Vitamin A and the interferon system

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Title:
Vitamin A and the interferon system
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vii, 74 leaves : ill. ; 29 cm.
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English
Creator:
Blalock, James Edwin, 1949-
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Subjects

Subjects / Keywords:
Interferons   ( mesh )
Vitamin A   ( mesh )
Immunology and Medical Microbiology Thesis Ph.D   ( mesh )
Dissertations, Academic -- immunology and medical microbiology -- UF   ( mesh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1976.
Bibliography:
Bibliography: leaves 68-72.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by James Edwin Blalock.

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University of Florida
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
    List of Tables
        Page iv
    List of Figures
        Page v
    Key to abbreviations
        Page vi
    Abstract
        Page vii
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
    Materials and methods
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
    Results
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
    Discussion
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
    Literature cited
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
    Biographical sketch
        Page 73
        Page 74
        Page 75
        Page 76
Full Text








VITAMIN A MA5D T-LHE1NRFO YSE





















By

JAMEZS EDWIN BTLALOCK/ 2W7
























A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL~ OF
THE UNIVERSITY OF FLORIDA IN P P \, FU-l-PI LVE:7P- oVl L'7 IEQIIUHT O DECREE Oil DOCTOR OF PHILOSOPHY





UNIVTERSITY OF FLORIDA 19-76









ACKNOWtEDGENiENTS


This dissertation is dedicated to my parents, Mr. Maury J. and

Mrs. Elizabeth B. Blalock and to other members of my family for their love and belief in me.

I extend my sincere appreciation to my teacher and friend, Dr.

Georg'e E. Cifford, for his constant willingness to help and guide me through my graduate years. I especially thank him for always encouraging me to be scientifically inquisitive and for firmly establishing, by example, that there is a right way to do science. I am especially grateful to Dr. Donna H. Duckworth for her friendship and genuine interest in my well-being. I also thank the other members of my advisory committee, Dr. J.W. Shands, Jr., Dr. L.W. Clem, Dr. Kenneth Ley, Dr. P.A. Klein and Dr. P.A. Small, Jr. for their suggestions and encouragement during my studies. Special thanks also go to the other members of the faculty, especially Dr. R.B. Crandall, and staff of the

Department of Immunology and Medical Microbiology, who have helped me in many ways.

I ai4 grateful to my former and to my present fellow graduate students for their suggestions and support throughout this investigation. In particular, I thank Rick Weber and Dave Dion for the many scientifically stimulating conversations.

Finally, I would like to express my gratitude to Mr. Mike Duke and Mr. Joe Brown for their excellent technical assistance and for making Dr. G[ford's laboratory such a fun place to work.









ii.











TABLE OF CONTENTS



A CK21OW LED GEMENT S .......... ...............ii

LIST OF TABLES................................................. iv

LIST OF FIGURES.................. .............................. v

KEY TO ABBREVIATIONS........................................... vi

ABSTRACT....................................................... vii

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

MATERIALS AN-D 'METHODS........................................... 5

M4aterials.................................................. 5

Methods................... ................................7

RESULTS......................................................... 11

Vitamin A and Interferon Action............................ 11

Vitamin A and Interferon Production........................ 29

Structural Requirements of Vitamin A for Suppression of Interferon Production and
Inhibition of its Action.................................. 56

DISCUTSSION..................................................... 5

LITERATURE CITED............................................... 68

BiOGRFAP11ICA:L SKETCH......................... ................... 73










TABLES






Table

1. Effect of Bovine Serum Albumin on the Simultaneous
Addition of Retinoic Acid and Interferon to Cells ...... 17

2. Effect of Retinoic Acid on Interfercn and
Cellular RNA Synthesis ................................... 30

3. Effect of Calf Serum on Suppression of Cellular
RNA Synthesis by Retinoic Acid .......................... 32

4. Effect of Retinoic Acid on L-929 Cell Cultures ......... 34

5. Effect of Retinoic Acid on L-929 Cell Proliferation .... 35

6. Effect of Retinoic Acid Concentration on Interferon Production by NDV ........................................ 36

7. Effect of Retinoic Acid on the Intracellular Level
of Interferon ............................................ 38

8. Effect of Retinoic Acid on NDV, SFV and Poly I:C Induction of Interferon ................................. 40

9. Effect of Time of Treatment with Retinoic Acid (20 pg/ml) after NDV Adsorption ........................ 42

10. Effect of Cycloheximide on Interferon Production ....... 50 11. Effect of Cycloheximide on Suppression of
Interferon Production by Retinoic Acid ................. 52

12. Structural Requirements of Vitamin A for
Suppression of Interferon Production and
Inhibition of Its Action ................................ 57
















iv









FIGURES

Figure

1. Effect of simultaneous addition of retinoic acid and
interferon on the assay of interferon..................... 13

2. Effect of calf serum on the simultaneous addition of
retinoic acid and interferon to cells..................... 16

3. Effect of treatment of interferon with retinoic acid
prior to assay for activity of interferon................. 20

4. Effect of calf serum on the treatment of interferon
with retinoic acid priot to assay for interferon
activity.................................................. 23

