Effect of dietary nucleotide supplementation on in vivo and in vitro immune function in protein-malnourished mice

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Effect of dietary nucleotide supplementation on in vivo and in vitro immune function in protein-malnourished mice
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Lin, Cheng-mao, 1960-
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Mice -- Immunology   ( lcsh )
Food Science and Human Nutrition thesis, Ph. D
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Thesis (Ph. D.)--University of Florida, 1995.
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Includes bibliographical references (leaves 115-129).
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by Cheng-mao Lin.
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Typescript.
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Vita.

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EFFECT OF DIETARY NUCLEOTIDE SUPPLEMENTATION
ON IN VIVO AND IN VITRO IMMUNE FUNCTION
IN PROTEIN-MALNOURISHED MICE















By

CHENG-MAO LIN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1995


UNIVERSITY OF FLORIDA LIBRARIES






























Copyright 1995

by

Cheng-mao Lin














ACKNOWLEDGMENTS


I wish to express my sincere gratitude to Dr. Harry S. Sitren, my major advisor,

for his guidance, encouragement, and patience throughout the course of this study.

Appreciation is extended to Dr. Cheng-i Wei, cochairman of my research committee, for

his support and invaluable advice. I am truly grateful to Drs. Thomas G. Baumgartner,

Robert J. Cousins, and Kuo-jang Kao for their willingness to serve on my research

committee and share their expertise.

I would also like to thank Ms. Juanita J. Bagnall, Dr. Tung-shi Huang, and all

my friends in the Department and IR-4 who have been generous with their friendship.

I am deeply indebted to my wife, Wei-fang, who braces me with her patience,

thoughtfulness, and endless love. My heartfelt gratitude goes to my parents, brother, and

sisters for their understanding and unconditional love.














TABLE OF CONTENTS


ACKNOWLEDGMENTS .................................. iii

LIST OF TABLES ...................................... vii

LIST OF FIGURES ..................................... viii

ABSTRACT ......................................... xi

INTRODUCTION ....................................... 1

Nutrition and Immune Function .......................... 1
Objectives and Specific Aims ............................ 3

LITERATURE REVIEW ...................................... 5

Biochemistry and Physiology of Nucleotides ................... 5
Metabolic Fate of Dietary Nucleotides .................. 5
Bioavailability of Dietary Nucleotides ................ 10
Is There a Requirement for Dietary Nucleotides? .............. 15
Dietary Nucleotides in Infant Nutrition ................ 15
Nucleotides in Enteral and Parenteral Nutrition ............ 17
Importance of Dietary Nucleotides ........................ 19
Maintenance and Enhancement of Immune Function ......... 19
Improvement of Infection Resistance ................. 26
Development and Maturation of Intestinal Tissues .......... 28
Protection and Regeneration of Internal Organs .......... 30
Proliferation and Differentiation of Cultured Cells .......... 31
Conversion and Composition of Fatty Acids ............ 32

MATERIALS AND METHODS ............................. 33

Animal Model and Dietary Regimen ...................... 33
Diet Preparation .............................. 33
Animal Model and Feeding Protocol ................. 34
Preparation of Bacterial Culture ........................ 35









Nutritional Assessment ............................... 36
Food Intake and Body and Organ Weights . 36
Plasma Albumin and Total Protein .................. 36
Immune Function Assay .............................. 37
Delayed-Type Hypersensitivity .................... 37
Popliteal Lymphoproliferation ..................... 38
Histological Analysis of the Popliteal Lymph Node ......... 39
Tritiated-Thymidine Incorporation ................... 39
Bactericidal Activity of Peritoneal Macrophages .......... 40
Containment of Intradermal Abscess Challenge ............ 42
Clearance of Intravenous Bacterial Challenge ............ 43
Statistical Analysis ................................. 44

RESULTS AND DISCUSSION .............................. 46

M odel System .................................... 46
Determination of Nutrition Deprivation Procedure .......... 46
Effect of Nutrition Deprivation on Body Weight and Blood Protein
Levels ... .. ....... ...... ....... ... .... 46
Effect of Nucleotide Supplementation on Food Intake ........ 48
Effect of Nucleotide Supplementation on Body and Organ
W eight .. ... .. ..... .. ..... ...... ...... 53
Effect of Nucleotide Supplementation on Blood Protein Levels 58
Immune Function .................................. 58
Time Course Development of Delayed-Type Hypersensitivity 58
Effect of RNA Supplementation on Delayed-Type
Hypersensitivity .......................... 58
Effect of Uracil Supplementation on Delayed-Type
Hypersensitivity .......................... 63
Time Course Development of Popliteal Lymphoproliferation 63
Effect of RNA Supplementation on Popliteal
Lymphoproliferation ....................... 66
Effect of RNA Supplementation on Histology of the Popliteal
Lymph Node ............................ 66
Effect of RNA Supplementation on In Vitro Proliferation of the
Popliteal Lymphocytes ...................... 68
Effect of Uracil Supplementation on Popliteal
Lymphoproliferation ....................... 74
Effect of RNA Supplementation on In Vitro Bactericidal Activity
of Peritoneal Macrophages .................... 74
Effect of Uracil Supplementation on In Vitro Bactericidal Activity
of Peritoneal Macrophages ................... 78
Effect of RNA Supplementation on Abscess Containment ..... 80









Clearance of Intravenous Bacterial Challenge . 80
Effect of RNA Supplementation on Clearance of Intravenous
Bacterial Challenge with Listeria monocytogenes ...... 83
Survival Rate of Mice after Intravenous Bacterial Challenge with
Streptomycin-Resistant Listeria monocytogenes ....... 86
Effect of RNA Supplementation on Clearance of Intravenous
Bacterial Challenge with Streptomycin-Resistant Listeria
monocytogenes ........................... 89
Effect of Uracil Supplementation on Clearance of Intravenous
Bacterial Challenge with Streptomycin-Resistant Listeria
monocytogenes ........................... 93

SUMMARY AND CONCLUSIONS ........................... 98

Sum m ary ...................................... 98
Conclusions ..................................... 100
Future Studies .................................... 104

APPENDIX A DIET COMPOSITION ......................... 106

APPENDIX B BACTERIAL CULTURE ........................ 109

APPENDIX C PROTEIN ASSAY ............................ 112

APPENDIX D IN VITRO BACTERICIDAL ASSAY ................ 114

LITERATURE CITED ................................... 115

BIOGRAPHICAL SKETCH ................................ 130














LIST OF TABLES


Table page


1. Food intake at different feeding periods of RNA supplementation study 51

2. Food intake at different feeding periods of uracil supplementation study 52

3. Final organ weights of mice in RNA supplementation study ....... 56

4. Final organ weights of mice in uracil supplementation study ........ 57

5. Tritiated-thymidine incorporation by lymphocytes of the popliteal lymph
nodes with or without antigen stimulation .................. 73

6. Summary of immune function assay results of RNA and uracil
supplementation experiments .......................... 102

7. Composition of purified diet ........................... 106

8. Composition of AIN-76 mineral and vitamin mixes ............ 107

9. Composition of stock diet .............................. 108














LIST OF FIGURES


Figure page


1. Effect of dietary nucleotide depletion on popliteal lymphoproliferation 47

2. Effect of dietary treatment on body weight .................. 49

3. Effect of dietary treatment on plasma albumin and total protein levels 50

4. Effect of RNA supplementation on body weight ............... 54

5. Effect of uracil supplementation on body weight .............. 55

6. Effect of RNA supplementation on plasma albumin and total protein
levels . . . . . 59

7. Effect of uracil supplementation on plasma albumin and total protein
levels . . . . .. 60

8. Time course development of ear swelling in healthy outbred mice fed a
stock diet .. . .. .. . 61

9. Effect of RNA supplementation on delayed-type hypersensitivity ..... 62

10. Effect of uracil supplementation on delayed-type hypersensitivity ..... 64

11. Time course development of popliteal lymphoproliferation in healthy
outbred mice fed a stock diet ........................... 65

12. Effect of RNA supplementation on popliteal lymphoproliferation ..... 67

13. Effect of RNA supplementation on histology of the popliteal lymph
nodes . . . . .. 69

14. Effect of uracil supplementation on popliteal lymphoproliferation ..... 75









15. Effect of RNA supplementation on in vitro bactericidal activity of
peritoneal macrophages . . . 77

16. Effect of uracil supplementation on in vitro bactericidal activity of
peritoneal macrophages ............................. 79

17. Effect of RNA supplementation on number of viable organisms recovered
24 hours after intradermal injection of 1.0 x 10 cells of Staphylococcus
aureus ........ ... ..... ....... ..... ....... .. .... 81

18. Model of natural and acquired resistance to Listeria monocytogenes in
sensitive and resistant strains of mice . . 82

19. Survival of organisms in blood and organs of mice fed a stock diet .. 84

20. Effect of RNA supplementation on number of viable organisms recovered
4 days after intravenous injection of 5.0 x 10' cells of Listeria
monocytogenes ................................... 85

21. Effect of RNA supplementation on number of viable organisms recovered
4 days after intravenous injection of 7.5 x 104 cells of Listeria
monocytogenes ................................... 87

22. Survival rate of mice fed a stock diet after intravenous injection of 3
different levels of streptomycin-resistant Listeria monocytogenes ...... 88

23. Effect of RNA supplementation on number of viable organisms recovered
4 days after intravenous injection of 1.0 x 107 cells of streptomycin-
resistant Listeria monocytogenes ........................ 90

24. Effect of RNA supplementation on number of viable organisms recovered
4 days after intravenous injection of 1.0 x 108 cells of streptomycin-
resistant Listeria monocytogenes ........................ 91

25. Effect of uracil supplementation on survival rate of mice fed different
diets after intravenous injection of 4.5 x 108 cells of streptomycin-resistant
Listeria monocytogenes ............................... 94

26. Effect of uracil supplementation on number of viable organisms recovered
4 days after intravenous injection of 7.5 x 107 cells of streptomycin-
resistant Listeria monocytogenes . . . 96

27. Growth curves for Listeria monocytogenes and Staphylococcus aureus 110









28. Standard curves for Listeria monocytogenes and Staphylococcus aureus 111

29. Flow chart of in vitro bactericidal assay of peritoneal macrophages .. .. 114














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

EFFECT OF DIETARY NUCLEOTIDE SUPPLEMENTATION
ON IN VIVO AND IN VITRO IMMUNE FUNCTION
IN PROTEIN-MALNOURISHED MICE

By

Cheng-mao Lin

December 1995


Chairman: Harry S. Sitren
Cochairman: Cheng-i Wei
Major Department: Food Science and Human Nutrition

Nucleic acids are not considered to be dietary essential nutrients since their

complete de novo synthesis has been well documented. However, diets without naturally

occurring nucleic acids (i.e., RNA) diminish immunity in animals. It is thought that

under physiological stresses such as tissue injury or infection, nucleotide synthesis is

insufficient to meet requirements. The immune-stimulating effect of nucleotides may

have important clinical applications associated with the risk of development of

malnutrition-induced secondary complications. Dietary supplementation with different

levels of nucleotides on immune function in protein-malnourished mice was studied.

Adult male mice were fed a casein (nucleotide-free) diet for 12 days, resulting in

partial depletion of tissue nucleobase pools. Further nucleotide depletion and mild









protein malnutrition were induced by feeding a protein-free diet for the next 6 days.

Mice were then given different dietary treatments for the next 6 days: protein-free with

or without 0.25 % yeast RNA, casein, or casein supplemented with graded levels of RNA

(0.1-1.5%) or the individual nucleobase uracil (0.025-0.375%). Two in vivo cell-

mediated immune tests were initiated: delayed-type hypersensitivity (DTH) assessed by

ear swelling in response to 2,4-dinitrofluorobenzene challenge and lymphoproliferation

assessed by enlargement of the popliteal lymph node (PLN) induced by footpad injection

of sheep erythrocytes. In other studies, infection resistance was evaluated by intravenous

challenge with Listeria monocytogenes or intradermal abscess using Staphylococcus

aureus. Viability of the organisms served as the criterion of immune strength. Finally,

in vitro immunity was measured by the intracellular killing of Listeria by peritoneal

macrophages.

The DTH and PLN enlargement tests indicate that suppressed immune function

induced by protein malnutrition was partially restored by protein repletion; addition of

RNA, but not uracil, further improved the immune response with a trend toward a dose-

related response. Resistance to either bacterial challenge was improved in mice refed

diets containing protein; addition of nucleotides was without benefit. Macrophage

bactericidal activity was unaffected by addition of either dietary protein or nucleotides.

Thus, repletion from protein malnutrition with diets enriched with nucleotides

strengthened cell-mediated immunity but failed to improve infection resistance and in

vitro macrophage bactericidal activity in this animal model.














INTRODUCTION


Nutrition and Immune Function


Nutritional status of the host is one of the most significant variables in

determining immune function. The body responds to injury or infection by inducing a

serious physiological drain on vital organs and tissues via complex metabolic reactions.

When the diet does not provide adequate nutrients and fuel, the deficiency must be made

up from the body's reserves. Therefore, if malnutrition occurs, the capacity of the

subject to heal or resist infection is lessened.

Associated with an increased susceptibility to infection, malnutrition is one of the

major causes of increased morbidity and mortality among hospitalized patients. The

prevalence of malnutrition has been reported to be 20 to 30% in hospitalized patients.

A substantial portion of these patients become malnourished as a result of illness,

inadequate dietary regimens, or the combination of both insults. Additionally, there is

an interaction between nutrition and infection. Malnutrition reduces resistance to

infection, and infection, in turn, adversely affects nutritional status. This synergistic

effect that suppresses host defense mechanisms and thereby leads to increased morbidity

and mortality from infectious complications has been documented in patients suffering

from AIDS, cancer, trauma, and thermal injuries.









2

Adequate protein status is a crucial factor for immune function. Several immune

mechanisms have been shown to be impaired in both dietary protein restricted laboratory

animals and severely protein malnourished human individuals. Protein malnutrition has

a particularly significant influence on cell-mediated immunity while humoral immunity

is less affected. Protein malnutrition results in an immune deficiency characterized by

thymic atrophy and impaired delayed-type hypersensitivity (DTH), reduced lymphokine

production, low T-cell counts, and decreased T-cell responses to specific or nonspecific

mitogenic stimuli. Although serum immunoglobulin levels (IgG or IgA) are normal or

even elevated, and antibody responses to most vaccines are normal or only slightly

altered in protein malnourished subjects, secretary IgA levels, number of IgA cells in

jejunal mucosa, and specific IgA antibody levels are significantly decreased. Neutrophil

chemotaxis and serum opsonic activity are normal but bacterial killing capacity is

markedly suppressed.

Under certain physiological stresses such as injury to tissues or exposure to

infectious agents, de novo synthesis of nucleotides may not be rapid enough to meet the

body's requirements for tissue repair and immune function. A certain amount of

nucleotides added as a supplement may enhance immune function and improve

maintenance of rapidly proliferating cells such as the pancreas and gut mucosa.

Therefore, exogenous nucleotides may have therapeutic or prophylactic applications

associated with the risk for development of malnutrition-induced secondary

complications.









3

Various nutrients have been identified to have important roles in modulating

specific aspects of immune function by maintaining gut integrity, stimulating the immune

system, and preventing bacterial translocation from the gut. Among these, arginine,

glutamine, nucleotides, and omega-3 fatty acids have demonstrated potential clinical

usefulness. Studies indicate that T-cell function is particularly sensitive to many

nutritional deficits or abnormalities and may compromise immunological responses.

