who died o n day 7 or were killed o n that day after infectious challenge was examined for viable organisms. This was d o n e by 10-fold serial d i l u t i o n o f o r g a n h o m o g e n a t e s o n to tryptose agar. Spleen from infected a n i m a l s was removed a n d weighed to examine the development of splenomegaly. The spleen index was calculated as the weight of the spleen divided by the weight of the animal. All results were expressed as the m e a n a n d s t a n d a r d error of values obtained. Delayed hypersensitivity response was d e t e r m i n e d o n day 5 after infectious challenge, slightly m o d i f i e d from the m e t h o d of C z u p r y n s k i et al. [1]. Soluble Listeria a n t i g e n was prepared from 24-h bacterial culture in tryptose p h o s p h a t e broth, c e n t r i f u g a t i o n at 1 0 0 0 x g for 20 min. The bacterial pellet was susp e n d e d in 10 ml pyrogen-free saline a n d sonicated. T h e filter-sterilized s u p e r n a t a n t was assayed for protein a n d a d j u s t e d to 1.24 m g / m l . 5 #g of the a n t i g e n in 0.03 ml o f saline was injected into one footpad a n d a n equal v o l u m e of saline into the other footpad. The thickness o f each f o o t p a d was measured with a sensitive a n d accurate micrometer. Results were expressed as the m e a n _+ SEM increase in thickness o f antigen-injected footpad c o m p a r e d with the one injected with saline. N a t u r a l killer (NK) cell activity was assessed in 16 mice in each o f the three feeding groups. The m e t h o d has been described by us recently [2]. Mice were anesthetized with ether, killed by cervical dislocation a n d the spleen collected a n d weighed. Spleen cells were o b t a i n e d by teasing in RPMI-1640 (Flow Laboratories, McLean, VA) with two curved needles. Large cellular debris was removed by s e d i m e n t a t i o n ,
and a single cell suspension was obtained by passing the material through graded needles. The lymphocytes were counted with a standard hemocytometer. The suspension was layered over Ficoll H y p a q u e with a specific gravity of 1.09 and centrifuged at 400 x g for 30 min at room temperature. The interface cells were collected and washed 3 times, counted and adjusted to a concentration of 20 x 106 cells/ml in RPMI-1640 supplemented with bicarbonate, penicillin, streptomycin, fungizone and 10% fetal calf serum. This was the effector cell population. The target ceils were Moloney virusinduced mouse l y m p h o m a cell line YAC-1 (American Type Tissue Culture, Rockville, MD) maintained in continuous culture in complete culture medium. Labeling was achieved with 200/~Ci 51Cr as sodium chromate added to 5 x 106 cells per ml in 0.5 ml culture medium. NK cell activity was determined by a microwell modification of a well-standardized Cr release assay, using 50:1 effector:target cell ratio. Spontaneous release of Cr was less than 10070. Percentage lysis was calculated as follows: (mean cpm experimental rdease) - (mean cpm spontaneous release) x 100 (mean cpm total release) - (mean cpm spontaneous release)
4. Statistics
Data were analyzed using the statistical package SAS. Analysis of variance was used to determine the
presence of treatment effect. Differences between two or more groups were determined by the Duncan's multiple-range test. The significance level used was 0.05. 5. Results
The two formulas had no significant impact on the general health and growth of the animals. Weight gain was not affected. The mortality among animals challenged with Listeria is shown in Table 2. IMPACT ® -fed mice had a significantly higher survival or reduced mortality on days 5 and 7 after challenge. This was associated with reduced number of viable organisms in the spleen on day 7 after challenge. There was no difference in spleen index between the different groups. Delayed cutaneous hypersensitivity was slightly higher in the IMPACT ®group but this was statistically not significant. Natural killer cell activity was significantly higher in the IMPACT ® group compared with the other two feeding regimens. 6. Discussion
Both cell-mediated immunity and NK cell activity are critical determinants of immune responses and of morbidity following challenge with facultative intracellular pathogens such as L. monocytogenes. Our observations suggest that selective manipula-
TABLE 2 Effect of fo rmula feeding on mortality and immune responses following challenge with L. monocytogenes. Parameter
IMPACT ®
Mortality (n = 60) Day 2 Day 5 Day 7 Viable organisms In spleen (day 7) Delayed cutaneous hypersensitivity (ram) (n = 16) NK cell activity (°7o) (n = 16) S p l e e n / b o d y weight index Day 2 Day 5 Day 7
4 10a 17a 814 + 43 a
OSMOLITE ®
6 18b 27 b
Chow
7 22 b 34 b
p
NS < 0.05 < 0.05
l 106 +_57 b
1234 _+64 c
34_+5
29_+4
30_+6
0.1 > p > 0 . 0 5
48_+6 a
33_+5 b
30+_5 b
<0.01
0.0043 0.0117 0.0033
0.0052 0.0129 0.0031
0.0056 0.0119 0.0028
<0.01
NS NS NS
Values with different superscript letters in the same horizontal line different significantly from one another.