5. Effect of temperature on treatment of interferon with 2
retinoic acid..............................................2

6. Effect of time at 37'C on the loss of interferon
activity in the presence of retinoic. acid................. 28

7. Effect of time of addition of retinoic acid after NDV
adsorption................................................. 4

8. Effect of time of addition of retinoic acid after addition of poly PC....................................... 46

9. Kinetics of interferon production by control and 4
retinoic acid treated cells...............................4

10. Kinetics of interferon action in the presence of 5
retinoic acid..............................................



























v










KEY TO ABBREVIATIONS


BSS..................... Balanced salt solution

CS. ..................... Calf serum

DEAE-dextran.......... Diethylaminoethyl dextran

DMSO .............. ... Dimethyl sulfoxide

DNA. ..................... Deoxyribonucleic acid

HA. ...................... Hemagglutination

MEM .................... Minimal essential medium

m-RNA. ................... Messenger ribonucleic acid

NDV..................... Newcastle disease virus

PBS..................... Phosphate buffered saline

PDD50. .................. 50% plaque depressing dose

PFU..................... Plaque forming unit

Poly I:C................ Polyriboinosinate:polyribocytidilate

RNA ..................... Ribonucleic acid

SFV. ..................... Semliki Forest virus

TCA...................... Trichloroacetic acid

VSV. ..................... Vesicular stomatitis virus

V/V. .................... Volume per volume





















vi










Abstract of Dissertation Presented to the Graduate Council
of th,,2 University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy



VITAMIN A AND THE INTERFERON SYSTEM

By

James Edwin Blalock

June, 1976


Chairman: Dr. George E. Gifford
Major Department: Immunology and Medical Microbiology


Vitamin A was shown to suppress both interferon action and production. The mechanisms of the two inhibitory effects of vitamin A appeared to be different and to occur by different moieties of the vitamin A molecule. The inhibition of interferon action seemed to result from an interaction of vitamin A with the interferon molecule. The loss of interferon activity was characterized by a dependence on time and temperature and was prevented by calf serum. The suppression of interferon production was due to an effect of vitamin A on the cell. The kinetics of the suppression atid results from experiments with metabolic inhibitors are consistent with transcription of the interferon gene being blocked by a vitamin Ainduced protein. Our data, therefore, point to a site of action of vitamin A at the genetic level and our system provides a potential model for the study of control of gene expression by vitamin A.















Vii









INTRODUCTION

Vitamin A is an essential itutriertt for vertebrates. Many of the

known physiological action.- of this vitamin have been elucidated by studying the effects of vitamin A deficiency. In its absence, animals suffer from blindness, I retarded growth, 2 impaired reproductive capacity 3 and ultimately death. 2 At the tissue and cellular level some of the most profound alterations associated with hypovitaminosis A involve epithelial structures. About 50 years ago Wolbach and Howe4 observed that vitamin A deficiency caused a replacement-of mucus-secreting epithelia by stratified keratinizing epithelia. In this classic study, vitamin A was postulated to induce and control epithelial differentiation. This hypothesis was strengthened when in 1957 Fell substantiated the findings of Wolbach and Howe by demonstrating that excess vitamin A in vitro caused keratinizing tissues to become mucus-secreting. More recently, vitamin A has been observed to have both a prophylactic and therapeutic effect on several types of experimental tumors. For instance, Saffiotti et al. 6 and Bollag showed a suppressive effect of vitamin A on the induction and development of tumors in response to chemical carcinogens. In another system, Felix et al. 8 demonstrated an anti-tumor action of vitamin A in mice inoculated with murine melanoma cells. Although other explanations are possible, the anti-tumor quality of vitamin A might also be interpreted as a reflection of the ability of vitamin A to induce and control differentiation. Our laboratory has, in fact, observed an apparent restoration of the control of proliferation of a transformed cell line in vitro by this vitamin.9




2




In spite of the impressive amount of knowledge on the physiological effects of vLtamin A, 10 its mechanism(s) of action remains obscure, with the excepLion of its role in vision. 11 At the biochemical level a number of systems have been studied in the search for the mechanism(s) of vitamin A action. For instance, there is no doubt that vitamin A labilizes membranes. 12 A hallmark of this labilization is the extracellular release of lysosomal enzymes by cells treated with vitamin A. In vitro, fetal cartilage or bone underwent resorption in the presence of vitamin A. 13 This resorption was surmised to be caused by a lysosomal acid protease, whose extracellular release was shown to be increased by vitamin A. 14 Isolated lysosomes from liver, also, rapidly released proteases upon addition of vitamin A. 15 Similar effects occur in vivo. Hypervitaminosis A in rabbits resulted in dissolution of cartilage. 16 This condition also caused release of the liver lysosomal enzyme, -glucuronidase, into serum. 17 These effects, while indicating a role in membrane function, only seem applicable in a hypervitaminotic state.

Vitamin A has also been postulated to serve a coenzyme function and that enzymatic conversion oF essential substances is suppressed in vitamin A deficiency. 18 Hence, vitamin A deficiency resulted in decreased activities of gulonolactone oxidase, 19 codeine demethylase, 20 A5-3 -hydroxysteroid dehydrogenase, 21 sulfate transferase, 22 and ATP-sullurylase. 23 However, in the ATP-sulfurylase system, 23 which was the most rigorously studied, it seems that the decreased activity

was the result of less of the enzyme rather than the absence of a coenzyre function of vitamin A. Whether the diminished amount of enzyme was due to increased catabolism or decreased synthesis is not known.