Focusing on the relative significance of specific nutrients, research in clinical

nutrition has advanced the concept of disease-specific nutrition support. Although

traditional nutrition support has reduced the morbidity and mortality associated with

uncomplicated malnutrition, the complications of critical illness associated with immune

suppression (e.g., infections, sepsis, and multiple organ failure) continue to be a major

consequence despite the use of aggressive nutritional therapy. Therefore, research in

identifying specific nutrients which potentially have both a nutritional value and a role

of modulating immune function is of great interest and clinical relevance.


Objectives and Specific Aims


Protein deficiency produces impairment of the immune system and lack of dietary

nucleotides is also thought to impair immunity. The objective of this project was to

evaluate the immune-stimulating effects of dietary nucleotides using protein-malnourished

mice as the animal model. Different dietary levels of intact yeast RNA and an individual

nucleotide, uracil, were examined for their effects on the immune system via both in vivo

and in vitro immune function tests. Specific aims for the study were as follows:









4

1. to determine the minimum level of RNA supplementation required to observe a

significant immune-stimulating effect;

2. to determine the level of RNA supplementation that provides the maximum

immune-stimulating effect; and

3. to examine the immune-stimulating effects of an individual nucleotide, uracil,

when supplemented in graded dose levels of the diet.

To address the hypothesis that dietary nucleotides supplementation enhances

immune response, in vivo and in vitro immune functions were measured in mice. An

experimental protein malnutrition was produced in outbred young adult Swiss albino

mice. Moderate protein depletion was induced by lack of dietary protein in otherwise

isocaloric conditions. Groups of these protein-malnourished mice were then randomly

assigned to various purified isocaloric diets supplemented with RNA and then tested for

their response to various immune challenges.

This project is relevant to our understanding of nutritional factors involved in

enhancing immune activity. The results may be beneficial in the formulation of more

optimal diets for individuals exposed to stressor agents which signal the immune system.














LITERATURE REVIEW


Biochemistry and Physiology of Nucleotides


Metabolic Fate of Dietary Nucleotides

Other than the commonly recognized major nutrients, the diet brings with it other

major constituents of the living system, such as nucleic acids. This group of substances,

chemically speaking, are polynucleotides. They are composed of nucleotide units joined

by sugar-phosphate linkages. Each nucleotide is composed of a purine or pyrimidine

base linked to a pentose sugar, either ribose or 2-deoxyribose, which in turn is linked to

one or more phosphate groups. In other words, nucleotides are phosphate esters of

nucleosides with the phosphate attached to at least one of the hydroxyl groups of the

pentose. In endogenous catabolism, nucleic acids are degraded by deoxyribonucleases

or ribonucleases to nucleotides, which are then dephosphorylated by alkaline phosphatase

to yield inorganic phosphate plus a nucleoside. Nucleosides are cleaved by nucleosidase

or phosphorylase to yield the free purine or pyrimidine bases plus ribose or deoxyribose

1-phosphate (Adams et al. 1986, Henderson and Paterson 1973, Mathews and Van Holde

1990, Stryer 1981).

Nucleotides are involved in most biochemical and physiological processes. They

are the activated precursors of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

As a carrier of genetic information, DNA serves as a template for the formation of RNA.








6

Most RNA molecules, in turn, direct the synthesis of cellular proteins. In addition to

serving as the structural units of DNA and RNA, nucleotides participate in a number of

metabolic reactions fundamental to cellular activity. Derivatives of nucleotides are

metabolic regulators and activated intermediates in many biosyntheses. Adenosine

triphosphate (ATP) is the major substance for the transfer of chemical energy in the

biological systems. Uridine diphosphate (UDP)-glucose and cytidine diphosphate (CDP)-

diacylglycerol serve as activated intermediates in synthesis of glycogen and

phosphoglycerides, respectively, and S-adenosylmethionine is a major methyl group

donor. In addition, cyclic adenosine monophosphate (cAMP) and cyclic guanosine

monophosphate (cGMP) serve as mediators of hormone actions. Adenine nucleotide is

also a component of three major coenzymes: nicotinamide adenine dinucleotide (NAD*),

flavin adenine dinucleotide (FAD), and coenzyme A (CoA). These coenzymes are

important in carbohydrate, lipid, and protein synthesis (Adams et al. 1986, Henderson

and Paterson 1973, Mathews and Van Holde 1990, Stryer 1981).

In mammalian tissues, the synthesis of nucleotides occurs by two pathways. The

first pathway is de novo biosynthesis of purine and pyrimidine bases through a series of

reactions to form respective mononucleotides, such as inosine 5'-monophosphate (IMP)

and uridine 5'-monophosphate (UMP). The second pathway is known as the salvage

pathway in which the preformed nucleobases that become available either from the diet

or through normal endogenous enzymatic breakdown of nucleic acids react with

phosphoribosylpyrophosphate (PRPP) to produce nucleoside 5'-phosphates (Adams et al.

1986, Henderson and Paterson 1973, Mathews and Van Holde 1990, Stryer 1981).









7
Nucleic acids are present in most foods and a mixed diet contains about 0.25%

of these substances. Dietary nucleic acids are unaffected by gastric enzymes. Digestion

occurs mainly in the duodenum, where nucleic acids are hydrolyzed to oligonucleotides

by pancreatic nucleases. Diesterases in the intestinal mucosa hydrolyze the

oligonucleotides to mononucleotides which are then hydrolyzed by intestinal phosphatases

or nucleotidases to form nucleosides. Phosphorolysis of the N-glycosyl linkage occurs

in reactions catalyzed by the specific nucleoside phosphorylases (Adams et al. 1986,

Henderson and Paterson 1973, Mathews and Van Holde 1990, Stryer 1981).

Of the two dietary purine bases, adenine is absorbed, while guanine is degraded

to uric acid (Clifford et al. 1976, Savaiano and Clifford 1978, Savaiano et al. 1980).

The dietary pyrimidine bases (cytosine, thymine, and uracil) are absorbed, and a

considerable amount is eventually excreted via the urine (Sonoda and Tatibana 1978).

Both dietary purines and pyrimidines decrease de novo pyrimidine biosynthesis in humans

(Zollner and Grobner 1977).

Studies indicate that diets supplemented with yeast RNA or uridine increased

uracil levels in urine, plasma, and erythrocytes in rats (Heaf and Davies 1976). Purine

and pyrimidine bases or nucleosides are constantly being converted into DNA, RNA, or

other nucleotides, and are reutilized through salvage pathways or degraded when not

required and excreted.

Nucleosides and free nucleobases are believed to be the chemical forms for

absorption because intact nucleotides are not able to permeate cell membranes. Although

the mechanisms of intestinal absorption are not well defined, passive absorption has been









8
reported (Khan et al. 1975, Oh et al. 1967, Schanker et al. 1963, Wilson and Wilson

1962). Several in vitro studies using rats and hamsters showed that dietary nucleobases

were rapidly and completely absorbed from the small intestine (Khan et al. 1975, Wilson

and Wilson 1962). On the other hand, some studies indicated that nucleobases may in

fact be secreted, rather than be absorbed, by the gut (Berlin 1971, Berlin and Hawkins

1968a and 1968b). In isolated sacs of small intestine, secretion occurred against a

concentration gradient abolished by metabolic inhibitors.

How dietary nucleotides affect de novo nucleotide synthesis and the body's

nucleobase pool is not understood (Seegmiller et al. 1977). In isolated chick liver cells,

adenine and guanine has been demonstrated to reduce purine synthesis while adenosine

5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) show a stimulatory

effect (Badenoch-Jones and Buttery 1976). In contrast, Grobner and Zollner (1977) have

concluded that dietary purines are almost completely excreted as uric acid and

consequently have no effect on de novo purine synthesis in humans. A study with mice

fed radioactive yeast nucleic acids indicated that while much of the dietary nucleic acids

were excreted in the urine, a small amount was retained in the tissues (Burridge et al.

1976). In another study, rats intubated with radiolabeled free purines bases showed

efficient absorption but rapid excretion with little tissue incorporation (Savaiano and

Clifford 1978). These results agree with those of Simmonds et al. (1973a and 1973b)

who fed free guanine to pigs and found less than 1% incorporation into tissues.

Bennett (1953) injected labeled adenine intraperitoneally into mice and found that

49% of it was incorporated into tissues. The greater incorporation of intraperitoneally









9

injected adenine compared with orally intubated adenine indicates that some adenine may

be metabolized during absorption and thus remain unavailable for incorporation into

tissue. Similar amounts of adenine recovered by body tissues in rats have been reported

by Tomlinson and Sbarsky (1957).

Sonoda and Tatibana (1978) studied the metabolic fate of purines in radiolabeled

nucleic acids added to a mouse diet. Results showed that the metabolism of purines is

distinguishable from that of endogenously synthesized purine bases. The metabolism of

the absorbed base is carried out mainly in the gastrointestinal and liver tissues. Most of

the exogenous nucleic acid bases appear to be removed, both by degradation and

utilization, from the portal circulation immediately after absorption. Since intestinal and

liver tissues are the first two organs which have initial access to these bases before entry

into the general circulation, only a small amount of dietary purine bases actually reaches

other tissues. Thus, utilization of ingested purines as precursors for nucleotide synthesis

may be limited in tissues other than stomach and intestines. Under normal conditions,

the endogenous synthesis of purines in other tissues may not be affected by the

availability of exogenous bases.

In a more conclusive in vivo study, Savaiano et al. (1980) compared the results

of orally and intravenously administered radiolabeled purines in rats. Orally

administered purine bases are extensively excreted in urine while intravenously

administered purines are retained in all body tissues, mainly in glandular and lymphoid

tissues. The difference in tissue retention associated with the route of administration

suggests that the gut and/or its microflora may influence the fate of exogenous purines.









10
Among all of the intravenously administered purines, adenine is incorporated most

extensively into body tissues. Similar results from oral administration have been reported

in other studies (Ho et al. 1979, Savaiano and Clifford 1978). Results of individual

purine absorption by isolated rat gut sacs showed that intestinal metabolism of dietary

adenine is uniquely different from that of guanine, hypoxanthine, and xanthine. A

significant proportion of unaltered adenine was found to cross the mucosal to serosal

barrier of intestinal sacs.

The final metabolic fate of exogenous purines in vivo is not well understood,

except that allantoin is the major end product of orally and intravenously administered

adenine in the rat (Ho et al. 1979, Savaiano and Clifford 1978). Whereas, in primates,

the major product is uric acid due to absence of urate oxidase. Uric acid formation

occurs in the liver and intestinal mucosa. The major route of exogenous purine excretion

is via the kidney. Dietary intakes of nucleobases, nucleotides, intact RNA and DNA,

and protein directly affect uric acid output (Clifford et al. 1976, Griebsch and Zollner

1974, Rauch-Janssen et al. 1977, Zollner and Griebsch 1974). Clifford et al. (1976)

showed that when given orally to humans, the individual purines yield different amounts

of uric acid in urine. However, the mechanisms underlying the characteristic effects of

individual purines on serum uric acid are still unknown.


Bioavailability of Dietary Nucleotides

The estimated daily turnover for purines and pyrimidines is 450 to 700 mg per

day in adult humans (Bono et al. 1964, Smith 1973). Since the body can synthesize

nucleotides, they are considered as nonessential dietary components. However, studies









11
of nucleotide biosynthesis suggest that little or no de novo biosynthesis occurs in certain

tissues such as bone marrow, leukocytes, and erythrocytes because the enzymes

necessary for the pathway are absent (Lajtha and Vane 1958, Lowy and Williams 1960,

Murray 1971, Scott 1962, Smellie et al. 1958). In addition, certain rapidly proliferating

cells, such as T-lymphocytes and intestinal epithelial cells, appear to lack the ability to

synthesize nucleotides. These cells meet their requirements for nucleotides by

reutilization of preformed bases released by other tissues into the systemic blood

circulation (Henderson and LePage 1959, Murray et al. 1970).

Inasmuch as digestion of food provides free nucleobases, the question arises

whether these free nucleobases contribute significantly to nucleotide synthesis in the cells

of the body. Studies of some diseases resulting from deficiency of enzymes for purine

and pyrimidine metabolism indicate the importance of these dispensable nutrients. For

example, the Lesch-Nyhan syndrome, a hereditary neurological disease is characterized

by the absence of hypoxanthine-guanine phosphoribosyltransferase (HG-PRT). It is

revealing in that the availability of dietary nucleobases may be important even to those

tissues of Lesch-Nyhan patients with demonstrable de novo nucleotide biosynthesis

because the reaction catalyzed by HG-PRT is the only pathway by which exogenous

hypoxanthine or guanine could be converted to nucleotides and utilized by the cells

(Henderson 1968, Rosenbloom et al. 1968).

The importance of dietary nucleotides in influencing protein metabolism and

immune function has been demonstrated (Chiba et al. 1985, Ogoshi et al. 1989, Pizzini

et al. 1990, Van Buren et al. 1983b). It has been demonstrated that dietary nucleotides









12
increase protein synthesis in mammalian tissues (Chiba et al. 1985). When protein intake

is relatively low, dietary nucleotides may also serve as precursors of DNA and RNA.

It appears that salvage pathways as well as typical intakes of dietary nucleotides are

inadequate to allow a strong response to severe metabolic stress such as trauma, burns,

and sepsis, and may contribute to immune dysfunction present in these conditions. In

effect, those cells which depend on the salvage pathway or typical dietary sources for

sufficient amounts of nucleotides may not be able to synthesize proteins and proliferate

upon increased demand for nucleotides. Therefore, dietary nucleotide supplementation

may have an important role in providing sufficient precursors for optimal synthesis of

nucleic acids.

The source of circulating preformed nucleobases has not been conclusively

established. With its great capacity for de novo nucleotide synthesis, the liver seems to

be the most likely source of nucleic acids supply (Henderson and LePage 1959, Lajtha

and Vane 1958, Mager et al. 1967, Pritchard et al. 1970, Smellie et al. 1958). Lajtha

and Vane (1958) obtained indirect evidence for the involvement of the liver in the

formation of purine nucleotides of rabbit bone marrow. In hepatectomized animals, the

labeled purine in marrow was originally synthesized from format released by other

tissues rather than from nucleobases as in normal animals. It was suggested that under

normal conditions there is a transfer of purines or derivatives from organs with a high

capacity for de novo purine nucleotide synthesis to tissues with insufficient capacity for

de novo synthesis. As a result, the mammalian liver is a major source of purines for

bone marrow cells and possibly for cells of most peripheral tissues (Lajtha and Vane 1958).









13
Henderson and LePage (1959) reported that radioactive adenine from prelabeled

rat liver is transferred to erythrocytes and also from washed prelabeled mouse

erythrocytes to tissue. Pritchard et al. (1970) provided more definitive evidence that the

liver is a major source of nucleic acids for other tissues. They demonstrated the

continuous transfer of liver purines to erythrocytes in vivo and apparently also to other

tissues. The quantity of purines supplied by the liver to erythrocytes was sufficient to

account for most, if not all, of their hypoxanthine-guanine turnover.