47
tion of feeding formulas can be used to advantage to enhance immune responses and to improve survival after infectious challenge. Twenty years ago, nutrition was suggested as an important determinant of immune responses [3]. The fundamental and applied significance of interactions between nutrients and the immune system is now generally recognized [4]. It is known that moderate to severe protein-energy malnutrition results in profound and consistent abnormalities in cell-mediated immunity, phagocyte function, complement system and mucosal antibody response [57]. Individual nutrients exert variable influence on different aspects of immunocompetence [8, 9]. A logical corollary to these observations is the development of nutrient mixtures that may be expected to stimulate or suppress selected aspects of immunity. The choice of nutrients and their relative concentrations may be a key factor in the i m m u n o m o d u l a t ing influence of a particular parenteral or enteral solution. There is much recent interest in micronutrients [10], amino acids such as arginine [11], fatty acids [12], and nucleotides [13]. Most of the data are derived from in vitro observations and work in laboratory animals. It has been shown that a moderate excess of arginine intake is associated with protection from stress-induced thymic involution, and enhanced thymic lymphocyte response to mitogens. There are less consistent effects on cytokine production and protection from viral infections. Fatty acids can influence immune responses by three separate routes [14]: effects on leukotriene production, alterations in cell membrane structure and stability, and proliferative ability of lymphocytes. The relative amounts of w-3 and w-6 fatty acids and of medium-chain and long-chain triglycerides alter immune responses of the host. Finally, moderate excess of nucleotides enhances lymphocyte responses to mitogens and alloantigens, and increases natural killer cell activity. One reason for considering nucleotides as immunoprotective molecules is their higher concentration in h u m a n breast milk: breast milk is known to have antiinfective and anti-inflammatory properties [15] and exclusively breast-fed infants tend to have fewer and less severe infections. The era of "designer" cocktails for enteral and parenteral nutrition of critically ill patients and those with chronic problems has finally arrived. It 48
is likely that the next decade will see the production and marketing of a variety of such products putatively indicated for specific disorders. However, the path to the development and utilization of these formulas should be trodden with caution. Imbalance of nutrients and excessive intakes are also detrimental for the immune system [16]. Several questions remain unanswered. What would be the influence of feeding Impact ® prior to or some time after infectious challenge? What are the changes in additional antigen-specific immune responses, and would a similar beneficial effect be obtained after challenge with other organisms that differ in the type of immune responses required for their inactivation and elimination? The quantitative contribution of each of the unique components of IMPACT ® - arginine, RNA, ~-3 fatty acids - should be determined. Moreover, the present study involved well-nourished mice rather than undernourished ones. Further observations on animals in restricted diets or those fed on diets deficient in selected nutrients would be useful. Finally, the results of the present study require confirmation and extension by carefully designed human studies which are in progress. References [1] Czuprynski, C. J., Brown, J. E, Young, K. M. and Cooley, A. J. (1989) Infect. Immun. 57, I00. [2] Chowdhury, B. A. and Chandra, R. K. (1989) Immunol. Len. 22, 287. [3] This week's Citation Classic (1987) Curr. Contents 30, 15. [4] Chandra, R. K. (1989) Nutrition 5, 297. [5] Chandra, R. K. and Newberne, P. M. (1977) Nutrition, immunity and infection: mechanisms of interactions. Plenum Press, New York. [6] Gerhwin, M. E., Beach, R. S. and Hurley, L. S. (1984) Nutrition and Immunity. Academic Press, New York. [7] Watson, R. R. (1984) Nutrition, Disease Resistance and Immune Function. Marcel Dekker, New York. [8] Chandra, R. K. and Dayton, D. H. (1982) Nutr. Res. 2, 32. [9] Beisel, W. R. (1982) Am. J. Clin. Nutr. 35,417. [10] Bendich, A. and Chandra, R. K. (1990) Ann. NY Acad. Sci. 687, 9. [11] Barbul, A. (1986) J.P.E.N. 10, 227. [12] Chandra, R. K. (1989) Health Effects of Fish and Fish Oils. ARTS Biochemical Publishers, St. John's. [13] Kulkarni, A. D., Fanslow, W. C., Rudolph, F. B. et al. (1987) Transportation 44, 847. [14] Johnston, P. V. and Marshall, L. A. (1984) Prog. Food Nutr. Sci. 8, 3. [15] Ogra, P. L. and Green, H. L. (1982) Pediatr. Res. 16, 266. [16] Chandra, R. K. (1984) J. Am. Med. Assoc. 252, 1443.