3



Interestingly, the decreased activity of codeine dei-iethylase 2

obsercved in vitamin A deficiency was reversed by the addition of vitamin A and actinomycin D inhibited the reversal. Since actinoinycin D blocks DNA dependent RNA synthesis, this data suggests de novo synthesis of the enz yme in response to the vitamin and could be interpreted as genetic transcription being one possible site of vitamin A action. Consistent with this idea, is the observation that vitamin A stimulateI RNA synthesis of intestinal mucosa 24 and liver 25 from vitamin A deficient rats. Incorporation of radio-labeled uridine was especially increased in the nuclear fraction and the increase occurred within 5 minutes. 25 Decreased protein synthesis was also associated with vitamin A deficiency. 26 This, however, could be explained as a secondary effect of blocked transcription. Perhaps, a transcriptional block by vitamin A deficiency could account for all of the depressed enzyme activities. There have also been reports of vitamin A induced glycoprotein synthesis. 27,28 The specific biological functions of these glycoproteins are unknown.

The obvious question which arises from the above mentioned apparently diverse observations is whether they are mediated by a number of modes of action of vitamin A or by a single action on a cardinal function, such as transcription. A key role for vitamin A in control of gene expression seems quite feasible and is certainly attractive. It would, therefore, be advantageous to study the effect of vitamin A on an inducible cell system with a known biological function.

The interferon system is a normal defense mechanism of an animal or aniMal Cell to virus infection. Virus infection of cells leads to the induction, Synthesis, and release of a new cellular protein, interferon. 29 InterFeron molecules then interact with neighboring, uninfected





4



cells and induce another cellular protein, the antiviral protein. 30,31 It is the antiviral protein, in turn, that causes these cells to become resistant to Subsequent virus infection. One can reasonably conclude, a priori that control at the transcriptional level exists for interferon since it is synthesized de novo. Additionally, a cell culture after one exposure to an inducer, will not produce the usual amount of interferon upon reexposure to an inducer during the next several days. This hyporeactivity or feedback inhibition of interferon synthesis may also represent transcriptional control. Employing the same reasoning as for interferon synthesis, the synthesis of the antiviral protein seems to be controlled at the transcriptional level since it is synthesized de novo. The interferon system can, therefore, be divided into interferon production and interferon action which share the common feature of inducibility. This common feature provides a unique means of study of the effect of vitamin A on the induction of cellular functions.

Initially, we observed an inhibitory effect of vitamin A on human interferon action. 32 in the present study, we have extended this observation to include the inhibitory effect of vitamin A on mouse interferon action. We have further shown a suppressive effect of this vitamin on mouse interferon production. The mechanisms of these two effects on the mouse interferon system were shown to be different and appeared to involve different moieties of the vitamin A molecule.









MATERIALS AND METHODS


Materials


Virus Strains

Vesicular Stomatitis Virus (VSV). A large plaque variant of the
v
Indiana strain was obtained from Dr. Jan Vilcek, New York University.

Newcastle Disease Virus (NDV). A lentogenic strain was obtained from Dr. R.P. Hanson, University of Wisconsin.

Semliki Forest Virus (SFV). Kumba strain was obtained from Dr.

J. Porterfield, National Institute for Medical Research, London, England.


Cell Cultures

Mouse L cell cultures. Strain 929


Media

L cell growth media. Eagle's Minimal Essential Medium (MEM), obtained from International Scientific Industries (Cary, Illinois), was supplemented with 10% calf serum (CS). This medium contained 125 pg streptomycin and 250 units of penicillin/ml.

-Mthyl cellulose overlay. The overlay medium for plaque assay

consisted of MEM containing 1% methyl cellulose (1500 centipoise, Fisher Scientific Company), 5% CS, 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 125 og/ml streptomycin and 250 units/ml of penicillin.

Balanced salt solution. Gey's balanced salt solution (BSS) was employed.

Phosphate buffered saline. Gey's BSS without calcium and magnesium salts was employed as phosphate buffered saline (PBS).






5





6



Reagents

Polyribonucleotides. The homopolymer pair polyriboinosinate: polyribocytidilate (poly I:C) was purchased from Sigma Chemical Company, St. Louis, Missouri. A stock solution of the polymer at a concentration of 1 mg/ml was prepared in MEM and stored at -20'C.

Radioisotope. Uridine-5-3H was obtained from Schwarz/Mann, Orangeburg, New York. The specific activity was 20 Ci/mM.

Scintillation fluid. Radioactive samples were diluted in scintillation fluid containing 8.25 g of 2,5-diphemyloxazole (Fisher Scientific Company), 0.25 g of 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene (Packard), 1,000 ml of toluene (J.T. Baker Chemical Company) and 500 ml of Triton X-100 (Packard).

Actinomycin D. A stock solution of 100 pg/ml of actinomycin D (Grand Island Biological Company) was prepared in MEM and stored at -200C.

Cycloheximide. Cycloheximide (Grand Island Biological Company) was dissolved at 1 mg/ml in HEM and stored at -200C.

Diethylaminoethyl (DEAE) dextran. DEAE-dextran (Pharmacia Fine

Chemicals, Uppsala, Sweden) was dissolved at 10 mg/ml in MEM and stored at -200C.