Rabbit and human erythrocytes exhibit turnover of adenine nucleotide during their

life-span (Lowy and Williams 1960, Mager et al. 1967) despite the inability for de novo

synthesis and the further inability of the human erythrocyte to convert IMP to AMP

because of the absence of adenylosuccinate synthetase (Bishop 1960, Lowy and Dorfman

1970, Lowy et al. 1962). In order to account for the maintenance of nucleotide levels

and for the observed turnover, it is probable that one or more precursors of nucleotides

is derived from an external source. The study done by Lerner and Lowy (1974)

indicated that hepatically derived adenosine can serve as a precursor of adenine

nucleotide of human and rabbit erythrocytes and may be involved in the mechanism for

their renewal.

Considerable quantities of purines exchange in the nucleobase pools of

erythrocytes. These cells take up free nucleobases and convert them into nucleotides.

Transport of hepatically synthesized purines by erythrocytes to tissues with limited or no

capacity of de novo synthesis has been reported (Henderson and LePage 1959, Lajtha and

Vane 1958,). For this function, erythrocytes should be able to perform a rapid uptake









14

and release of nucleic acids operating selectively under different conditions at suitable

points in the circulatory system.

Kinetics of radioactivity release from 14C-hypoxanthine-loaded human erythrocytes

suggest that hypoxanthine transport into cells could be mediated by a two-component

system, only one being saturable (Lassen 1967). This hypothesis is confirmed by Muller

and Falkner (1977) who devised a double reciprocal plot that revealed saturation kinetics

of hypoxanthine uptake and showed a competitive inhibition by adenine. Both saturation

kinetics and inhibition pattern could be regarded as evidence for the existence of a special

transport system for purines in the cell membrane of human erythrocytes.

De Bruyn and Oei (1977) studied the uptake of radioactive purine bases in intact

erythrocytes and reported the dependence of the transport rate on the concentration of

purine, which suggests a two-component mechanism. At low concentration, purine base

uptake depends mainly on the saturable carrier system. At higher concentration, a

nonsaturable diffusion system prevails. It was concluded that the saturable, nucleotide-

forming system plays a major role in the transport of purines in vivo.

The uptake of hypoxanthine mediated by its incorporation into IMP is dependent

on the activity of HG-PRT and on intracellular availability of PRPP (Gutensohn 1975).

Phosphoribosylpyrophosphate formation in erythrocytes is stimulated by a sufficiently

high inorganic phosphate level in the suspending medium (Hershko et al. 1969).

Therefore, nucleotide synthesis plays an important role in the translocation of purines

across the human erythrocyte membrane. The rate of hypoxanthine uptake by human









15

erythrocytes depends on the inorganic phosphate concentration in the medium and the

time of preincubation (Giacomello and Salerno 1983, Salerno and Giacomello 1979).

The existence of hypoxanthine binding receptors on the outer surface of the

human erythrocyte membrane has been suggested to regulate the transport of purine bases

through the cell membrane (Capuozzo et al. 1986). In the absence of inorganic

phosphate, the hypoxanthine receptor appears to be saturated by a relatively low purine

base concentration. When inorganic phosphate is present, PRPP becomes available, and

hypoxanthine is phosphoribosylated to IMP. Under these conditions the receptor releases

hypoxanthine across the cell membrane and takes up new exogenous purine base.


Is There a Requirement for Dietary Nucleotides?


Dietary Nucleotides in Infant Nutrition

Dietary nucleotides may have significant effects in the infant. Due to its role in

the synthesis of protein and other macromolecules, nucleotide supplementation of

formulas has been of research interest in infant nutrition. Human milk contains relatively

high amounts of nucleotides (accounts for approximately 0.1 to 0.15% of the total

nitrogen content), mainly CMP, AMP, UMP, IMP, and UDP sugars, compared with

those in cow's milk and infant formulas made from milk proteins. In addition, dietary

nucleotides have been shown to modulate metabolic function in gut and liver (Barness

1994), decreasing the incidence of diarrheal disease in infants (Brunser et al. 1994), and

ensuring normal growth and immune function of newborn infants (Carver et al. 1991).









16
Dietary nucleotides are involved in fatty acid metabolism. Studies of the

polyunsaturated fatty acid (PUFA) composition of plasma and erythrocyte membrane

phospholipids in term neonates indicated that dietary nucleotides play a role in the in vivo

regulation of desaturation and elongation of essential fatty acids. The relative content of

omega-3 and omega-6 PUFA greater than 18 carbon atoms was significantly reduced in

infants fed regular formula compared with those fed human milk or the same formula

supplemented with nucleotides at a level similar to that present in human milk (DeLucchi

et al. 1987, Gil et al. 1986). Since linoleic acid in plasma lipid fractions was increased

in infants fed formula compared with those fed human milk or nucleotide-supplemented

formula, it was suggested that supplementation of dietary nucleotides may be used to

reverse the partial inhibition of delta 5-desaturase induced by excess linoleic acid in the

diet (Gil et al. 1988). The enhancement of lipoprotein synthesis or secretion during the

early neonatal period by dietary nucleotide supplementation is even more significant in

preterm infants (Morillas et al. 1994, Pita et al. 1988, Sanchez-Pozo et al. 1994).

Despite extensive modification by infant formula manufacturers, fecal flora of

bottle-fed babies remains substantially different from that of breast-fed babies (Wharton

et al. 1994). Most differences in fecal flora were found at 2 weeks of age when breast-

fed babies had bifidobacteria, lactobacilli, and staphylococci as the dominant organisms,

whereas formula-fed babies were predominantly colonized with enterococci, coliforms,

and bacteroides.

Bifidobacteria have been found to be more abundant in the feces of breast-fed

babies compared with those of formula fed babies. Although nucleotides have been









17

suggested as cofactors for the growth of bifidobacteria in vitro, results of some studies

do not support their addition for the enhancement of bifidobacteria in the faecal flora as

growth of bifidobacteria was hindered in infants fed a formula supplemented with

nucleotide salts (Balmer et al. 1994, Uauy 1994).


Nucleotides in Enteral and Parenteral Nutrition

The primary factor which prevents delivery of adequate nutrition to sick patients

is the inability to tolerate food by the oral route. Patients with malabsorption syndromes,

pancreatitis, colitis, intestinal obstruction, advanced cancer, burn injury, and those

subjected to chemical and radiation therapy usually require some form of forced nutrient

delivery. If the intestinal tract is functional, then patients are prepared with an enteral

tube through which commercially available, defined-formula diets are passed. If enteral

delivery is contraindicated or insufficient, then patients are nourished by intravenous

feeding.

Most of the commercially available enteral formulas are based on milk proteins

such as casein and whey, which supply very little usable nucleotides. Nutrient formulas

for total parenteral nutrition (TPN) are compounded from commercially available purified

products which include dextrose, synthetic amino acid solutions, lipid emulsions, fat and

water soluble vitamin preparations, and electrolyte and trace element combinations. With

their extreme purity, TPN products are completely free of nucleotides.

Although considerable progress has been made in efforts to formulate optimal

parenteral and enteral solutions to meet patient needs, these feeding techniques are

associated with altered metabolic functions, such as decrease in gastrointestinal function









18

(Sitren and Stevenson 1980, Stevenson et al. 1980), pancreatic hypoplasia (Callegari et

al. 1985), liver dysfunction (Sitren and Stevenson 1980), altered immune function

(Birkhahn and Renk 1984), hyperammonemia (Seashore 1980), disruption of the normal

gut flora population (Bryant et al. 1988), and increased permeability of gut to

translocation of microorganisms into the circulation (Klimberg et al. 1989), thereby

increasing the risk for infection.

The use of total enteral nutrition in the critically ill patient has been promoted

aggressively due to its lower cost, similarity to normal physiological responses to eating,

and lower complication rate when compared to parenteral feeding. Various components

such as glutamine, arginine, RNA (nucleotides), refined menhaden oil (omega-3 fish

oils), and fiber have been suggested to be important in maintaining gut integrity,

stimulating the immune system, and preventing bacterial translocation from the gut

(McClave et al. 1992). Evidence from recent studies on parenteral and enteral nutrition

suggest that nucleotides may be beneficial in reducing feeding-related abnormalities of

hospitalized patients (Cerra et al. 1991a and 1991b).

Preliminary evidence suggests that Impact* (Sandoz Pharmaceuticals, East

Hanover, NJ), a commercially available enteral formula supplemented with arginine,

yeast RNA, and omega-3 fatty acids, improved immunologic, metabolic, and clinical

outcomes in surgical and intensive care unit patients (Alexander 1993, Cerra et al. 1991a,

Daly et al. 1992). Recently, in a prospective, randomized, double-blind, multicenter trial

with early enteral feeding, intensive care unit patients who received this specialized

enteral formula showed a substantial (approximately 75%) reduction in hospital stay









19
along with a significant (more than 20%) reduction in the frequency of acquired

infectious complications when compared with feeding a common use enteral formula,

Osmolite HN" (Ross Laboratories, Columbus, OH) (Bower et al. 1995). However, in

the animal studies, the same product showed no advantage in improving survival from

experimental E. coli peritonitis and was no more effective than standard enteral feeding

products or rat Chow* in maintaining intestinal morphology or restoring it following

fasting (Grant et al. 1994). The intravenous infusion of specific nucleosides and

nucleotides has been documented to improve liver function in rats with liver injury

induced by D-galactosamine (Ogoshi et al. 1988) and to reduce intestinal atrophy

associated with total parenteral feeding in rats (Shenkin 1994).


Importance of Dietary Nucleotides


Maintenance and Enhancement of Immune Function

The importance of nucleotide-free feeding in suppressing immune responses has

been demonstrated in recent investigations (Kulkarni et al. 1994). Interest in a possible

connection between dietary nucleotides and immune function was first aroused from

observing decreased allograft rejection in renal transplant patients receiving TPN (Van

Buren et al. 1983a). Subsequent work in animals confirmed that deprivation of

exogenous nucleotides is definitely beneficial under certain conditions in which immunity

must be depressed. However, it is common that immune function needs to be enhanced

in order to decrease risk for infections (Rudolph et al. 1984, Van Buren et al. 1990a).









20

Dietary nucleotides are important for the maintenance of cell-mediated responses

in mice (Rudolph et al. 1990). Absence of a dietary source of preformed nucleotides

suppress T-lymphocyte function. Suppressed sensitivity to xenoantigens in mice fed a

diet which lacks nucleotides indicates that nucleotides are vital to the activity of T

effector cells in DTH (Kulkarni et al. 1982 and 1987). Dietary nucleotide restriction also

delays the onset of primary murine cardiac allograft rejection and acute graft-versus-host

disease followed H-2-incompatible bone marrow transplantation (Kulkarni et al. 1984,

Van Buren et al. 1983b and 1987). The proliferative response of spleen cells to mitogens

(Kulkami et al. 1984) and phagocytic activity of macrophages (Kulkarni et al. 1986a) are

also reduced. Addition of 0.25% RNA and 0.06% uracil (a level equal to that in 0.25%

RNA) restores tests of T-cell mediated immune function to normal or near-normal

whereas addition of 0.06% adenine is without benefit. This suggests a need for

pyrimidines rather than purines in these types of responses.

The target cell of dietary nucleotide deprivation appears to be the helper/inducer

T-lymphocytes which appear to require exogenous nucleotides to respond optimally

following immune stimulation. Failure to provide nucleotides selectively suppresses

helper T-cells and suppresses interleukin (IL) production (Fanslow et al. 1988).

Production of IL-1, IL-2, IL-3, and IL-4 in response to mitogen in spleen cell cultures

from mice fed a nucleotide-free diet was decreased. Supplementation with RNA or uracil

was able to restore production of lymphokines (Kulkarni et al. 1984).

Restriction of dietary nucleotides also affected phenotypes of T-lymphocytes.

While naive animals fed diets with no nucleotides or with varying nucleotide content









21

showed no differences in splenic lymphocyte subpopulations following complete Freund's

adjuvant stimulation, splenic lymphocytes from nucleotide-depleted mice showed fewer

Thy 1.2+ cells and a significant lower level of Lyt 1+ cells (Van Buren et al. 1985).

The fact that diets supplemented with RNA or uracil significantly augment expression of

IL-2 receptor, Mac-1, and Lyt 1 suggests that dietary nucleotides are effective in the

macrophage activation of the helper T-cell population.

Dietary nucleotides may also play a critical and regulatory role in immuno-

hematologic responses. A nucleotide-free diet has been found to decrease in vivo and in

vitro production of hemopoietic growth factor and IL-3, a cytokine which is normally

produced by activated T-cells to enhance the differentiation of early T-cell precursors,

promote lymphocyte proliferation, and induce the T-cell receptor surface markers on T

precursor cells in bone marrow. Decreased production of hemopoietic growth factor,

rendering the host splenic environment deficient for stem cell growth, was reversed by

supplementation of diets with 0.25% RNA and partially recovered by 0.06% uracil

supplementation. Addition of 0.06% adenine had no significant effect (Kulkami et al.

1992).

While some investigators reported that B-cell function does not appear to be

influenced by dietary nucleotide levels and there is no change in antibody formation after

antigen challenge (Rudolph et al. 1984), RNA supplementation to culture media does

affect in vitro production of antibodies in response to T-cell-dependent antigens

(Jyonouchi et al. 1992 and 1994). It is possible that supplementation of RNA may

enhance antibody production by augmenting the production of various soluble factors









22

(e.g., interleukins) by T helper cells. When supplemented to culture media, whole yeast

RNA has also been shown to potentiate the proliferation of murine spleen lymphocytes

while certain mononucleotides exhibited less, but similar, action. The enhancing activity

of RNA appears to be dose-dependent and is significantly reduced by RNAse treatment

(Jyonouchi et al. 1992).

Humoral immune responses to T-cell-dependent antigen were depressed in mice

fed a nucleotide-free diet. In vivo and in vitro antibody production and numbers of

immunoglobulin M and G secreting cells in the spleen in response to T-cell-dependent

antigens in mice fed a nucleotide-free diet were significantly lower while responses to

T-cell-independent antigens remained intact (Jyonouchi et al. 1994). Intraperitoneal

injection of a mononucleotide-nucleoside mixture restored T-cell function but had no

effect on in vitro antibody production and did not further increase humoral immune

responses in mice fed a stock diet (Jyonouchi et al. 1994). It was suggested that the in

vivo actions of polynucleotides on humoral immune responses may only reflect local

immune responses while mononucleotides and nucleosides may be incorporated into the

tissue nucleobase pool fairly rapidly and help to restore T-cell-dependent humoral

immune responses.

The decline in immune function induced by restriction of dietary nucleotides has

not been well categorized but is thought to be mediated by decreased T helper activity,

diminished T-effector function, suppressed IL-2 production, and depressed macrophage

function. These immunosuppressive effects are directly related to the dependence of T-









23

lymphocytes for circulating nucleotides, which appear to be necessary for T-cell

maturation and expression of phenotypic markers (Kulkarni et al. 1989).

It has been demonstrated that a nucleotide-free diet delays lymphocyte maturation

thereby impairing T-cell function (Rudolph et al. 1986, Van Buren et al. 1985). The

results of terminal deoxynucleotidyl transferase (TdT) analysis demonstrated that mice

maintained on nucleotide-free diet have a higher percentage of immature lymphocytes

(Rudolph et al. 1986). Removal of nucleotides from the diet may block maturation of

lymphocytes by decreasing the number of cells that enter the S phase of the cell cycle.