Effect of two feeding formulas on immune responses and mortality in mice challenged with Listeria monocytogenes R. K. C h a n d r a , M a r y a n n e Baker, Shengli W h a n g a n d Bing Au Departments of Pediatrics, Medicine and Immunology, Memorial University of Newfoundland, St. John's, Newfoundland, Canada (Received 15 June 1990; revision received 28 July 1990; accepted 26 September 1990)
1. Summary
2. Introduction
Cell-mediated immunity and natural killer cells play an important role against facultative intracellular organisms. The effect of two commercially available tube feeding formulas used for patients with acute or chronic debilitating and life-threatening illnesses was studied in mice challenged with Listeria monocytogenes. C57BL/6 x DBA/2 F t hybrid mice were given ad libitum access to one of two formulas or to chow. Sixty mice in each of the feeding groups were challenged with 4.8 × 103 organisms intraperitoneally. Mortality was significantly less in animals fed Impact ® , a formula enriched with arginine, R N A and selected fatty acids. This was associated with reduced number of viable organisms in the spleen on day 7 after challenge. There was no difference in the spleen/body weight index between the different groups. Delayed cutaneous hypersensitivity was slightly higher in the Impact ® group but this was not statistically significant. Natural killer cell activity was significantly higher in the Impact ® group compared with the other two feeding regimens. These observations suggest that selective manipulation of the composition of tube feeding formulas may have a significant impact on immune responses and on morbidity and mortality following infectious challenge.
Malnutrition is the commonest cause of immunodeficiency worldwide. The frequent presence of nutritional deficiencies in developing countries is well known. It has now been revealed that mild to moderate undernutrition is not uncommon, even in industrialized countries. For example, iron deficiency is seen in at least 9°7o of the North American population. In hospitalized patients, obvious protein-energy malnutrition is frequent. The combination of malnutrition and infection is responsible for morbidity and mortality in all age groups. In patients with a variety of primary diseases, such as cancer and Crohn's inflammatory bowel disease, nutritional deficiencies further worsen the picture and increase the risk of infectious complications. Although much of the initial work on nutrition and immunity was done on young children in developing countries, the general principle that nutrition is a critical determinant of immunocompetence is applicable universally. The enormous cost of health care required by patients with trauma, burns, cancer and a variety of other surgical and medical conditions has led to attempts at prevention of infectious and other complications. One such approach is the design and use of feeding formulas that may be expected to enhance immunocompetence and reduce morbidity and mortality due to infection. We have studied the impact of feeding two different formulas in mice challenged with an intracellular organism, Listeria mono-
Key words." Listeria; Enteral feeding; Immune response Correspondence to: Professor R. K. Chandra, Health Sciences
cytogenes.