Vitamin A and related compounds. All trans forms of retinoic acid (vitamin A acid), retinol (vitamin A alcohol), retinal (vitamin A aldehyde), and retinyl acetate (acetate ester of vitamin A) were purchased from Sigma Chemical Company. VitaminK1 was obtained from Schwarz/Mann. Trans B-carotene and citronellol were obtained from Aldrich Chemical Company, Milwaukee, Wisconsin. Stock solutions were prepared by making each compound 6.7 X 10-3 M in dimethyl sulfoxide (DMSO, Fisher Scientific




7



Company) and stored at -20'C. For experimental purposes, the compounds were diluted to 6.7 X 10-5 M (equivalent to 20 ig/ml of retinoic acid) in culture medium.



Methods


Cell Cultures

Mouse L cells (Strain 929). Cell cultures were maintained and propagated in 32 oz prescription bottles in MEM with 10% CS. These cells were passaged weekly. The growth medium was decanted, the cell sheet was washed with PBS and treated with 0.1% trypsin and 0.04% versene in PBS for 30 seconds. When the cells had detached from the glass surface they were diluted in fresh growth medium and dispensed into 32 oz bottles forfurther propagation or into 2 oz glass bottles for production and assay of interferon, assay of viruses and 3H-uridine incorporation.

Cell counts. Cell monolayers were treated with 1 ml of 0.1% trypsin in phosphate buffered saline (PBS) containing 0.04% versene. After 30 seconds, the trypsin solution was removed, and 3-4 minutes later, when cells detached from the glass surface, 4 ml of MEM with 5% CS was added. After addition of 1 ml of 0.5% trypan blue in buffered saline, to determine cell viability, an aliquot of the cell suspension was counted in a haemocytometer. Cells unable to exclude trypan blue dye were counted as dead.


Growth and Assay of Viruses

Preparation. NDV and VSV were grown in the allarntoic cavity of

10-day-old chick embryos by inoculation of 0.2 ml of a virus suspension. After 48 hours incubation at 37C, the eggs were chilled and the allantoic










fluid collected. The allantoic fluid was centrifuged at low speed (1,000 rpm) to sediment red blood cells and tissue fragments. The supernatant fluids were dispensed in sealed glass ampules and stored at

-70'C. SFV was propagated in the brains of newborn mice. The newborn outbred mice were intracerebrally inoculated. The infected brains were harvested 48 to 72 hours after infection and a 10% suspension of brains in medium was made with a Potter-Elvehjem homogenizer. The suspension was centrifuged to remove coarse material. The supernate was dispensed in sealed glass ampules and stored at -70'C.

Assay. VSV and SFV were assayed on monolayers of L-929 cells by plaque assay. Medium was aspirated from monolayer cultures and cells were infected with 0.2 ml of a suitable dilution of virus. After 1 hour incubation at room temperature to permit virus adsorption, each monolayer was overlaid with 5 ml of methyl cellulose overlay. During the virus adsorption period cultures were rocked every 15 minutes to evenly distribute the virus suspension. The cultures were incubated foT 48 hours at 37'C and monolayers were then stained with crystal violet. Assays were performed in triplicate or quadruplicate. Plaques were enumerated after X6.5 magnification of the monolayers by useof a photographic enlarger. By this assay the pools of VSV and SFV contained 3.0 X 109 and 2.1 X 108 plaque forming units (PFU)/ ml respectively. NDV was assayed by hemagglutination with chicken red blood cells. Following an initial 1 to 10 dillution, two-fold serial dilutions of the virus suspension were made in hemagglutination buffer (Difco Laboratories, Detroit, Michigan). To 0.5 ml of diluted virus was added 0.5 ml of a 0.5% suspension of red blood cells. One hemagglutination (HA) unit was defined as the





9



highest dilution of virus which gave total hemagglutination. The pool of NDV contained 320 IA units/il.


Production and Assay of Interferon

Production. Interferon was prepared by inoculating monolayer cultures of L--929 cells with NDV or with SFV. Triplicate cultures were used for all determinations. After adsorption at room temperature for one hour, residual virus was removed, cultures were washed and fresh medium added. Culture fluids, unless specified, were harvested at 24 hours after infection, the triplicate samples pooled, and clarified by low speed centrifugation. Culture fluids were then dialyzed against pH 2 buffer for five days at 9'C and then against Gey's balanced salt solution (BSS) to restore pH to neutrality.

Interferon was also induced by treatment of confluent monolayers of L-929 cells for various times with medium containing 10 pg/ml of poly I:C and 100 pg/ml of DEAE dextran. The medium was then removed, cells washed and refed fresh medium. Culture fluids were harvested 24 hours after poly I:C treatment and assayed for interferon.

Assay. A plaque reduction assay using VSV and L-929 cells was

employed to determine the interferon content of an interferon preparation. Confluent monolayers in 2 oz glass bottles were treated overnight at 37'C with twofold serial dilutions of interferon preparation. Dilutions of interferon were made in MEM supplemented with 5% CS. Supernatant fluids were then aspirated and cells infected with 0.2 ml of a dilution of VSV containing about 300 PFU. The remainder of the assay is identical with that used for titration of VSV. Assays were performed in triplicate or quadruplicate. The 50% plaque depressing dose (PDD50)





10



was defined as the amount of an interferon preparation, in ii, that inhibited 50% of the plaques from developing as compared to the controls. The PDD50 was calculated according to the method of Lindenmann and Gifford. 33


3H-Uridine Incorporation

Twenty pCi of 3H-uridine in 0.1 ml of MEM were added for 1 hour

to monolayer cultures in 2 oz glass bottles containing 2 ml of medium. Incorporation was stopped by decanting the supernates and placing the cultures on ice. Unincorporated label was removed by 5 successive washes of the cell sheets with 5 ml of cold 5% trichloroacetic acid (TCA). Cultures were drained by inverting on absorbent towels and then 2 ml of 5% TCA were added to each culture. Incorporated 3H-uridine was hydrolyzed by heating the cultures at 80C for 1 hour. Aliquots (0.2 ml) of hot TCA extracts were placed in 10 ml of scintillation fluid and counted in a Beckman LS-230 liquid scintillation counter.