When activated by an immune stimulus, lymphocytes are transferred into a state of high

biochemical activity and appear to have a requirement for an exogenous supply of purines

and pyrimidines for maximal function. Although whole yeast RNA has been shown to

be moderately mitogenic to mature spleen lymphocytes, it does not potentiate the

proliferation of thymocytes (immature lymphocytes). Since the large thymocytes in S

phase are capable of de novo synthesis of nucleotides, whereas mature lymphocytes in

G, stage are largely dependent upon exogenous nucleotides, certain components of

exogenous RNA may enhance lymphocyte function depending upon the stages of

proliferation or maturation of cells.

Enzyme defects in the purine salvage pathways have revealed the importance of

nucleotides to maintenance of a normal immune response in human beings. Two

differentiation-induced enzymes critical for T lymphocyte function and maturation,

adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), have been

associated with inherited immunodeficiency diseases (Giblett et al. 1972 and 1985). The









24

induction of ADA and PNP in popliteal lymph nodes (PLNs) during proliferative

response to in vivo antigen stimulation appears to require dietary sources of certain

nucleotides (Kulkarni et al. 1986c).

Lack of induction or decreased ADA and PNP activity in animals fed a

nucleotide-free diet is associated with decreased in vivo lymphoproliferation response

because these enzymes are critical for T-lymphocyte function and maturation of a normal

immune response. Although supplementation of a nucleotide-free diet with yeast RNA

or uracil has been demonstrated to enhance the host immune response in mice (Kulkarni

et al. 1992), in the clinical situation, using parenteral deoxycytidine therapy as treatment

for ADA deficiency and combined immunodeficiency disease in humans showed only

transient improvement in the in vitro immune response and no clinical improvement in

long-term treatment (Cowan et al. 1985).

Nucleotides are natural components of human milk that may contribute to the

enhanced immunity of the breast-fed infant. When healthy term infants were either

breast-fed or fed standard formula (NT-) or standard formula supplemented with

nucleotides (NT+) for 2 months, in vitro natural killer (NK) cell cytotoxicity was

significantly higher in the breast-fed and the NT+ groups compared with the NT-

group. Interleukin-2 production by stimulated mononuclear cells was also higher in the

NT+ group compared with the NT- group (Carver et al. 1991).

Dietary nucleotides may also be important in accelerating recovery from

malnutrition. A nucleotide-free diet supplemented with 0.25% yeast RNA has been

shown to reverse the immunosuppression induced by a protein-free diet in mice (Pizzini









25

et al. 1990, Robin 1990, Van Buren et al. 1990b). Both helper T-cells and macrophage

population appeared to benefit from RNA supplementation. After starvation for 7 days,

animals previously maintained on the nucleotide-free diet supplemented with RNA

showed a significant increase in mitogen-stimulated blastogenesis (Pizzini et al. 1990).

These studies indicate that exogenous nucleotides may have therapeutic or

prophylactic applications. Under certain conditions, a supplement may enhance immune

function and improve maintenance of rapidly proliferating cells such as epithelial cells

and T-cells. The effect of dietary nucleotides on immune function should be of great

importance in a number of clinical situations such as management of organ transplant

patients, recovery from malnutrition, various chemotherapeutic regimens, and treatment

of T-cell derived leukemias.

Although the administration of dietary nucleotides at levels exceeding those

normally present in diets may have immune-enhancing properties, a very high level of

nucleotides may also cause toxic effects. NK cell activity, macrophage activation, and

spleen weight (as percent of body weight) were higher in mice fed up to 0.035% (w/w)

nucleotides than those in mice fed a nucleotide-free diet (Carver et al. 1990). However,

macrophage activation was slightly decreased by feeding over 0.35% compared to those

receiving a nucleotide-free diet. The recommended maximum safe limit for adult humans

of nucleic acids from all dietary sources is 4 g per day (PAG 1975). Studies are needed

in finding the optimal dose which results in maximal immunological response without

undesirable side effects.










Improvement of Infection Resistance

Infection and sepsis remain major causes of death in medical and surgical

services, despite the availability of potent antibiotics and close monitoring of patients in

intensive care units. Host resistance to infection is adversely affected by restriction of

dietary nucleotides in animals. Mice maintained on a nucleotide-free diet exhibited a

significant increase in mortality from candidiasis and staphylococcal sepsis compared with

mice fed diet supplemented with nucleotides at 0.25% (Fanslow et al. 1988, Kulkarni et

al. 1986a and 1986b). In addition, the number of viable organisms recovered in the

reticuloendothelial system was significantly increased following intravenous injection of

Candida albicans or Staphylococcus aureus. The mechanism for the increased

susceptibility to infection appears to be decreased ability of the host to destroy

microorganisms. Splenic macrophages ingest, but do not necessarily kill, the

microorganisms. Addition of 0.25% RNA or 0.06% uracil decreased the susceptibility

of the host to a microbial challenge by restoring normal immune responses. However,

addition of 0.06% adenine did not improve the resistance to infection (Fanslow et al.

1988, Kulkarni et al. 1986a and 1986b).

When challenged with an intravenous injection of 1.0 x 10' cells of S. aureus

(ATCC 25923), mice fed a nucleotide-free casein diet had a 100% mortality while there

were survivors in mice fed Chow (60%) or casein diet supplemented with 0.25% RNA

(48%). Addition of 0.06% uracil to the nucleotide-free diet resulted in mortality similar

to that of the 0.25% RNA group whereas addition of 0.06% adenine showed a mortality









27

close to that of the nucleotide-free diet group. Certain dietary nucleotides appear to be

essential for improving the resistance to bacterial challenge (Kulkarni et al. 1986a).

In vitro macrophage phagocytic activity as measured by the uptake of radiolabeled

bacteria was significantly decreased in mice on nucleotide restricted diets. However,

there was no difference in antibody production in response to S. aureus among the

various diets (Kulkarni et al. 1986b). These suggests that B-cell immunity is not

responsive to dietary nucleotide levels. When challenged intravenously with graded

inocula of C. albicans, mice fed a nucleotide-free diet had a significantly lower mean

survival time and higher viable organism recovery in the spleen compared with mice fed

diets supplemented with either 0.25% RNA or 0.06% uracil (Fanslow et al. 1988).

Intraperitoneal administration of a nucleoside-nucleotide mixture to mice

maintained on a nucleotide-free diet was effective for the recovery from the infection.

After challenge with an intravenous inoculation of methicillin-resistant S. aureus

(8985N), survival rate was significantly higher and the viable organisms recovered from

the spleen and kidney of the surviving mice were fewer in the nucleoside-nucleotide

group than that of the control (saline) group (Adjei et al. 1992 and 1993a). Furthermore,

this infection recovery effect cannot be fully achieved with the individual components of

the mixture, namely inosine, GMP, uridine, thymidine, and cytidine (Yamamoto et al.

1993), although improvement was noted.

Following bacterial challenge by an intravenous injection of methicillin-resistant

S. aureus, intraperitoneal administration of a nucleoside-nucleotide mixture containing

inosine, GMP, uridine, thymidine, and cytidine increased the peripheral blood neutrophil









28

numbers and stimulated bone marrow cell proliferation as evidenced by incorporation of

intraperitoneally administered bromodeoxyuridine, an thymidine analogue, into the DNA

of bone marrow cells while oral administration of the same mixture elicited similar but

weaker responses (Matsumoto et al. 1995). Supplementation of the nucleotide-free diet

with RNA at 0.5% did not further improve survival of mice injected with the nucleoside-

nucleotide mixture (Adjei et al. 1993b).


Development and Maturation of Intestinal Tissues

Optimal nutritional support is essential to preserve the integrity of the gut mucosal

barrier. Dietary nucleotides may play an important role in the development and

maturation of intestinal tissues (Uauy et al. 1990). Removal of dietary purines and

pyrimidines has been shown to result in a dramatic decrease of intestinal and colonic total

RNA in rats (LeLeiko et al. 1987). In adult rats, deprivation of dietary nucleotides

affects the maturation status of small intestine epithelium by decreasing the content and

specific activity of the brush border enzymes such as alkaline phosphatase, leucine-

aminopeptidase, maltase, sucrase, and lactase. These enzymes serve as maturation

markers of the intestinal cells (Ortega et al. 1995). In different subpopulations of

epithelial cells separated sequentially from the villus tip-to-crypt axis of the small

intestine, nucleotide deprivation showed little effect on enzymatic activity within the crypt

zone but had progressively increasing effect towards the tip of the villus.

Because rapid renewing of intestinal mucosal cells may benefit from an external

source of nucleotides for their optimal growth and maturation, external provision of

nucleotides may be necessary to sustain mucosal function and prevent the progression of









29

TPN-associated gut mucosal atrophy by compensating for a relatively insufficient delivery

from liver. The addition of a mixture containing inosine, GMP, cytidine, uridine, and

thymidine to a standard TPN solution improved mucosal growth and maturity in rats by

increasing the proliferating activity of crypt cells as evidenced by increased villus height,

mucosal wet weights, bromodeoxyuridine labeling index, diamine oxidase activities, and

protein and DNA content (lijima et al. 1993, Tsujinaka 1994, Tsujinaka et al. 1993).

Dietary nucleotides appear to be modulators of intestinal development after

chronic diarrhea and may have important implications in the growth of enterocytes.

They have been shown to be critical for intestinal repair in intestinal diseases in which

the mucosa is altered (Nunez et al. 1990). Dietary nucleotide supplementation has also

been shown to promote recovery of the intestinal mucosa from experimental chronic

diarrhea induced by a lactose enriched diet in the weanling rat (Bueno et al. 1994, Nunez

et al. 1990). In a newborn swine model, perfusion of the intestinal lumen with

nucleotides decreased the inflammatory response to ischemia-reperfusion. In the

presence of nucleotides, the number of leukocytes accumulating in the intestine was

lower and the degree of protein leakage and the production of nitric oxide during

ischemia were reduced (Bustamante et al. 1994).

In protein-malnourished mice tested for endotoxin-induced bacterial translocation,

dietary supplementation with a nucleoside-nucleotide mixture at 0.5% of the diet led to

a reduced number of gram-negative enteric bacteria in the mesenteric lymph node and

spleen. Histologically, the damage to the gut mucosal barrier induced by endotoxin was

less pronounced in the supplemented group (Adjei and Yamamoto 1995).










Protection and Regeneration of Internal Organs

Dietary nucleotide supplementation has been shown to affect hepatic growth and

composition in weanling mice (Novak et al. 1994). Hepatic cholesterol was significantly

higher, but liver fatty acid composition and distribution of phospholipid subclasses were

not affected by dietary nucleotide supplementation. Liver weight and glycogen were

lower in animals fed a nucleotide-free diet compared with mice fed the same nucleotide-

free diet supplemented with either a mixture of 5 nucleotides (0.21% w/w) or just AMP

(0.0425% w/w).

Nucleotides have also been demonstrated to have protective effects on liver injury

induced by carbon tetrachloride (CC14) administration in rats (Kitajima et al. 1994).

Other studies also showed that nucleotide supplementation is able to prevent a fall in

metabolic function in physiologically compromised rats. For example, rats subjected to

70% hepatectomy or chemically induced liver injury showed more rapid recovery when

provided with intravenous uridine and thymidine (Ogoshi et al. 1985, 1988, and 1989).

In other studies, partially hepatectomized, parenterally fed rats were used as a

model of patients undergoing extensive hepatectomy due to liver cancer.

Supplementation with a nucleoside-nucleotide mixture (0.4% w/v) to the TPN formula

in rats after 70% hepatectomy resulted in restoration of nitrogen balance within 3 days

(Ogoshi et al. 1985 and 1989) as compared with 5 to 7 days in rats without the

supplementation. Rates of protein turnover and protein synthesis were also significantly

higher in the rats supplemented with a nucleoside-nucleotide mixture (13% v/v) than in

the control group. An increase in protein synthesis, rather than change in protein









31

breakdown, was thought to be responsible for the better nitrogen balance of the

nucleotide-supplemented group (Ogoshi et al. 1989).

In a study that focused on preventing loss of adenine nucleotides from heart cells,

an event that causes ischemic myocardial damage in surgical patients, the effect of a

parenteral nucleotide formula on myocardial mechanical function and energy metabolism

was examined in stunned myocardium of dogs subjected to coronary artery ligation.

With a positive inotropic action, infusion of a nucleoside-nucleotide mixture resulted in

a marked cardioprotective effect in association with the restoration of tissue ATP levels

(Satoh et al. 1993).


Proliferation and Differentiation of Cultured Cells

Under normal culture conditions, supplementation of the medium with a

nucleotide mixture containing equal amounts (10 mg per L) of AMP, CMP, GMP, IMP,

and UMP has been shown to enhance proliferation and differentiation of normal rat small

intestinal crypt cells (IEC-6) whereas human colon tumor cells (Caco-2) were not

affected. However, when the level of glutamine in culture media was deprived,

nucleotide supplementation promoted growth and maturation of both cell lines (He et al.

1993). With a more active endogenous nucleic acid metabolism as evidenced by greater

cellular pools of nucleotides and their metabolites, the de novo nucleotide biosynthesis

in tumor cells is sufficient to meet the requirements for proliferation. Exogenous

nucleotides appear to be needed only when the supply of glutamine is limited. On the

other hand, nucleotide supplementation is essential for growth and maturation of normal

enterocytes (Sanderson and He 1994).









32

In another study, addition of thymidine or a nucleotide mixture containing inosine,

GMP, cytidine, uridine, and thymidine (4:4:4:3:1 in molar ratio) did not enhance the

proliferation of a human gastric cancer cell line (KATO III). However, the antitumor

effect of 5-fluorouracil was enhanced by the coadministration of thymidine (Wang et al.

1994).


Conversion and Composition of Fatty Acids

Dietary nucleotides affect fatty acid composition via the conversion of essential

fatty acids into their long chain polyunsaturated derivatives. Weanling rats fed a

semipurified diet supplemented with equal amounts (250 mg per 100 g diet) of AMP,

CMP, GMP, IMP, and UMP showed significant increases in levels of the n-6 series of

long chain polyunsaturated fatty acids (LC-PUFA) in both plasma and the red blood cell

membrane (Boza et al. 1992, Jimenez et al. 1992). Although the concentrations of the

n-3 series of LC-PUFA in plasma were also increased, there was minimal change in the

red blood cell membrane. Similar results were obtained in phospholipids of liver

microsomes of weanling rats fed diets supplemented with equal amounts (50 or 250 mg

per 100 g diet) of AMP, CMP, GMP, IMP, and UMP (Nunez et al. 1993). Since the

cholesterol:phospholipid phosphorus ratio was maintained fairly constant, dietary

nucleotides may modify PUFA metabolism through a lowering of delta-9 desaturase and

increasing delta-4 and delta-5 desaturases activities. Furthermore, the blood levels of

two major eicosanoids, namely prostacyclin (PGI2) and thromboxane (TXA2), were also

increased in weanling rats fed the diet supplemented with nucleotides (Ramirez et al.

1991).