Centre, Memorial University of Newfoundland, St. John's, Newfoundland AIB 3V6, Canada. 0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
45
3. Materials and Methods Six- to eight-week-old C 5 7 B L / 6 x D B A / 2 F 1 hybrid male mice were housed singly u n d e r controlled e n v i r o n m e n t a l c o n d i t i o n s ( 2 2 - 2 4 ° C temperature, 4 4 - 5 5 % relative humidity, 0 8 : 0 0 - 2 0 : 0 0 lighting hours). Three groups were studied: O n e was given a n o n - p u r i f i e d diet (Rodent Chow No. 5012, TABLE 1 Composition of feeding formulasa. Nutrient
IMPACT~
OSMOLITE®
Energy Proteins Arginine From intact protein Added free L-arginine Carbohydrate Fat MCT Linoleic acid EPA and DHA Total ~-3 fatty acids Yeast RNA Vitamin A Vitamin D Vitamin E Vitamin C Niacin Pantothenic acid Vitamin B6 Riboflavin Thiamine Folic Acid Biotin Vitamin KI Vitamin B12 Sodium Potassium Chloride Calcium Phosphorus Magnesium Zinc Iron Manganese Copper Iodine Selenium Chromium Molybdenum
420 kJ 5.6 g 1358 mg 158 mg 1200 mg 13.2 g 2.8 g 0.7 g 0.22 g 0.17 g 0.22 g 135 mg 670 IU 27.0 1U 6.0 IU 8.0 mg 2.0 mg 0.67 mg 0.15 mg 0.17 mg 0.20 mg 0.04 mg 0.020 mg 0.0067 mg 0.0008 mg 110 mg 130 mg 130 mg 80 mg 80 mg 27.0 mg 1.50 mg 1.20 mg 0.20 mg 0.17 mg 0.010 mg 0.001 mg 0.001 mg 0.002 mg
445 kJ 4.5 g 164 mg 164 mg 0 14.2 g 3.7 g 1.8 g 1.2 g 0 0.03 g 0 380 IU 30.6 IU 3.5 IU 22.8 mg 2.28 mg 1.14 mg 0.23 mg 0.2 mg 0.17 mg 0.046 mg 0.035 mg 0.0055 mg 0.0007 mg 94 mg 157 mg 145 mg 76 mg 76 mg 30.4 mg 1.71 mg 1.37 mg 0.38 mg 0.15 mg 0.012 mg * * *
a Amounts are shown per 100 ml. * Concentration not provided in available literature. EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid. 46
Ralston P u r i n a , St. Louis, MO). The second group was fed a n experimental f o r m u l a enriched with arginine, R N A a n d n-3 fatty acids, a n d recently marketed as I M P A C T ® (Sandoz N u t r i t i o n , M i n n e a p o l i s , MN). The third group received O S M O L I T E H N ® ( A b b o t t Laboratories, Montreal, PQ). The composition o f the two formulas is given in Table 1. Sixty mice in each o f the three feeding groups were challenged with 4.8 × 103 L. monocytogenes given intraperitoneally. The onset o f feeding the two form u l a s a n d the infectious challenge occurred simultaneously o n the same m o r n i n g . Virulence of the m i c r o o r g a n i s m was m a i n t a i n e d by c o n t i n u o u s passage t h r o u g h m o u s e spleens. The a n i m a l s were exa m i n e d a n d weighed daily. T h e spleen of those a n i m a l s who died o n day 7 or were killed o n that day after infectious challenge was examined for viable organisms. This was d o n e by 10-fold serial d i l u t i o n o f o r g a n h o m o g e n a t e s o n to tryptose agar. Spleen from infected a n i m a l s was removed a n d weighed to examine the development of splenomegaly. The spleen index was calculated as the weight of the spleen divided by the weight of the animal. All results were expressed as the m e a n a n d s t a n d a r d error of values obtained. Delayed hypersensitivity response was d e t e r m i n e d o n day 5 after infectious challenge, slightly m o d i f i e d from the m e t h o d of C z u p r y n s k i et al. [1]. Soluble Listeria a n t i g e n was prepared from 24-h bacterial culture in tryptose p h o s p h a t e broth, c e n t r i f u g a t i o n at 1 0 0 0 x g for 20 min. The bacterial pellet was susp e n d e d in 10 ml pyrogen-free saline a n d sonicated. T h e filter-sterilized s u p e r n a t a n t was assayed for protein a n d a d j u s t e d to 1.24 m g / m l . 