RESULTS


Vitamin A and Interferon Action


Effect of Simultaneous Addition of Retinoic Acid and Interferon on the

Assay of Interferon.

Separate interferon assays were simultaneously performed in the presence or absence of different concentrations of retinoic acid (vitamin A acid). Dilutions of interferon and retinoic acid in MEM with 3% calf serum (CS) were mixed and added to L cell monolayers which were then incubated overnight at 37'C. Residual interferon and retinoic acid were removed and cultures challenged with approximately 300 P F U of VSV. Overlay medium was added and cultures incubated at 37'C for 48 hours. Plaques in experimental and control cultures were counted and the percent inhibition of plaque formation was plotted against the logarithm of the dose of interferon in 4l. A series of approximately parallel dose response lines were obtained. These were used to estimate the PDD50 units of interferon measured with each concentration of retinoic acid. The percentage of control PDD50 units obtained in the presence of various concentrations of retinoic acid were calculated for two separate experiments and are shown in Figure 1. Increasing concentrations of retinoic acid resulted in decreasing activity of interferon. A marked reduction (93%) in measurable interferon was obtained when 20 1g/ml of retinoic acid was employed (from 6.7 PDD50 units in the control to 0.5 units). This concentration of retinoic acid was not demonstrably toxic for the cells since the same number (and similar size) of VSV plaques were found with all concentratLons of retinoic acid (and controls) in the absence of




11




































Figure 1. Effect of simultaneous addition of retinoic acid and
interferon on the assay of interferon. Separate
interferon assays were simultaneously performed in the presence or absence of different concentrations of retinoic acid in MEM with 3% CS. The percentage
of control PDD50 units (6-7 units) obtained in the
presence of various concentrations of retinoic acid
were calculated for two separate experiments (X or





13











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14



interferon. Greater concentrations of retinoic acid could not be used since they were toxic for the L cells. Effect of Calf Serum or Bovine Serum Albumin on the Simultaneous

Addition of Retinoic Acid and Interferon to Cells.

Preliminary experiments indicated that the effect of retinoic acid on the interferon assay was markedly influenced by the concentration of calf serum employed. Various concentrations of calf serum (%, v/v) were employed with 20 Pg/ml of retinoic acid or 1% DMSO and a dose of interferon which would normally result in approximately 80% inhibition of VSV plaques. Controls without interferon received an equivalent amount of retinoic acid or DMSO. These mixtures were then assayed for determination of resultant interferon activity. These results (Figure 2) indicate that concentrations of calf serum from 2.5 to 20% had only a slight effect on the interferon assay as previously reported by Vilcek & Lowy. 34 However, increasing concentrations of calf serum decreased the inhibitory effect of retinoic acid on interferon action.

In a similar experiment, bovine serum albumin was substituted for calf serum. Table 1 shows that increasing concentrations of bovine serum albumin also decreased the inhibitory effect of retinoic acid on interferon action.


Effect of Treatment of Interferon with Retinoic Acid Prior to Assay

for Activity of Interferon.

A loss of measurable interferon activity, when the interferon is assayed in the presence of retinoic acid, could be due to either a combination of interferon with retinoic acid resulting in an inactive
































Figure 2. Effect of calf serum on the simultaneous addition of
retinoic acid and interferon to cells. Vertical bars
indicate the standard deviation of quadruplicate
determinations; interferon; Q(- -O, interferon
plus retinoic acid (20 ug/ml).





16









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product or to some intracellular event, influenced by retinoic acid, which prevented the expression of interferon activity. The previous data showing that increasing concentrations of calf serum or bovine serum albumin prevented the effect of retinoic acid on interferon activity suggested that the event was extracellular. To test this possibility, interferon and retinoic acid were mixed, incubated, and then diluted beyond the effective range of retinoic acid but with sufficient interferon remaining to significantly inhibit virus replication. Thus, a 1:10 dilution of stock interferon in MEM was mixed with various concentrations of retinoic acid. Control interferon received an equivalent amount of DMSO. Since the stock interferon was made in 10% calf serum, the resultant concentration of calf serum employed was 1%. Following overnight incubation (22 hours) at 37'C, interferon activity was assayed. Prior to assay, the interferon samples were sufficiently diluted in MEM with 5% CS so that the residual retinoic acid was diluted beyond the concentration needed to interfere with the assay (i.e. 0.4 to 0.003 g/l). The control activity represented 400 PDD50 units of interferon. Figure 3 shows that retinoic acid treatment of interferon resulted in a loss of interferon activity under these conditions. This data indicates that interferon and retinoic acid must interact in some fashion to inhibit interferon activity and that this interaction was not rapidly reversible. Effect: o[ Calf Secum on the Treatment of Interferon With Retinoic Acid

Prior to Assay.