MATERIALS AND METHODS


Animal Model and Dietary Regimen


Diet Preparation

The diets were based on the AIN-76 diet for laboratory animals (American

Institute of Nutrition 1977, Appendix A). Protein-free, casein, and nucleotide

supplemented diets consisting of relatively pure sources of nutrient ingredients (BioServ,

Holton Industries Co., Frenchtown, NJ and Dyets Inc., Bethlehem, PA) were prepared

in the laboratory. Casein is very low in naturally occurring nucleotides and thus the diet

is considered to be essentially nucleotide-free (Kulkarni et al. 1986b). To stabilize the

corn oil, 0.02% antioxidant, butylated hydroxytoluene (BHT, Eastman Kodak Co.,

Rochester, NY) was added. In the nucleotide supplemented diets, specific amounts of

yeast RNA (type VI, Sigma Chemical Co., St. Louis, MO) or uracil (Sigma Chemical)

were added to the basal diets (casein or protein-free) and the relative percentage of each

ingredient in the diet was maintained by the removal of the same amount of corn starch.

Analysis of yeast RNA and uracil by atomic absorption spectrophotometry showed they

are essentially free of zinc, an immune function stimulator.

Ingredients for the semi-purified diets were measured with an electronic

toploading balance (7204A, Denver Instrument Co., Denver, CO), well mixed with a

blender (Univex Mixer, Universal Industries), and pelleted to 1/4 inch in diameter using

33









34

a food extruder (Wenger Manufacturing, Sabetha, KS). Commercial food coloring was

used to aid identification of diets. The diets were then air dried at room temperature for

3 days, put in zipped plastic bags, and stored at -20*C.

Stock diet (Laboratory Rodent Diet 5001, Purina Mills, Inc., St. Louis, MO) is

a commercially available laboratory animal diet that consists of nonpurified ingredients,

such as ground whole grains, soybean meal, fish meal, brewers yeast, etc., plus added

vitamins and minerals (Appendix A). The nucleotide content of the stock diet has been

measured to be approximately 0.25% (Kulkarni et al. 1986b). It was stored at room

temperature.


Animal Model and Feeding Protocol

To study the effect of dietary nucleotide supplementation on immune function

under conditions of nutritional stress, an experimental protein malnutrition model was

produced in mice. Partial protein depletion was induced by restriction of protein in

otherwise isocaloric conditions with all other essential nutrients for mice. This model

resembles human protein malnutrition with respect to weight loss, hypoproteinemia, and

hypoalbuminemia (Barone et al. 1993).

Outbred young adult male Swiss albino (ICR) mice weighing 21 to 25 g (4 to 6

weeks of age from Harlan Sprague Dawley, Inc. Indianapolis, IN) were used for all

experiments. The animals were housed in stock cages (6 mice per cage) containing wood

shaving bedding and given free access to the stock diet and water. All animals were

maintained in a temperature-controlled (approximately 24C) animal facility with a

constant 12-hour light and dark cycle.









35

Mice were allowed to acclimate to the laboratory for at least 1 week prior to

experiments. They were then given free access to the nucleotide-free casein-based diet

for 12 days followed by a protein-free diet for 6 days to induce moderate protein

malnutrition and to partially deplete tissue nucleobase pools evidenced by changes in

immune function (Van Buren et al. 1983b). Groups of these protein-malnourished mice

were then randomly assigned to the following dietary regimens for the next 6 days:

protein-free with or without 0.25% RNA, stock, casein, or casein supplemented with

graded levels of RNA (0.1, 0.25, 0.5 or 1.5%) or uracil (0.025, 0.0625, 0.125, or

0.375%). In addition, three groups of mice without protein malnutrition were also

included in the study. One of these groups received casein diet whereas the other two

groups were fed stock diet throughout the experiment. One of the stock diet groups

served as the well-nourished control while the other group was injected intraperitoneally

3 days before immune test with an immunosuppressive agent, methotrexate (MTX,

+Amethopterin, Sigma Chemical). Methotrexate was diluted to 10 to 20 mg per ml with

sterile 2.2% sodium bicarbonate and injected intraperitoneally at 85 mg per kg body

weight. This group served as immunosuppressed positive control as a check on the

efficacy of the immune function tests.


Preparation of Bacterial Culture


In the preparation of bacterial culture for immune assays, a single colony was

obtained from a Trypticase* soy agar (TSA, Becton Dickinson, Cockeysville, MD) master

plate and grown in Trypticase soy broth (TSB, Becton Dickinson) supplemented with









36

0.6% yeast extract (YE, Becton Dickinson) for 16 hours to reach the logarithmic phase

of growth (Appendix B). Five ml bacterial culture were centrifuged at 1200 x g for 10

minutes (IEC Clinical Centrifuge, Fisher Scientific, Pittsburgh, PA). The pelleted cells

were then washed twice with 10 ml sterile phosphate buffered saline (PBS, pH 7.4) and

then suspended in 10 ml PBS. Concentration of the bacterial culture was estimated with

the optical density at 540 nm using a spectrophotometer (DU-40, Beckman Instruments,

Inc., Fullerton, CA) and adjusted to a selected concentration with sterile PBS according

to the standard curve (Appendix B).


Nutritional Assessment


Food Intake and Body and Organ Weights

Food intake and body weight of animals were measured with an electronic

toploading balance (Denver Instrument) to ascertain basic nutritional parameters.

Animals were weighed every 3 days. Since the mice were gang-caged, daily food

consumption (g per mouse per day) was obtained from the average food intake every 3

days for each group. At the end of the experiment, selected organs from each animal

were removed, weighed, and expressed as percent of body weight as another indicator

of health status.


Plasma Albumin and Total Protein

A blood sample from inferior vein cava of each animal was collected using a

syringe containing about 30 ld heparin (1000 units per ml, LyphoMed, Inc., Rosemont,

IL) and centrifuged at 250 x g for 10 minutes (Eppendorf 5415, Brinkmann Instruments,









37

Inc., Westbury, NY). Plasma was then harvested and transferred to a 500 Al centrifuge

tube and kept at -200C until assessed. Plasma albumin was assessed by the bromocresol

green (BCG) method (Gulyassy et al. 1981). Total protein was determined by the biuret

method (Lubran 1978). The assay methods are described in Appendix C.


Immune Function Assay


Delayed-Type Hypersensitivity

Delayed-type hypersensitivity reaction, a peripheral expression of cell-mediated

immunity, remains a crucial in vivo immunological test. The DTH assay selected for this

study is a sensitive and reproducible method of quantitating the degree of contact

sensitivity elicited by the antigen 2,4-dinitrofluorobenzene (DNFB, Sigma Chemical).

Direct applications of DNFB to the skin of the abdomen result in local cell-mediated

immunity upon antigenic rechallenge of the ear. Dinitrofluorobenzene and other

dinitrobenzene compounds conjugated to soluble proteins at physiologic pH (Parker et

al. 1983). The contact hypersensitivity reaches a maximum at 4 days after sensitization

(Cho and Hough 1986, Phanuphak et al. 1974).

The assay consists of a two-step procedure. First, the mouse is sensitized by

painting the shaved abdomen with 50 p1 of 0.5 % DNFB (in 4:1 acetone:olive oil) for two

consecutive days. Four days later, one ear is challenged with 20 1 of 0.2% DNFB in

the same vehicle. The other ear is painted with the vehicle and serves as a nonstimulated

control ear. The degree of immunocompetence is assessed by measuring ear thickness

(swelling) with a micrometer (Fisher Scientific). The level of DTH response can be









38

quantified by the difference of ear swelling between the stimulated and nonstimulated

ears in the same animal.


Popliteal Lymphoproliferation

The status of cell-mediated immunity can be assessed by quantitating the

spontaneous proliferation of the PLN cells induced by local subcutaneous injection of

various antigenic substances such as sheep erythrocyte (SE) in the hind-leg footpad

(Papadimitriou et al. 1983, Twist and Barnes 1973). Antigens that migrate into the

lymphatic system are sequestered by the PLN in the leg which drains that foot. This

induces formation of new (secondary) follicles in the PLN. Efficient follicle formation

is associated with particulate and high molecular weight antigens which are liable to be

phagocytized (Hoshi et al. 1986). Immune cells present in the lymph node proliferate

in response to the antigen, which will lead to an increase in the size of the lymph node.

One hind-leg footpad of each animal was injected subcutaneously with 1.0 x 101

cells of sheep erythrocytes (Sigma Chemical) in 50 jd sterile PBS. The contralateral

footpad was injected with the same volume of sterile PBS and served as the

nonstimulated control. Since there is no crossover effect, the injected antigen stimulates

only the PLN in the leg which drains that foot and does not affect the other PLN. On

the sixth day after the challenge, the PLNs were excised, cleaned of adherent fat, and

weighed. In healthy animals that are challenged, the affected lymph node size increases

markedly (about 300%) whereas in immunocompromised animals, the change is

insignificant. Differences in size of the stimulated and nonstimulated PLNs in the same

animal were taken as the degree of immune stimulation.










Histological Analysis of the Popliteal Lymph Node

Lymph nodes respond to antigens by developing a reactive structure known as the

germinal center. Thus, to confirm the immune-stimulating effect of dietary nucleotide

supplementation, formation of lymph follicles in draining PLN after antigen (i.e., SE)

injection was examined in groups of mice fed stock diet or casein diets supplemented

with 0.25 or 1.5% RNA. One group of mice without SE immunization was also

included in the study and served as a nonimmunized control.

Six days after immunization with a footpad injection of 1.0 x 10' SEs, both PLNs

of each mouse were removed. Popliteal lymph nodes were fixed in 10% phosphated-

buffered formalin (Fisher Scientific) at room temperature for 2 days. The specimens

were then submitted for routine histology sectioning and stained with hematoxylin-eosin

(Sigma Chemical). Each section was examined and photographed with a light

microscope to identify the presence of germinal centers.


Tritiated-Thymidine Incorporation

An in vitro cell proliferation assay was used to quantify the growth response of

lymphocytes of PLN upon stimulation by SE. Cellular proliferation is quantified by

measuring tritiated-thymidine (3H-thymidine) incorporation into DNA (Corradin et al.

1977). Each time a cell divides it replicates its DNA; as such, the total amount of de

novo DNA in a cell culture is a proportionate indicator of the amount of cell growth that

has taken place. De novo DNA production is most easily monitored by adding 3H-

thymidine, a radioactive DNA precursor, to a cell culture system. The thymidine is

transported into the cell's nucleus where it is phosphorylated to thymidine triphosphate.









40
This nucleotide triphosphate is then incorporated into the cellular DNA at a rate that is

proportional to the rate of cell growth.

Six days after immunizing footpads with injection of 1.0 x 108 SEs, PLNs were

removed aseptically from groups of mice fed casein diets supplemented with 0, 0.25, or

1.5% RNA. Mice of the same dietary treatment without SE immunization were also

included as controls. A single cell suspension was prepared by scraping the lymph node

across a sieve. Cells were dispersed by repeating aspiration through reducing bore size

of needles (21, 23, and 25 gauges) and resuspended in RPMI-1640 (pH 7.4, Gibco BRL,

Life Technologies, Inc., Gaithersburg, MD). Cells were then washed with RPMI and

centrifuged at 1200 rpm for 10 minutes (RT6000, Sorvall Instruments, Du Pont Co.,

Newtown, CT). Cells were then resuspended in 0.5 ml RPMI. Cell concentration was

then determined by the propidium iodide staining method (Kao and Scornik 1989).

Triplicate cell suspensions containing 4.0 x 10s PLN cells were added to each well of a

96-well U-shape bottom microplate (Coming Glass Works, Coming, Inc., Corning, NY).

Cells were incubated with 4.0 x 105 SEs at 37C in a 5% CO2 atmosphere for 4 days.

Cells were then pulsed with 10 il 3H-thymidine (1 iCi per well) and incubated for 10

hours. Cells were harvested onto glassfiber filter papers. The total radioactivity of each

well was measured by a liquid scintillation counter (Wallac Inc., Gaithersburg, MD) for

1 minute. Results were expressed as counts per minute (cpm).


Bactericidal Activity of Peritoneal Macrophages

In vitro methods have been widely employed for the investigation of the roles of

various cell subpopulations in the generation and regulation of specific immunity. Tests









41

of antibacterial function are the most frequently employed assays to determine

microbicidal activities of macrophages. The bactericidal activity of macrophages can be

assessed by determining the number of organisms retaining viability after incubating these

immune cells with a pathogenic organism (Wilder and Edberg 1973).

To examine the antibacterial function of macrophages, a freshly isolated peritoneal

exudate consisting primarily of young macrophages was obtained from the mice.

Macrophages were assessed for their ability of in vitro phagocytosis and intracellular

killing of the fast-growing intracellular pathogen, L. monocytogenes. Bacterial culture

was prepared as described and adjusted to 2.5 x 10' cells per ml with Dulbecco's

Modified Eagle's Medium (DMEM, pH 7.4, Gibco BRL). Triplicate of appropriate

dilutions of bacterial suspension were plated out on TSA plates and incubated at 37C

for 24 hours.

Macrophages were harvested by peritoneal lavage by application of 5 ml aliquots

of ice-cold sterile Hanks' balanced salt solution (HBSS, pH 7.4, Gibco BRL) into the

peritoneum of mice. Cells from 3 mice in each group were pooled in a 50-ml centrifuge

tube. The cell suspension was then centrifuged at 250 x g for 10 minutes and

resuspended with 2 ml cold DMEM. The concentration of viable peritoneal macrophages

was determined by trypan blue exclusion. Briefly, 50 dl cell suspension were mixed with

400 l1 PBS and 50 jil of 2% trypan blue (Gibco BRL). After 3 to 4 minutes, an aliquot

(about 10 pl) of the cell suspension was transferred to a hemacytometer and examined

with an inverted microscope (Nikon TMS, Nikon Co., Japan) to assess the concentration

and viability of the macrophages.









42
After adjusting the concentration to 2.5 x 106 cells per ml with DMEM, 400 pl

cell suspension containing approximately 1.0 x 106 cells were transferred to each well of

a 48-well flat bottom cell culture plate (Coming) and 100 Il mouse serum from the same

group was added. The cell suspension was allowed to adhere to the plate at 37C in a

5% CO2 atmosphere for 20 hours. After washing twice with 500 l1 warm DMEM to

remove nonadherent cells, the attached cells were challenged by application of 5.0 x 106

cells of L. monocytogenes in the same medium.

After incubating for 1 hour with 10% mouse serum at 370C in a 5% CO2

atmosphere, the mixture of macrophage and L. monocytogenes was washed twice with

warm DMEM to remove the nonengulfed bacteria. One-half of the samples were lysed

by hypotonic shock with d-H20 and served as control. All samples were incubated for

2 hours with 10% mouse serum for intracellular killing of bacteria. After incubation,

the macrophages were lysed by disrupting them by hypotonic shock with d-H20 followed

by rapid freezing with dry ice. Triplicates of appropriate dilutions of each sample were

then plated on TSA plates and incubated for 24 hours. Plates were then enumerated for

viable organisms. The magnitude of bactericidal activity of the macrophages was

determined by the reduction in the percentage of viable bacteria as compared with the

control (Appendix D).


Containment of Intradermal Abscess Challenge

Elimination of bacterial contamination from wounds is a nonspecific resistance

mechanism against infection. S. aureus has remained an important cause of nosocomial

wound infections. The staphylococcal toxins are well known stimulators of T-lymphocyte









43
proliferative responses in both humans and mice (Kalland et al. 1991). The ability of

mice to contain an intradermal bacterial invasion was assessed by using an in vivo S.

aureus abscess model. Bacterial culture was prepared as described. A nonlethal dose

(1.0 x 106 cells) of viable S. aureus cells was injected intradermally on both sides of the

shaved flank (Latter et al. 1987, Tchervenkov et al. 1988). Twenty-four hours later,

using aseptic technique, the regions of formed abscesses were excised, homogenized, and

cultured by surface plating on TSA plates after appropriate dilutions for enumeration of

the viable organisms. The number of culturable organisms is directly related to the

degree of immune suppression, i.e., a higher number of viable bacteria indicates a

weaker immune system.