5 #g of the a n t i g e n in 0.03 ml o f saline was injected into one footpad a n d a n equal v o l u m e of saline into the other footpad. The thickness o f each f o o t p a d was measured with a sensitive a n d accurate micrometer. Results were expressed as the m e a n _+ SEM increase in thickness o f antigen-injected footpad c o m p a r e d with the one injected with saline. N a t u r a l killer (NK) cell activity was assessed in 16 mice in each o f the three feeding groups. The m e t h o d has been described by us recently [2]. Mice were anesthetized with ether, killed by cervical dislocation a n d the spleen collected a n d weighed. Spleen cells were o b t a i n e d by teasing in RPMI-1640 (Flow Laboratories, McLean, VA) with two curved needles. Large cellular debris was removed by s e d i m e n t a t i o n ,
and a single cell suspension was obtained by passing the material through graded needles. The lymphocytes were counted with a standard hemocytometer. The suspension was layered over Ficoll H y p a q u e with a specific gravity of 1.09 and centrifuged at 400 x g for 30 min at room temperature. The interface cells were collected and washed 3 times, counted and adjusted to a concentration of 20 x 106 cells/ml in RPMI-1640 supplemented with bicarbonate, penicillin, streptomycin, fungizone and 10% fetal calf serum. This was the effector cell population. The target ceils were Moloney virusinduced mouse l y m p h o m a cell line YAC-1 (American Type Tissue Culture, Rockville, MD) maintained in continuous culture in complete culture medium. Labeling was achieved with 200/~Ci 51Cr as sodium chromate added to 5 x 106 cells per ml in 0.5 ml culture medium. NK cell activity was determined by a microwell modification of a well-standardized Cr release assay, using 50:1 effector:target cell ratio. Spontaneous release of Cr was less than 10070. Percentage lysis was calculated as follows: (mean cpm experimental rdease) - (mean cpm spontaneous release) x 100 (mean cpm total release) - (mean cpm spontaneous release)
4. Statistics
Data were analyzed using the statistical package SAS. Analysis of variance was used to determine the
presence of treatment effect. Differences between two or more groups were determined by the Duncan's multiple-range test. The significance level used was 0.05. 5. Results
The two formulas had no significant impact on the general health and growth of the animals. Weight gain was not affected. The mortality among animals challenged with Listeria is shown in Table 2. IMPACT ® -fed mice had a significantly higher survival or reduced mortality on days 5 and 7 after challenge. This was associated with reduced number of viable organisms in the spleen on day 7 after challenge. There was no difference in spleen index between the different groups. Delayed cutaneous hypersensitivity was slightly higher in the IMPACT ®group but this was statistically not significant. Natural killer cell activity was significantly higher in the IMPACT ® group compared with the other two feeding regimens. 6. Discussion
Both cell-mediated immunity and NK cell activity are critical determinants of immune responses and of morbidity following challenge with facultative intracellular pathogens such as L. monocytogenes. Our observations suggest that selective manipula-
TABLE 2 Effect of fo rmula feeding on mortality and immune responses following challenge with L. monocytogenes. Parameter
IMPACT ®
Mortality (n = 60) Day 2 Day 5 Day 7 Viable organisms In spleen (day 7) Delayed cutaneous hypersensitivity (ram) (n = 16) NK cell activity (°7o) (n = 16) S p l e e n / b o d y weight index Day 2 Day 5 Day 7
4 10a 17a 814 + 43 a
OSMOLITE ®
6 18b 27 b
Chow
7 22 b 34 b
p
NS < 0.05 < 0.05
l 106 +_57 b
1234 _+64 c
34_+5
29_+4
30_+6
0.1 > p > 0 . 0 5
48_+6 a
33_+5 b
30+_5 b
<0.01
0.0043 0.0117 0.0033
0.0052 0.0129 0.0031
0.0056 0.0119 0.0028
<0.01
NS NS NS
Values with different superscript letters in the same horizontal line different significantly from one another.