Since the concentration of calf serum markedly influenced the

effect of retinoic acid on the interferon assay when assayed immediately




































Figure 3. Effect of treatment of interferon with retinoic acid
prior to assay for activity of interferon. The
control activity represents 400 PDD50 units of
interferon.




20












100









40


06



0


0



0 10 20 30 40

Concentration of Retinoic Acid (aug/mI)





21



(see Figure 2), the effect of calf serum was then determined on the interaction of retinoic acid and interferon when incubated together in the absence of cells.

A dilution of the stock interferon was made in MEM with 1, 5 or

10% calf serum and with or without 20 jg/mi of retinoic acid. Following overnight incubation (22 hours) at 37'C, interferon activity was assayed after dilution (residual concentration of retinoic acid following dilution was less than 0.2 pg/mi). Figure 4 shows that calf serum again prevented an apparent interaction of interferon and retinoic acid under these conditions. In another experiment the calf serum concentration was reduced to 0.125%, and interferon activity was thereby reduced to 2.5% (10 units) of the control (400 PDD50 units/ml) when 40 idg/mil of retinoic acid was employed. Effect of Temperature on Treatment of Interferon With Retinoic Acid.

Since it appears that interferon and retinoic acid interact to result in an inactive product, the effect of temperature on this interaction was determined.

Retinoic acid was added to interferon in MEM (400 units/ml in 1% CaS) at a concentration of 20 ig/nl. One ml portions of the interferon and retinoic acid mixture, as well as control interferon prepared without retinoic acid, were placed at 5, 25 and 37C for 24 hours. The mixtures were then diluted 1:100 in MEM with 5% CS (reducing the retinoic acid concentration to 0.2 pg/[iLl and assayed for residual interferon activity. Figure 5 shows only a slight loss of interferon activity in the control at 37C, while there is a marked loss of interferon activity at this temperature in the presence of retinoic acid. No loss of interferon activity resulted when the mixture of interferon

































Figure 4. Effect of calf serum on the treatment of interferon
with retinoic acid prior to assay for interferon activity; interferon; Q- -'retinoic
acid-treated interferon.




23








400




/


300- /

: /
o O[
0 /



CO /
4




0 I0
1000o

o
0

M Interferon
0- --o Retinoic Acid Treated Interferon

0 I
0 5 10
Concentration of Calf Serum (%)

































Figure 5. Effect of temperature on treatment of interferon with
retinoic acid. Vertical bars indicate the standard
deviation of quadruplicate determinations; interferon; Q-- -Q retinoic acid-treated interferon.





25













100







8070g-r60CO)
>50
0
c
20
e 40- Interferon
C0- -'0 Retinoic Acid Treated Interferon


30



2010




5 1 0 1 2 0 25 3 10 3 5
Temperature (*C) for 24 hours





26



and retinoic acid were kept at 500 for 24 hours. This data would indicate that the interaction of interferon and retinoic acid resulting in the loss of interferon activity was temperature dependent. Effect of Time at 370C on the Loss of Interferon Activity in the

Presence of Retinoic Acid.

Retinoic acid (20 pg/ml) was added to 400 units/ml of interferon in HEM (1% CS). The mixture was placed at 370C and portions were removed at various times and placed at 50C (a temperature at which retinoic acid did not result in a loss of interferon activity). When all samples were collected (23 hours), each sample was diluted 1:100 in MEM with 5% CS (reducing the retinoic acid concentration to 0.2 Pg/ml) and assayed for residual interferon activity. As shown in Figure 6, there is a progressive loss of interferon activity in the presence of retinoic acid with increasing periods of time at 37C.




































Figure 6. Effect of time at 37%C on the loss of interferon
activity in the presence of retinoic acid. Vertical
bars indicate the standard deviation of quadruplicate
determinations; ,interferon; ~--.~,retinoic
acid-treated interferon.





28


























Interferon Cr 60- (9-- Retinoic Acid Treated Interferon

(I,

> 50C
.0
401 40
C


30



20



10




0 5 10 15 20 25
Time MHrs.) at 370C




29






Vitamin A and Interferon Production


Effect of Retinoic Acid on Interferon Synthesis and Cellular RNA

Synthesis.

Monolayer cultures of L-929 cells were infected with NDV as described. Following infection, cultures were treated with 20 pg/ml of retinoic acid in MEM with 3% CS and 1% DMSO. Controls received an equivalent amount of DMSO (1%) in culture media. Retinoic acid or the control fluid either remained on the cells for 24 hours or was washed out after

3 or 6 hours and replenished with fresh medium lacking retinoic acid and DMSO. Table 2 shows that retinoic acid suppressed interferonproduction. The same amount of suppression of interferon yield resulted regardless of whether the retinoic acid remained on the cultures for the entire production period or was washed out after 3 or 6 hours. Moreover, when replicate noninfected cultures were treated in a similar fashion with retinoic acid and labeled with 10 Pg/ml of 3H-uridine for 1 hour at 23 hours after infection, there was a 74% suppression of 3H-uridine incorporation by cell cultures treated for 24 hours with retinoic acid but no suppression by cultures treated for 3 or 6 hours. These results indicate the suppression of interferon production was not correlated with the suppression of 3H-uridine incorporation. Part of the suppression of interferon yield could be explained by inactivation of interferon by retinoic acid after the interferon was produced as previously shown (Figure 3). However, 20 Vg/ml of retinoic acid in 3% CS would inactivate only about 50% of the interferon and therefore would not totally explain the suppression (see Figure 4). More