Clearance of Intravenous Bacterial Challenge

The reticuloendothelial system (RES) plays an important role in the surveillance

of organisms that enter the circulation. Specialized immune cells residing in the liver

and spleen filter out foreign material from the blood for subsequent destruction. A

functional measure of the RES would be the response to injection of a viable pathogen.

The model assay selected for clearance is intravenous injection of Listeria monocytogenes

(Dean et al. 1980, Tran et al. 1990). Evidence exists that resistance of mice to infection

by L. monocytogenes involves a biphasic response (Schultheis and Kearns 1990). The

first phase of the bacterial resistance mechanism responsible for early clearance of

infectious agents is a nonspecific immune response that comprises the first 48 hours after

infection, during which there is multiplication of L. monocytogenes in the liver and

spleen. Macrophages and polymorphonuclear leukocytes are the effector cells involved









44
in controlling bacterial multiplication. In the second phase which involves the induction

of acquired immune function responsible for the late clearance of the bacteria, cell-

mediated immunity develops, during which multiplication of L. monocytogenes is

prevented by macrophages possessing increased microbicidal activity that is mediated

through the action of lymphokines released by immunologically committed T-

lymphocytes.

Mice were injected in a lateral tail vein with a sublethal dose of viable L.

monocytogenes. For comparison, two strains of L. monocytogenes were tested at various

doses. The pathogenic strain (ATCC 19114, American Type Cell Collection, Rockville,

MD) had been shown to be lethal at 1.0 x 106 cells per mouse (Ziprin and McMurray

1988) while the streptomycin-resistant strain was much less pathogenic. The lethal dose

is 1.0 x 109 cells per mouse (Fensterbank 1986a and 1986b, Linde et al. 1991).

Four days after bacterial injection, using aseptic technique, blood, liver, spleen,

and kidneys of the animals were removed, weighed, homogenized, and cultured for

enumeration of the viable organisms by surface plating on TSA plates supplemented with

0.6% YE. Immunosuppressed animals are expected to kill fewer organisms and allow

the bacteria to multiply extensively, which is revealed by more extensive colonization in

the tissues 72 hours after infection.


Statistical Analysis


Data are expressed as mean SEM (the standard error of the mean) for each

group. The analysis of variance (ANOVA) was used to identify the presence of









45

significant difference among the groups. Significance of differences was assessed using

the Least Significant Difference (LSD) test for multiple comparisons. Dunnett's t test,

which compares all treatment groups to a control group, was also performed (Dunnett

1955). A P-value of 0.05 was taken as the level of significance. All statistics were done

using the Statistic Analysis System (SAS 6.04, SAS Institute Inc., Cary, NC) on a

personal computer.














RESULTS AND DISCUSSION


Model System


Determination of Nutrition Deprivation Procedure

A combination of a casein-based diet and a protein-free diet was used to accelerate

nucleotide depletion and protein malnutrition. Adult male ICR (outbred) mice were

acclimated to the laboratory and then given free access to a nucleotide-free casein-based

purified diet for 12 to 16 days. This was believed to result in partial depletion of body

tissue nucleobase pools (LeLeiko et al. 1987). Further depletion of the nucleobase pools

as well as protein malnutrition was induced by feeding a protein-free diet for the next 6

days. The effect of diminished tissue nucleobase pools on immune function was

demonstrated by the suppressed immune response assessed by a lymphoproliferation

assay (Figure 1). Since no further decrease in immune response was observed with

lengthened casein diet feeding, the combination of 12 days on a casein diet plus 6 days

on a protein-free diet was used as the nutrition deprivation model thereafter.


Effect of Nutrition Deprivation on Body Weight and Blood Protein Levels

Body weight of animals at various stages of nutrition deprivation and repletion

was measured to ascertain basic nutritional parameters. Blood samples were collected

to assess the status of protein malnutrition. Changes in plasma albumin and total protein






































12 26


Time on casein diet (days)


Figure 1. Effect of dietary nucleotide depletion on popliteal lymphoproliferation. Mice
were fed a nucleotide-free casein diet for various intervals followed by 6 days
on a protein-free diet and refed with casein diet. Values are means SEM.
Bars with different letters are significantly different (P < 0.05).


a


b


b


b
4-
_L T









48
levels were examined at different time points in mice fed casein diet for 12 days and

followed by protein-free diet for 6 days. The nutrition deprivation resulted in a 18% loss

of body weight (Figure 2) and hypoalbuminemia as evidenced by a 35% reduction of

plasma albumin concentration and a 21% decrease in total protein level (Figure 3).

Groups of these protein-malnourished mice were then fed 4 different diets

(protein-free, stock, casein, or casein supplemented with 0.25% RNA) for 11 days. In

the repletion period, mice fed protein-containing diets had statistically higher body

weight, with a corresponding improvement in plasma albumin and total protein levels,

than did protein-deprived mice (MN) (Figures 2 and 3). All groups that were repleted

with protein-containing diets regained most or all of the lost body weight after 4 days

while mice that remained on the protein-free diet continued on a linear decline in body

weight (Figure 2). The addition of 0.25 % RNA to the casein diet (NS) did not show any

additional benefit on weight gain and blood protein levels (Figures 2 and 3). The results

demonstrated that a nucleotide-free casein diet is adequate for maintaining normal growth

and blood protein levels under these experimental conditions.


Effect of Nucleotide Supplementation on Food Intake

No significant difference in food intake was observed among the groups (Tables

1 and 2). The addition of RNA at levels of 0.1 to 1.5% or uracil at levels of 0.025 to

0.375% did not affect the amounts of food consumed. The slightly higher consumption

in groups fed stock diet (IS, NR, and WN) was mainly due to the lower percentage

(76%) of digestible ingredients (Anonymous 1992), necessitating an increase in food

intake to compensate for a lower energy yield.









49
40
I I








I I I I
I I
SI
38 2 4













30 "






SProtein
ANucleoide depletion retrict
4I I
I ,











Time of dietary treatment (days)




---r---. NS: refed with casein diet containing 0.25 % RNA
-d ND: refed with casein diet
B-- lNR: refed with stock chow
-A MN: refed with protein-free diet
u WN: on stock diet throughout experiment





Figure 2. Effect of dietary treatment on body weight. Groups of mice (except WN)
were fed a casein diet for 12 days followed by a protein-free diet for 6 days
and refed with different diets for 11 days.








- Plasma albumin
-defg efg


abcde


III
fg~v^1 ^


4 6 8 11


Time of refeeding (days)
m WN: on stock diet throughout experiment
m NF: nucleotide depletion with casein diet for 12 days
O PF: protein restriction with protein-free diet for 6 days
after nucleotide depletion for 16 days
O MN: refed with protein-free diet
ND: refed with casein diet
M NS: refed with casein diet containing 0.25 % RNA
NR: refed with stock diet

Figure 3. Effect of dietary treatment on plasma albumin and total protein levels.
Groups of mice (except WN) were fed a casein diet for 12 days followed by
a protein-free diet for 6 days and refed with different diets for 11 days.
Values are means SEM. In each panel, bars with different letters are
significantly different (P < 0.05).


abcd
ab ab


I


abc
S cdef cdef


II II














Table 1. Food intake at different feeding periods of RNA supplementation study

6 days 12 days 6 days 6 days
Group casein casein protein-free refeeding


4.2 0.1a

4.1 0.1

4.2 0.2a

4.1 0.1l

4.1 0.08

4.1 0.02

4.1 0.0

4.2 0.2a

4.0 0.2"

5.1 0.2b

4.9 + 0.1b


3.3 0.1a

3.3 0.1

3.4 0.0a

3.3 0.22a

3.4 0.1a

3.3 0.02

3.3 0.1a

3.3 0.1P

3.3 0.la

5.8 0.1b

5.8 0.2b


3.5 0.2a

3.1 0.0

3.1 0.2a

4.5 0.5b

4.9 0.4b

3.7 0.02

3.3 0.12

3.4 0.1-

3.1 0.0a

4.8 0.4b

4.5 0.4b


4.4 0.1a

4.8 0.4b

4.7 0.1b

4.4 0.2a

4.1 0.0O

4.2 0.7"

3.1 0.2a

3.9 0.2a

3.5 0.0

5.7 0.3b

5.3 0.2b


Note: Values are means SEM


(g/mouse/day). Within a column, values


with different


letter superscripts are significantly different (P < 0.05).
Group Abbreviations: HS, refed with casein diet containing 1.5% RNA; HR, refed with
casein diet containing 0.5% RNA; NS, refed with casein diet containing 0.25% RNA;
LR, refed with casein diet containing 0.1 % RNA; ND, refed with casein diet; NR, refed
with stock diet; MR, refed with protein-free diet containing 0.25% RNA; MN, refed
with protein-free diet; CA, on casein diet throughout experimentation; WN, on stock diet
throughout experimentation; IS, on stock diet throughout and treated with Methotrexate.


HS

HR

NS

LR

ND

NR

MR

MN

CA

WN

IS



















Table 2. Food intake at different feeding periods of uracil supplementation study

6 days 12 days 6 days 6 days
Group casein casein protein-free refeeding


US 3.8 0.1a 3.6 0.la 4.5 0.2a 3.6 0.2a

UR 3.7 0.08 3.6 0.0a 4.2 0.22 3.5 0.2a

UN 3.7 0.la 3.5 0.1 4.1 0.3a 3.7 0.0a

UL 3.8 0.1a 3.5 0.1P 4.1 0.2a 3.6 0.2a

ND 3.9 0.1a 3.6 0.1a 3.7 0.1P 4.1 0.1P

NR 3.9 0.1a 3.7 0.1P 4.4 0.2a 5.1 0.5b

WN 5.3 0.2b 5.2 0.0b 5.6 0.0b 5.1 0.7b


Note: Values are means SEM (g/mouse/day). Within a column, values with different
letter superscripts are significantly different (P < 0.05).
Group abbreviations: US, refed with casein diet containing 0.375% uracil; UR, refed
with casein diet containing 0.125% uracil; UN, refed with casein diet containing 0.063%
uracil; UL, refed with casein diet containing 0.025% uracil; ND, refed with casein diet;
NR, refed with stock diet; WN, on stock diet throughout experimentation.










Effect of Nucleotide Supplementation on Body and Organ Weight

During the nucleotide depletion period, weight gains were similar among all the

groups and all mice appeared healthy and normal. Six days of protein-free diet resulted

in an average of 20% body weight loss for all mice (Figures 4 and 5). All groups

repleted with diet containing protein regained body weight after 6 days while mice that

remained on the protein-free formula (MN and MR) continued on a linear decline in

weight. Nucleotide supplementation with RNA (0.1 to 1.5%) or uracil (0.025 to

0.375%) had no effect on weight gain, nor did addition of 0.25% RNA.

By the end of the feeding period, organ weights (expressed as percent of body

weight) were not significantly different among the groups repleted with diet containing

protein. Mice that remained on the protein-free formula (MN and MR) had significantly

lower organ weights (Table 3). In comparison to the protein-malnourished groups (MN

and MR), refeeding with protein-containing diets significantly increased the response of

the liver (34%), spleen (44%), and thymus (41%) while the change in kidneys was only

13% (Table 3). Spleen weight was higher in groups supplemented at the three highest

levels of RNA, i.e., 0.25, 0.5, and 1.5%, compared with the casein group (ND).

However, no significant difference was observed in groups supplemented with uracil at

levels equal to the amounts found in RNA (Table 4). The significant increase in organ

weights of major immune organs (liver, spleen, and thymus) with higher levels (0.5 and

1.5 %) of RNA supplementation may be a basic mechanism underlying improved immune

function with exogenous RNA.










36






SI I
34 4#








I i
I I
I I
I

















--r--- eS:refedwithcaseindietcontaining0.25%RNA


-x-- LR: refed with casein diet containing 0.10% RNA
S NR refed with stock chow
22 -



















-A.-- MR: refed with protein-free diet containing 0.25% RNA
24 NProtin : refed with protein diet







-*- CA: on casein diet throughout experiment
WNucleo: on stockide deplet throughout expestrimection Repletionnt
------ IS: refed withroughout oand treated with MethtreateRNA
---*---. HR: refed with casein diet containing 0.50% RNA
---A--. NS: refed with casein diet containing 0.25 % RNA












Figure 4. Effect of RNA supplementatiet containin body weight. Groups of mice (except
CA, IS, and WN) were fed witha casein diet for 12 days followed by a protein-
free diet for 6 days and refed for 6 days with different diets.
free diet for 6 days and refed for 6 days with different diets.











36 :-
III
I II
I I I
SI I



I I


















I .. UR refed with casein diet containing 0.125% uracil
28

















.. M .... UN: refed with casein diet containing .063% uracil
......*...... UL: refed with casein diet containing 0.025% uracil
-e- ND: refed with casein diet
-B- NR: refed with stock chow
S WN: on stock diet throughout experiment





Figure 5. Effect of uracil supplementation on body weight. Groups of mice (except
WN) were fed a casein diet for 12 days followed by a protein-free diet for
6 days and refed for 6 days with different diets.














Table 3. Final organ weights of mice in RNA supplementation study

Group Liver Kidney Spleen Thymus


HS

HR

NS

LR

ND

NR

MR

MN

CA

WN

IS


5.74

5.76

5.88

6.64

6.29

6.33

3.98

4.13

6.36

5.84

5.49


0.14k

0.18b

0.17b

0.16a

0.093

0.13"

0.11d

0.10d

0.09a

0.14k

0.08c


1.37

1.42

1.46

1.40

1.38

1.48

1.25

1.21

1.40

1.47

1.40


0.03a

0.06"

0.02'b

0.05"cd

0.03bc

0.03"

0.03e

0.02e

0.02d

0.03cd

0.04~"


0.51

0.55

0.51

0.43

0.45

0.53

0.29

0.27

0.39

0.41

0.41


0.02b

0.02a*

0.02 a

0.02 a



0.01C
0.02.

0.01C



O.Ol12

0.02a

0.02*'


0.10

0.11

0.12

0.12

0.11

0.10

0.07

0.06

0.12

0.12

0.11


0.00b

0.01

0.01c

0.01a

0.01O

0.01b



0.00O

0.01at

0.01"

0.01j


Note: All organ weights were expressed as percent of body weight. Values are means
SEM. Within a column, values with different letter superscripts are significantly
different (P < 0.05).
Group Abbreviations: HS, refed with casein diet containing 1.5% RNA; HR, refed with
casein diet containing 0.5% RNA; NS, refed with casein diet containing 0.25% RNA;
LR, refed with casein diet containing 0.1% RNA; ND, refed with casein diet; NR, refed
with stock diet; MR, refed with protein-free diet containing 0.25% RNA; MN, refed
with protein-free diet; CA, on casein diet throughout experimentation; WN, on stock diet
throughout experimentation; IS, on stock diet throughout and treated with Methotrexate.



