47
tion of feeding formulas can be used to advantage to enhance immune responses and to improve survival after infectious challenge. Twenty years ago, nutrition was suggested as an important determinant of immune responses [3]. The fundamental and applied significance of interactions between nutrients and the immune system is now generally recognized [4]. It is known that moderate to severe protein-energy malnutrition results in profound and consistent abnormalities in cell-mediated immunity, phagocyte function, complement system and mucosal antibody response [57]. Individual nutrients exert variable influence on different aspects of immunocompetence [8, 9]. A logical corollary to these observations is the development of nutrient mixtures that may be expected to stimulate or suppress selected aspects of immunity. The choice of nutrients and their relative concentrations may be a key factor in the i m m u n o m o d u l a t ing influence of a particular parenteral or enteral solution. There is much recent interest in micronutrients [10], amino acids such as arginine [11], fatty acids [12], and nucleotides [13]. Most of the data are derived from in vitro observations and work in laboratory animals. It has been shown that a moderate excess of arginine intake is associated with protection from stress-induced thymic involution, and enhanced thymic lymphocyte response to mitogens. There are less consistent effects on cytokine production and protection from viral infections. Fatty acids can influence immune responses by three separate routes [14]: effects on leukotriene production, alterations in cell membrane structure and stability, and proliferative ability of lymphocytes. The relative amounts of w-3 and w-6 fatty acids and of medium-chain and long-chain triglycerides alter immune responses of the host. Finally, moderate excess of nucleotides enhances lymphocyte responses to mitogens and alloantigens, and increases natural killer cell activity. One reason for considering nucleotides as immunoprotective molecules is their higher concentration in h u m a n breast milk: breast milk is known to have antiinfective and anti-inflammatory properties [15] and exclusively breast-fed infants tend to have fewer and less severe infections. The era of "designer" cocktails for enteral and parenteral nutrition of critically ill patients and those with chronic problems has finally arrived. It 48
is likely that the next decade will see the production and marketing of a variety of such products putatively indicated for specific disorders. However, the path to the development and utilization of these formulas should be trodden with caution. Imbalance of nutrients and excessive intakes are also detrimental for the immune system [16]. Several questions remain unanswered. What would be the influence of feeding Impact ® prior to or some time after infectious challenge? What are the changes in additional antigen-specific immune responses, and would a similar beneficial effect be obtained after challenge with other organisms that differ in the type of immune responses required for their inactivation and elimination? The quantitative contribution of each of the unique components of IMPACT ® - arginine, RNA, ~-3 fatty acids - should be determined. Moreover, the present study involved well-nourished mice rather than undernourished ones. Further observations on animals in restricted diets or those fed on diets deficient in selected nutrients would be useful. Finally, the results of the present study require confirmation and extension by carefully designed human studies which are in progress. References [1] Czuprynski, C. J., Brown, J. E, Young, K. M. and Cooley, A. J. (1989) Infect. Immun. 57, I00. [2] Chowdhury, B. A. and Chandra, R. K. (1989) Immunol. Len. 22, 287. [3] This week's Citation Classic (1987) Curr. Contents 30, 15. [4] Chandra, R. K. (1989) Nutrition 5, 297. [5] Chandra, R. K. and Newberne, P. M. (1977) Nutrition, immunity and infection: mechanisms of interactions. Plenum Press, New York. [6] Gerhwin, M. E., Beach, R. S. and Hurley, L. S. (1984) Nutrition and Immunity. Academic Press, New York. [7] Watson, R. R. (1984) Nutrition, Disease Resistance and Immune Function. Marcel Dekker, New York. [8] Chandra, R. K. and Dayton, D. H. (1982) Nutr. Res. 2, 32. [9] Beisel, W. R. (1982) Am. J. Clin. Nutr. 35,417. [10] Bendich, A. and Chandra, R. K. (1990) Ann. NY Acad. Sci. 687, 9. [11] Barbul, A. (1986) J.P.E.N. 10, 227. [12] Chandra, R. K. (1989) Health Effects of Fish and Fish Oils. ARTS Biochemical Publishers, St. John's. [13] Kulkarni, A. D., Fanslow, W. C., Rudolph, F. B. et al. (1987) Transportation 44, 847. [14] Johnston, P. V. and Marshall, L. A. (1984) Prog. Food Nutr. Sci. 8, 3. [15] Ogra, P. L. and Green, H. L. (1982) Pediatr. Res. 16, 266. [16] Chandra, R. K. (1984) J. Am. Med. Assoc. 252, 1443.