30






TABLE 2

Effect of Retinoic Acid on Interferon

and Cellular RNA Synthesis


Hour of Treatment with Percent Suppression
Retinoic Acid (20 vg/ml)
Following NDV Adsorptiona Interferon Yieldb 3H11-Uridine
(24 hours) Incorporationc



3 94 0

6 94 0

24 98 74





a Retinoic acid was allowed to remain on the cells for the indicated
times after NDV adsorption, cultures were washed to remove the
vitamin and replenished with fresh medium and allowed to incubate
for a total of 24 hours.

b Interferon yield was determined by pooling the supernates from
triplicate cultures. Four dilutions of each pooled supernate
were assayed in quadruplicate.

c 3H-uridine incorporation was measured after 1 hour of labeling
at 23 hours after initial exposure to retinoic acid (non-infected
cultures). Incorporation of 3H-uridine was determined by two
counts of duplicate aliquots of hot 5% TCA extracts from triplicate
cultures.





31



importantly, in those experiments where the retinoic acid was removed after 3 or 6 hours and the interferon harvested after 24 hours, one would expect no inhibition of interferon activity due to the retinoic acid since extracellular interferon did not appear until 8 hours after infection Ivide infra).


Effect of Retinoic Acid on L-929 Cells.

Although the suppression of interferon production was apparently not correlated with the suppression of 3H-uridine incorporation by retinoic acid, we were concerned that the suppression of interferon production might result from some non-specific toxicity of vitamin A. Studies were initiated to determine conditions for vitamin A treatment of cells which were relatively non-toxic. We had previously shown that the inhibition of interferon activity by vitamin A was dependent on the concentration of calf serum (Figure 2 and 4), apparently because a serum component bound the vitamin A. The effect of calf serum on suppression of 3H-uridine incorporation, therefore, was also investigated.

To determine the effect of CS on suppression of RNA synthesis by vitamin A when cells were in contact with vitamin A for 23 hours, confluent cell monolayers were treated at 370C with 20 g/ml of retinoic acid in MEM with 2.5, 5 or 10% CS. After retinoic acid treatment for 23 hours, cell cultures were washed and labeled for 1 hour with 2 ml of MEM containing 10 pg/ml 3H-uridine. Table 3 shows that increasing CS concentrations prevented the inhibitory effect of retinoic acid on 3H-uridine incorporation.






32











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33



Since 20 pg/ml of retinoic acid did not significantly suppress 3l-uridine incorporation in medium containing 10% CS, we studied the effects of 24 hours treatment with 5, 10, or 20 vg/ml of retinoic acid in HEM with 10% CS on other parameters of cell viability. As can be seen in Table 4, the concentrations of retinoic acid used did not alter: a) cell number; b) cell viability as determined by trypan blue dye exclusion; or c) the capacity of these cells to allow vesicular stomatitis virus plaque formation. In other experiments we have shown that L-929 cells are able to proliferate when seeded and grown in 20 pg/ml of retinoic acid, although they do not reach as high a cell density as controls (Table 5).

These criteria indicated that retinoic acid at the concentrations employed was not toxic to cells when medium containing 10% CS was employed. Therefore all subsequent experiments were performed with 10% CS.


Effect of Retinoic Acid Concentration on Interferon Production by NDV.

Confluent cell monolayers were infected with NDV, washed and fed

medium with 1% DMSO or medium containing 5, 10 or 20 pg/ml of retinoic acid and 1% DMSO. Triplicate cultures were used for each concentration of retinoic acid. Supernates were harvested 24 hours after NDV infection, virus inactivated, and assayed for interferon. Table 6 shows that increasing concentrations of retinoic acid resulted in decreasing yields of interferon. Twenty Pg/ml of retinoic acid was used in all subsequent experiments since it gave the most marked suppression and yet was apparently non-toxic for the cells.






34.













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36



TABLE 6

Effect of Retinoic Acid Concentration on Interferon Production by NDV


Retinoic Acid Interferon Yield
(ig/ml)
PDD50 Units/ml % Inhibition


20 7,500 79

10 10,000 72

5 15,000 58

0 36,000 -





37



Effect of Retinoic Acid on the Intracellular Level of Interferon.

Since we routinely assayed interferon in the supernates of NDV infected cultures, it was possible that the suppressive action of retinoic acid on interferon production was due to interference with the release process. To test this possibility, extracellular and intracellular interferon levels were determined in retinoic acid treated and control cultures 24 hours after NDV infection. Treatment with retinoic acid reduced the amount of interferon recovered from extracts of NDV infected cells (Table 7). However, the ratio of extracellular/intracellular interferon was 6 in both the control and retinoic acid treated group. This data shows that retinoic acid did not suppress interferon production by impeding its release.


Effect of Retinoic Acid on NDV, SFV and Poly I:C Induction of Interferon.