Table 4. Final organ weights of mice in uracil supplementation study

Group Liver Kidney Spleen Thymus


US 5.93 0.138 1.34 0.02' 0.41 0.02c 0.11 0.01b

UR 5.56 0.16b 1.41 0.05c 0.38 0.01 0.09 0.01"

UN 5.66 0.16'* 1.45 0.04c 0.42 0.02a 0.09 0.01l

UL 5.70 0.16a' 1.43 0.03a 0.43 0.03A 0.12 0.01-'

ND 5.69 0.1181 1.47 0.06a' 0.44 0.03' 0.10 0.00-

NR 5.98 0.09" 1.48 0.048 0.47 0.02-b 0.12 0.012

WN 5.89 0.08A' 1.53 0.05a' 0.45 0.02a 0.10 0.01l


Note: All organ weights were expressed as percent of body weight.


Values are means


SEM. Within a column, values with different letter superscripts are significantly
different (P < 0.05).
Group abbreviations: US, refed with casein diet containing 0.375% uracil; UR, refed
with casein diet containing 0.125 % uracil; UN, refed with casein diet containing 0.063 %
uracil; UL, refed with casein diet containing 0.025% uracil; ND, refed with casein diet;
NR, refed with stock diet; WN, on stock diet throughout experimentation.











Effect of Nucleotide Supplementation on Blood Protein Levels

Protein-malnourished groups (MN and MR) had significantly lower levels of

plasma albumin and total protein (Figure 6). Repletion with diet containing protein for

6 days restored the levels of plasma albumin and total protein to normal (Figures 6 and

7). There was no significant difference between the casein group (ND) and any of the

nucleotide-supplemented groups. The addition of 0.25% RNA did not show any

beneficial effects on blood protein levels.


Immune Function


Time Course Development of Delayed-Type Hypersensitivity

Delayed-type hypersensitivity was assessed by ear swelling in response to an

antigen, DNFB. A time course study was carried out to determine the proper time point

for assessment of ear swelling after DNFB challenge. Results showed that ear swelling

of sensitized animals reached its peak at 24 hours after challenge with DNFB whereas

nonsensitized control animals showed no significant swelling after DNFB challenge

(Figure 8). The results are in agreement with the reported data of Phanuphak et al.

(1974). Thus, the reliability of the assay was deemed satisfactory.


Effect of RNA Supplementation on Delayed-Type Hypersensitivity

Mice repleted with the casein diet (ND) showed improvement in the immune

response to DNFB compared to mice remained on the protein-free formula (MN) (Figure

9). All groups repleted with RNA-supplemented casein diets displayed higher mean

values than mice repleted with the casein diet with P < 0.05 achieved for groups on the










3
Plasma albumin
2 a ab ab ab ab b ab ab ab
C C
-



6
0 Total protein
5
abcd bed abcd cd a cd d
4

3 e e

2

1

0
HS HR NS LR ND NR MR MN CA WN IS

HS: refed with casein diet containing 1.50% RNA
HR: refed with casein diet containing 0.50% RNA
NS: refed with casein diet containing 0.25 % RNA
LR: refed with casein diet containing 0.10% RNA
ND: refed with casein diet
NR: refed with stock chow
MR: refed with protein-free diet containing 0.25% RNA
MN: refed with protein-free diet
CA: on casein diet throughout experiment
WN: on stock diet throughout experiment
IS: on stock diet throughout and treated with Methotrexate


Figure 6. Effect of RNA supplementation on plasma albumin and total protein levels.
Groups of mice (except CA, IS, and WN) were fed a casein diet for 12 days
followed by a protein-free diet for 6 days and refed with different diets for
6 days. Values are means SEM. In each panel, bars with different letters
are significantly different (P < 0.05).










Plasma albumin
a a a a a a b




mmmmm Fm m


Total rotein


b


UN


ND


NR


WN


US: refed with casein diet containing 0.375% uracil
UR: refed with casein diet containing 0.125% uracil
UN: refed with casein diet containing 0.063% uracil
UL: refed with casein diet containing 0.025% uracil
ND: refed with casein diet
NR: refed with stock chow
WN: on stock diet throughout experiment



Figure 7. Effect of uracil supplementation on plasma albumin and total protein levels.
Groups of mice (except WN) were fed a casein diet for 12 days followed by
a protein-free diet for 6 days and refed with different diets for 6 days.
Values are means SEM. In each panel, bars with different letters are
significantly different (P < 0.05).









61




30

n Sensitized

25 Control



8 20



15



10-



5 -
10
*



0 12 24 36 48
Time after DNFB challenge (hours)


Figure 8. Time course development of ear swelling in healthy outbred mice fed a stock
diet. Mice were sensitized with 2,4-dinitrofluorobenzene (DNFB) for 2
consecutive days by application of 50 l1 0.5% DNFB to the shaved
abdomen. Four days later, one ear was challenged with 20 il 0.2% DNFB.
The other ear was challenged with the vehicle (acetone) and served as the
baseline for comparison. The control mice were not sensitized.











a
"1-"


* a a
-r


HR NS LR


bc


C


d
"-


e
T


ND NR MR MN CA WN IS


HS: refed with casein diet containing 1.50% RNA
HR: refed with casein diet containing 0.50% RNA
NS: refed with casein diet containing 0.25 % RNA
LR: refed with casein diet containing 0.10% RNA
ND: refed with casein diet
NR: refed with stock chow
MR: refed with protein-free diet containing 0.25 % RNA
MN: refed with protein-free diet
CA: on casein diet throughout experiment
WN: on stock diet throughout experiment
IS: on stock diet throughout and treated with Methotrexate


Figure 9. Effect of RNA supplementation on delayed-type hypersensitivity. Groups of
mice (except CA, IS, and WN) were fed a casein diet for 12 days followed
by a protein-free diet for 6 days and refed with different diets for 6 days.
Values are means + SEM. Bars with different letters are significantly
different (P < 0.05).









63

0.25, 0.5 and 1.5% RNA formulas. Refeeding with stock diet (NR) significantly

increased the response to DNFB. However, the 0.25% RNA supplemented protein-free

diet group (MR) failed to show any significant improvement. Therefore, suppression of

DTH response induced by protein malnutrition can be partially restored by protein

repletion and the addition of RNA at levels of 0.25% and higher further increases the

immune response. In addition, DTH responses to DNFB were markedly suppressed in

the chemically induced immune suppressed group (IS). These otherwise well-nourished

animals showed no sign of sickness, such as diarrhea, usually seen with MTX treatment.


Effect of Uracil Supplementation on Delayed-Type Hypersensitivity

In comparison to the response of the casein group (ND), there was no

improvement in any of the 4 uracil-supplemented groups (Figure 10). Supplementation

of uracil up to 0.375% (equal to that in 1.5% RNA) did not lead to any better response

than the nonsupplemented casein group. Therefore, uracil is not solely responsible for

the increase in the DTH response demonstrated in the RNA supplementation study. This

is in contrast to the results of other researchers (Fanslow et al. 1988, Kulkarni et al.

1987, Van Buren et al. 1985) who have shown improvement in immune response with

diet supplemented with 0.06% uracil, a level equal to that in 0.25% RNA.


Time Course Development of Popliteal Lymphoproliferation

Results of this study in normal healthy mice showed that the difference in size of

the stimulated and nonstimulated PLN reaches a maximum at 6 days after SE injection

(Figure 11). Therefore, this time point was used for all subsequent assays.













a
-F


ab
"'1"-


ab
F


b
--1-


b
"1--


b
..-7


ab
"'1-


ND NR


WN


US: refed with casein diet containing 0.375% uracil
UR: refed with casein diet containing 0.125% uracil
UN: refed with casein diet containing 0.063% uracil
UL: refed with casein diet containing 0.025 % uracil
ND: refed with casein diet
NR: refed with stock chow
WN: on stock diet throughout experiment


Effect of uracil supplementation on delayed-type hypersensitivity. Groups
of mice (except WN) were fed a casein diet for 12 days followed by a
protein-free diet for 6 days and refed with different diets for 6 days. Values
are means + SEM. Bars with different letters are significantly different (P
< 0.05).


Figure 10.















ab
T


ab
T


be
7-


be
T


c
T


Time after SE injection (days)


Time course development of popliteal lymphoproliferation in healthy
outbred mice fed a stock diet. Mice were injected subcutaneously with 1.0
x 108 sheep erythrocytes (SEs) in 50 p1 sterile phosphate buffered saline
(PBS) into one of the hind footpads. The contralateral footpad was injected
with the same volume of sterile PBS. Values are means SEM. Bars
with different letters are significantly different (P < 0.05).


Figure 11.










Effect of RNA Supplementation on Popliteal Lymphoproliferation

Protein repletion with the casein diet (ND) led to a 30% increase in PLN weight

in comparison to the group that remained on the protein-free diet (MN) (Figure 12). All

groups fed RNA-supplemented casein diets showed a stronger response to SE challenge

than the group on the casein diet, with the 1.5% RNA group at 56% above the ND group

and a dramatic 102% increase compared to the mice maintained on the protein-free diet.

When compared to the protein-free group (MN), mice fed protein-free diet supplemented

with 0.25% RNA (MR) showed a 33% increase, a level similar to the group refed with

casein diet (ND), but was not statistically different from that of protein-free group.

Thus, repletion from malnutrition with diets enriched with RNA appeared to strengthen

T-cell mediated immunity in this model system. In order to proliferate, lymphocytes

synthesize DNA but must rely on a salvage pathway of circulating nucleotides released

by the liver and other tissues for synthesis of RNA. When high rates of mitosis are

required, e.g., as in infection, exogenous RNA may be essential to allow for elevated

proliferation levels. The beneficial effect of dietary nucleotide supplementation on

improving immune function after protein deprivation is in agreement with that of Pizzini

et al. (1990) who showed that after feeding a protein-free diet for 7 to 10 days, the

immune response assessed by the PLN assay was only restored in mice repleted with a

stock diet or a nucleotide-free casein diet supplemented with 0.25% yeast RNA.


Effect of RNA Supplementation on Histology of the Popliteal Lymph Node

To further identify the immune-enhancing effect of dietary nucleotide

supplementation observed in the popliteal lymphoproliferation experiment, the histology


















cd bed

IT


bc
-


bed
T


cd


HR NS LR ND


cd


d
_T


NR MR MN
NR MR MN


HS: refed with casein diet containing 1.50% RNA
HR: refed with casein diet containing 0.50% RNA
NS: refed with casein diet containing 0.25% RNA
LR: refed with casein diet containing 0.10% RNA
ND: refed with casein diet
NR: refed with stock chow
MR: refed with protein-free diet containing 0.25% RNA
MN: refed with protein-free diet
CA: on casein diet throughout experiment
WN: on stock diet throughout experiment
IS: on stock diet throughout and treated with Methotrexate


Effect of RNA supplementation on popliteal lymphoproliferation. Groups
of mice (except CA, IS, and WN) were fed a casein diet for 12 days
followed by a protein-free diet for 6 days and refed with different diets for
6 days. Values are means SEM. Bars with different letters are
significantly different (P < 0.05).


41-


a


ab

T


cd


CA WN


HS


Figure 12.









68

of PLNs from mice fed a stock diet or casein diet supplemented with different levels

(0.25 and 1.5%) of RNA were examined. Compared with the contralateral PLNs, the

lymph nodes draining the immunized feet from all three dietary treatment groups showed

significant enlargement at 6 days after footpad injection of 1.0 x 108 SEs (Figure 13).

Secondary follicles containing germinal centers were formed in the stimulated

lymph nodes of all groups while only primary follicles were observed in the contralateral

and nonimmunized PLNs (Figure 13). Secondary follicles in PLNs of mice fed a casein

diet supplemented with 1.5% RNA (HS) were significant larger than that of 0.25% RNA-

supplemented group (NS) and were comparable to that of stock diet group (WN).


Effect of RNA Supplementation on In Vitro Proliferation of the Popliteal Lymphocytes

The dose-related response of dietary nucleotide supplementation observed in the

histological study was further investigated by measuring the ability of cellular

proliferation in response to antigenic challenge in mice immunized by footpad injection

of 1.0 x 108 SEs. Mice without immunization were also included and served as controls.

Results showed that, regardless of dietary treatment, PLN cells from nonimmunized mice

showed no difference in thymidine incorporation with or without the presence of SE in

the medium. Popliteal lymph node cells from immunized mice fed the casein diet

supplemented with 1.5% RNA (HS) had a 4-fold increase in the uptake of thymidine

upon stimulation by SE while no increase was observed in PLN cells from mice fed the

casein diet (ND) or casein diet supplemented with 0.25% RNA (NS) (Table 5). These

results demonstrate that when mice are fed a diet containing a higher level of RNA, the

immunological response to rechallenge of an antigen is substantially enhanced.















































Effect of RNA supplementation on histology of the popliteal lymph nodes
(PLNs). Part A. The PLNs without immunization in mice fed a stock diet.
a) The normal PLNs (140 x). b) Primary follicle (arrow) in the normal
PLNs (1400 x).


Figure 13.


.,*<




























c d















Figure 13--continued. Effect of RNA supplementation on histology of the popliteal
lymph nodes (PLNs). Part B. The PLNs 6 days after immunization with
1.0 x 108 sheep erythrocytes (SEs) in mice fed a stock diet. a) The
nonimmunized PLNs (140 x) with primary follicles (arrows). b) Primary
follicles (arrows) in the nonimmunized PLNs (1400 x). c) The immunized
PLNs (140 x) with secondary follicles (arrows). d) Secondary follicles
(arrows) in the immunized PLNs (1400 x).










a b

















c d














Figure 13--continued. Effect of RNA supplementation on histology of the popliteal
lymph nodes (PLNs). Part C. The PLNs 6 days after immunization with
1.0 x 10' sheep erythrocytes (SEs) in mice fed a casein diet for 12 days
followed by a protein-free diet for 6 days and refed with a casein diet
supplemented with 0.25% RNA for 6 days. a) The nonimmunized PLNs
(140 x) with primary follicles (arrows). b) The nonimmunized PLNs (1400
x). c) The immunized PLNs (140 x) with primary follicles (arrows). d)
Primary follicles (arrows) in the immunized PLNs (1400 x).















































Figure 13--continued. Effect of RNA supplementation on histology of the popliteal
lymph nodes (PLNs). Part D. The PLNs 6 days after immunization with
1.0 x 108 sheep erythrocytes (SEs) in mice fed a casein diet for 12 days
followed by a protein-free diet for 6 days and refed with a casein diet
supplemented with 1.5% RNA for 6 days. a) The nonimmunized PLNs
(140 x) with primary follicles (arrows). b) The nonimmunized PLNs (1400
x). c) The immunized PLNs (140 x) with secondary follicle (arrows). d)
Secondary follicle (arrow) in the immunized PLNs (1400 x).























Table 5. Tritiated-thymidine incorporation by lymphocytes of the popliteal lymph nodes
with or without antigen stimulation

Nonimmunized lymph node Immunized lymph node

Treatment + antigen % antigen % + antigen % antigen %


HS 422 23 385 20 14182 229 3438 40

NS 724 39 1080 56 1695 27 1684 20

ND 1845 100 1914 100 6206 100 8631 100


Note: Values are medians of triplicates of total 3H-radioactivity (cpm) in each well.
Group abbreviations: HS, refed with casein diet containing 1.5% RNA; NS, refed with
casein diet containing 0.25% RNA; ND, refed with casein diet.










Effect of Uracil Supplementation on Popliteal Lymphoproliferation

In comparison to the casein group (ND), all groups except that supplemented with

0.06% uracil (UN) showed no significant difference in the PLN response, with the NL

and US groups displaying slightly lower responses (Figure 14). These results are in

partial agreement with those reported by Kulkarni et al. (1992) who showed that mice

fed a casein diet supplemented with 0.25% yeast RNA or 0.06% uracil had a

significantly enhanced immune response as assessed by a similar popliteal

lymphoproliferation assay. Supplementation with 0.06% adenine failed to show any

benefit.

The reason why refeeding with casein diet supplemented with 0.06% uracil, a

level equal to that present in 0.25% RNA (the amount normally found in stock diet),

showed a better response while supplementation with higher levels of uracil displayed a

suppressed immune response may be explained by a possible toxic effect induced by an

imbalance of nucleobase pools.


Effect of RNA Supplementation on In Vitro Bactericidal Activity of Peritoneal
Macrophages

Preliminary work was done to determine the optimal conditions for this in vitro

assay. The bactericidal activity of peritoneal macrophages was assessed under various

conditions, such as bacterial strain, mouse serum concentration, attachment time for

macrophage, and incubation time for intracellular killing. With numerous adjustments,

the final conditions provided an ideal environment to achieve maximal bactericidal

activity.










a


SIab

T


UN


b


ND


NR WN


US: refed with casein diet containing 0.375% uracil
UR: refed with casein diet containing 0.125% uracil
UN: refed with casein diet containing 0.063% uracil
UL: refed with casein diet containing 0.025% uracil
ND: refed with casein diet
NR: refed with stock chow
WN: on stock diet throughout experiment


Effect of uracil supplementation on popliteal lymphoproliferation. Groups
of mice (except WN) were fed a casein diet for 12 days followed by a
protein-free diet for 6 days and refed with different diets for 6 days. Values
are means SEM. Bars with different letters are significantly different (P
< 0.05).


3.0



2.5



2.0



1.5


b


b


UR


Figure 14.









76
The bactericidal activity of isolated peritoneal macrophages is expressed by the

reduction of viable bacteria compared with the control. Results showed that peritoneal

macrophages from all groups displayed similar intracellular killing activities toward the

streptomycin-resistant L. monocytogenes under the conditions studied (Figure 15). No

significant differences were found among the groups repleted with casein regardless of

RNA supplementation. However, peritoneal macrophages from the group fed the RNA-

supplemented protein-free diet (MR) showed a significantly stronger killing activity

toward L. monocytogenes compared with that of mice maintained on the protein-free diet

(MN). These unusual results may be explained by the conditions of the assay. The

relatively high killing capacity may be so overwhelming that the difference between

various dietary treatments became undetectable under these conditions.

The effect of dietary nucleotide supplementation on macrophage activity has been

studied by other investigators. Kulkarni et al. (1986a) measured the uptake (but not

killing) of radiolabeled bacteria by macrophages and showed that the phagocytic ability

of macrophages was enhanced in mice when they were fed a casein diet supplemented

with adenine (0.06%), uracil (0.06%), or RNA (0.25%). Carver et al. (1990) reported

that macrophage activity was stronger in mice fed a nucleotide-free diet but given water

containing 0.035% (w/w) nucleotides compared to those given just plain water.

However, macrophage activity was decreased by providing water with 0.35% (w/w)

nucleotides. In addition, macrophage activity was lower in mice fed a stock diet and

water containing 0.0035% (w/w) nucleotides for 6 weeks compared to mice fed a stock

diet and plain water.













ab ab ab
"]'-T -1-" 4.--


HS HR NS LR
HS HR NS LR


a
7 ~


NDNR
ND NR


ab
-r-


ab ab
-ET


MR MN CA WN IS


HS: refed with casein diet containing 1.50% RNA
HR: refed with casein diet containing 0.50% RNA
NS: refed with casein diet containing 0.25% RNA
LR: refed with casein diet containing 0.10% RNA
ND: refed with casein diet
NR: refed with stock chow
MR: refed with protein-free diet containing 0.25% RNA
MN: refed with protein-free diet
CA: on casein diet throughout experiment
WN: on stock diet throughout experiment
IS: on stock diet throughout and treated with Methotrexate


Effect of RNA supplementation on in vitro bactericidal activity of peritoneal
macrophages. Groups of mice (except CA, IS, and WN) were fed a casein
diet for 12 days followed by a protein-free diet for 6 days and refed with
different diets for 6 days. Values are means SEM. Bars with different
letters are significantly different (P < 0.05).


100


Figure 15.









78
Results obtained from this study contradict those of other researchers. Technique

and conditions of the assay may account for these differences. For example, in this

project, the killing activity rather than phagocytic activity of macrophages was used as

the indication of immune function. Certain bacteria such as L. monocytogenes have been

shown to be able to parasitize macrophages (Mackaness 1969). After being engulfed by

macrophages, some of the bacteria may survive and colonize macrophages. Thus, an

increase in phagocytosis only suggests that macrophages are able to engulf more bacteria.

It does not indicate an actual increase in bactericidal function. The functional test used

in this study provides more valuable information than that of phagocytosis alone.


Effect of Uracil Supplementation on In Vitro Bactericidal Activity of Peritoneal
Macrophages

Unlike the results showed in the RNA supplementation study, none of peritoneal

macrophages from the uracil-supplemented groups showed stronger killing activity toward

L. monocytogenes challenge than that of the casein group (ND). Peritoneal macrophages

obtained from mice fed casein diet supplemented with higher levels of uracil showed

lower bactericidal activity than to those from mice refed with casein or stock diet (NR).

These latter groups also had a level of bactericidal activity similar to that of the well-

nourished controls (WN) (Figure 16). Thus, repletion from malnutrition with diets

enriched with uracil at levels of 0.025 to 0.375% failed to improve peritoneal

macrophage activity against a pathogenic organism. These results contrast with an

enhanced response as reported by Kulkarni et al. (1986a) in which in vitro macrophage

activity was increased by supplementation with 0.06% uracil. However, rather than












ab
----


abc
---


c
C


a
T-


a
"-1"-


ab
-1I-


be
""T-


UL


ND


WN


US: refed with casein diet containing 0.375% uracil
UR: refed with casein diet containing 0.125% uracil
UN: refed with casein diet containing 0.063% uracil
UL: refed with casein diet containing 0.025% uracil
ND: refed with casein diet
NR: refed with stock chow
WN: on stock diet throughout experiment


Effect of uracil supplementation on in vitro bactericidal activity of peritoneal
macrophages. Groups of mice (except WN) were fed a casein diet for 12
days followed by a protein-free diet for 6 days and refed with different diets
for 6 days. Values are means SEM. Bars with different letters are
significantly different (P < 0.05).


0 "


Figure 16.









80

killing activity, phagocytic activity of peritoneal macrophages was used as the index of

immune strength in those studies.


Effect of RNA Supplementation on Abscess Containment

Results of the intradermal challenge with S. aureus showed no differences in

abscess area size among the various dietary treatment groups. However, the number of

viable bacteria in the abscess was higher in protein-malnourished groups (MN and MR)

compared with those maintained on diets containing protein (Figure 17). Thus, RNA

supplementation did not enhance bactericidal activity against this organism in any of the

dietary treatment groups. Therefore, RNA supplementation at levels of 0.1 to 1.5% has

no effect on the nonspecific immune response against a pathogenic intradermal infection.


Clearance of Intravenous Bacterial Challenge

Listeria resistance of the host involves a unique immune response controlled by

the existence of a Listeria-resistant (Lr) gene (Skamene and Kongshavn 1982). The

infected host activates several lines of defense in successive steps. Most of the invading

organisms are first taken up and destroyed by the RES, mainly the immune cells in the

liver and spleen. However, a small amount survive and colonize within the immune

cells. If the animal carries the Lr gene, macrophages are activated by T-cells and

subsequently eliminate the bacteria. If the animal does not carry the Lr gene, then

uncontrollable bacteria growth ensues which usually results in death in about 3 days

(Figure 18). Therefore, to study the specific T-cell mediated immune function, 4 days

after bacteria injection is used as the terminal observation.












a
77


bed
T


cd
-1


9.0




8.5


abc
-7


a


bcd

T


d

T


HS HR NS LR ND NR MR MN WN IS

HIS: refed with casein diet containing 1.50% RNA
HR: refed with casein diet containing 0.50% RNA
NS: refed with casein diet containing 0.25% RNA
LR: refed with casein diet containing 0.10% RNA
ND: refed with casein diet
NR: refed with stock chow
MR: refed with protein-free diet containing 0.25 % RNA
MN: refed with protein-free diet
WN: on stock diet throughout experiment
IS: on stock diet throughout and treated with Methotrexate

Effect of RNA supplementation on number of viable organisms recovered
24 hours after intradermal injection of 1.0 x 108 cells of Staphylococcus
aureus. Groups of mice (except IS and WN) were fed a casein diet for 12
days followed by a protein-free diet for 6 days and refed with different diets
for 6 days. Values are means + SEM. In each panel, bars with different
letters are significantly different (P < 0.05).


ab
-T


8.0


Figure 17.




















Sensitive




I
Resistant










\

Na Acquired
-1

i -- i __ i __ i __ i __


Time after bacterial infection (days)


Model of natural and acquired resistance to Listeria monocytogenes in
sensitive and resistant strains of mice. Graph was adapted from Skamene
and Kongshavn, 1982.


Figure 18.









83

An experiment was carried out to determine an effective, nonlethal dose of L.

monocytogenes which could be cleared by the RES within a certain time period. Mice

received a dose (8.0 x 103 cells) of L. monocytogenes (ATCC 19114) by tail vein

injection. At various time points, blood, liver, spleen, and kidney from groups of mice

fed a stock diet were removed and enumerated for total viable organisms (Figure 19).

Results similar to that of Skamene and Kongshavn (1982) were observed. There was a

dramatic increase in the number of viable bacteria recovered from the liver and spleen

between 4 and 24 hours after injection. The number of viable bacteria recovered from

liver and spleen were increased by 10-fold and 40-fold, respectively, compared to that

of injected. As the T-cell mediated immune function activated, the number of bacteria

in the liver and spleen started to decline. Less than 2% of bacteria were recovered in

liver and spleen by 4 days after injection.


Effect of RNA Supplementation on Clearance of Intravenous Bacterial Challenge with
Listeria monocytogenes

Four days after intravenous injection of 5.0 x 105 cells of a pathogenic strain L.

monocytogenes (ATCC 19114), 30 to 50% of the mice showed signs of sickness but no

mortality. Light-colored spots were observed on the livers and spleens. No bacteria

were found in the blood. However, substantial amounts of viable bacteria were

recovered from livers and spleens of the mice (Figure 20), whereas the number of

bacteria was relatively low in the kidneys. Regardless of dietary treatment, the number

of bacteria in the organs increased significantly above the initial dose. This indicated that

the mice were not able to overcome the challenge of this sublethal dose of the pathogen.


















liver
-0-

Spleen
-U-

Kidney
-e

Blood
-B-


24 48 72 96

Time after injection (hours)


Survival of organisms in blood and organs of mice fed a stock diet and
challenged with intravenous injection of 8.3 x 103 cells of Listeria
monocytogenes.


Figure 19.































a a
"7- r


a a


HS HR


NS LR ND NR


MR MN WN IS


HS: refed with casein diet containing 1.50% RNA
HR: refed with casein diet containing 0.50% RNA
NS: refed with casein diet containing 0.25% RNA
LR: refed with casein diet containing 0.10% RNA
ND: refed with casein diet
NR: refed with stock chow
MR: refed with protein-free diet containing 0.25% RNA
MN: refed with protein-free diet
WN: on stock diet throughout experiment
IS: on stock diet throughout and treated with Methotrexate


Effect of RNA supplementation on number of viable organisms recovered
4 days after intravenous injection of 5.0 x 105 cells of Listeria
monocytogenes. Groups of mice (except IS and WN) were fed a casein diet
for 12 days followed by a protein-free diet for 6 days and refed with
different diets for 6 days. Values are means + SEM. In each panel, bars
with different letters are significantly different (P < 0.05).


8 liver ab abc a abc
7 abc abc bc ab C
6 l
5
4
3


SKidney
8 1 Kidney


Figure 20.









86

Among the RNA-supplemented groups, only the 1.5% RNA group (HS) showed higher

immune strength when compared to the protein-malnourished (MN) or nucleotide-

depleted (ND) groups. Addition of 0.25% RNA to the protein-free diet (MR) did not

show any beneficial response.

When the mice were challenged with a lower dose, i.e., 7.5 x 104 cells of the

same pathogen, bacterial numbers in the organs were slightly lower than that injected

(Figure 21). No significant differences in bacterial counts in the livers were found

among the groups. Bacterial numbers in the spleen were lower in the 0.5 and 1.5%

RNA-supplemented groups (HR and HS) with a trend of dose-related response as

compared to the protein-malnourished (MN) or nucleotide-depleted (ND) groups. The

kidneys had about 1000 and 100-fold less bacteria compared to the numbers in the livers

and spleens, respectively. No bacteria were found in the blood. Since none of the mice

showed signs of sickness and bacterial levels in the organs were decreased, then a longer

time period may be required to recover from the challenge of the pathogenic strain L.

monocytogenes.


Survival Rate of Mice after Intravenous Bacterial Challenge with Streptomycin-Resistant
Listeria monocytogenes

Mice fed stock diet were challenged intravenously with three levels of

streptomycin-resistant L. monocytogenes (Figure 22). After intravenous injection of the

highest dose (8.7 x 10' cells) of bacteria, all mice died within 30 minutes. Of those mice

challenged with 8.7 x 10' cells, 75% died within 24 hours and 100% died within 48

hours. All mice injected with 8.7 x 10' cells of bacteria survived. Very few bacteria








87
7
6 Liver ab
ab a ab ab a) ab a ab
5b
4
3
2
1

7 Spleen

5 ab a a ab ab a a
4-c bc at* I



7
6 Kidney
5
4
3 a ab ab a
2 ab at b ab b

0
HS HR NS LR ND NR MR MN CA WN IS

HS: refed with casein diet containing 1.50% RNA
HR: refed with casein diet containing 0.50% RNA
NS: refed with casein diet containing 0.25% RNA
LR: refed with casein diet containing 0.10% RNA
ND: refed with casein diet
NR: refed with stock chow
MR: refed with protein-free diet containing 0.25% RNA
MN: refed with protein-free diet
CA: on casein diet throughout experiment
WN: on stock diet throughout experiment
IS: on stock diet throughout and treated with Methotrexate


Figure 21. Effect of RNA supplementation on number of viable organisms recovered
4 days after intravenous injection of 7.5 x 104 cells of Listeria
monocytogenes. Groups of mice (except CA, IS, and WN) were fed a
casein diet for 12 days followed by a protein-free diet for 6 days and refed
with different diets for 6 days. Values are means + SEM. In each panel,
bars with different letters are significantly different (P < 0.05).

















A AA A


-A- Low: i

Mediu

--"- IHigh: i


















I


injected with 8.7 x 107 cells

m: injected with 8.7 x 10' cells

injected with 8.7 x 109 cells


i


1


Time after bacterial infection (days)


Survival rate of mice fed a stock diet after intravenous injection of 3
different levels of streptomycin-resistant Listeria monocytogenes.


- -- -__-


Figure 22.




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