The results in Table 2 indicated that retinoic acid probably suppressed an early event in the production of interferon since there was no difference in the degree of suppression of interferon yield if the retinoic acid remained in contact with the cells for the 24 hours of production or was removed after three hours. The earliest event in the production cycle of interferon is thought to be the generation 35
of the inducer of interferon, double stranded RNA. If retinoic

acid interfered with virus replication, the generation of the interferon inducer would probably be adversely affected. Since NDV abortively infects L-cells we could not detect an effect of retinoic acid on NDV replication. SFV, however, productively infects L-cells and produces moderate amounts of interferon. We, therefore, investigated the effect of retinoic acid on SFV replication and interferon production.





38




TABLE 7

Effect of Retinoic Acid on the Intracellular Level

of Interferon


Treatmenta Interferon Yield (PDD50/ml)b

Extracellular Intracellular Total



Control 20,800 3,500 24,300

Retinoic Acid 6,000 1,000 7,000





a Retinoic acid (20 pg/ml) or DMSO (1%) in MEM with 10% CS was added
to NDV infected cultures at the end of virus adsorption.

b Culture fluids were collected 24 hours after addition of NDV.
Triplicate supernates were pooled and were centrifuged for 15 minutes
at 1,000 rpm to remove cells which had detached from the glass
surface. After dialysis of the clarified supernatant fluids, they
were assayed for the level of extracellular interferon. For the
determination of the intracellular interferon level, the cell
pellets from the supernates were resuspended in fresh medium and
added back to the cells which were still adherent to the glass
surface. The cells were frozen at -20'C, thawed and homogenized
by 10 strokes with a Dounce homogenizer. The cell homogenates were
dialyzed at pH 2 for 5 days and centrifuged (1,000 rpm for 15 minutes)
prior to interferon assay. Four dilutions of each interferon sample
were assayed in quadruplicate.





39



L-cell cultures were infected with NDV or SFV, as previously

described. Cultures were refed with fresh medium containing 20 pg/ml of retinoic acid or 1% DMSO (controls). After three hours, cultures were washed to remove the vitamin and DMSO and were refed with fresh medium. Supernates were harvested from these cultures 24 hours after virus adsorption, virus inactivated by dialysis at pH 2, and assayed for interferon. Additionally, SFV infected cultures were assayed for the yield of virus.

Table 8 shows that retinoic acid suppressed SFV induced interferon production without affecting the yield of virus. The degree of suppression of interferon yield was similar with both SFV and NDV. This data shows that retinoic acid did not suppress interferon production by interfering with virus replication and suggested that generation of the interferon inducer was not inhibited.

In a similar experiment, L-cell cultures were induced to make

interferon by treatment for 2 hours at 37'C with poly I:C (10 pg/ml) in medium containing 100 jig/ml of DEAE dextran. After poly I:C treatment, cells were washed and refed with medium containing retinoic acid (20 Pg/ml or DMSO (1%). Twenty-four hours later, supernatant fluids were assayed for interferon. Table 8 shows that retinoic acid suppressed poly I:C induced interferon production. Since poly I:C is a synthetic double stranded RNA molecule, the interferon inducer had been formed prior to interaction with the cell.. In this instance, retinoic acid could not have interfered with generation of the inducer.






40










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41




Kinetics of Suppression of Interferon Yield byRetinoic Acid.

To investigate further the period during which interferon production was sensitive to suppression, retinoic acid and DMSO were removed at various times after NDV adsorption, the cell cultures were washed and replenished with fresh medium without retinoic acid or DMSO. Table 9 shows that maximum suppression of interferon production required two hours treatment with retinoic acid after NDV adsorption. Again there was no difference in the amount of suppression whether the vitamin A was removed after 2 hours treatment or remained on cells for 24 hours.

Similar experiments (Figures 7 and 8) showed that even if 'addition of retinoic acid was delayed for up to 2 hours after the end of NDV adsorption, or poly I:G addition, there was still maximum suppression of interferon production. By 4 hours after NDV adsorption, or poly T:C addition, cells were becoming less sensitive to the suppressive effects of retinoic acid, and they were resistant to suppression by 8 hours. These data indicate that retinoic acid affected a step in the production of interferon which maximally occurred about 2 hours after NDV adsorption or poly I:C addition.

Kinetics of Interferon Production by Retinoic Acid Treated Cells.

When the kinetics of interferon production in HEM with 10% CS were examined (Figure 9), interferon was first detected in control cultures 8 hours after NDV infection, while interferon was not detected from cultures treated with 20 Pu'iml retinoic acid until 4 hours later. The apparent rate of synthesis of interferon was reduced in retinoic acid treated cultures, as was the total yield. This difference in apparent rates of synthesis did not result from inter-




42








TABLE 9

Effect of Time of Treatment with Retinoic Acid
(20 pg/ml) after NDV Adsorption



Length of treatment with Interferon Yieldb
Retinoic Acid (20 pg/ml)a
PDD50 units/ml % Inhibition


0 22,900 -0-0.5 hrs 21,000 9

0-1 hrs 15,900 31

0-2 hrs 6,000 74

0-4 hrs 5,600 76

0-24 hrs 5,600 76




a 0 time is immediately following adsorption of NDV for 1 hour. b Interferon yield was determined by pooling the supernates from
triplicate cultures. Four dilutions of each interferon sample
were assayed in quadruplicate.

































Figure 7. Effect of time of addition of retinoic acid after NDV
adsorption. Monolayer cultures were infected with NDV
as described in Materials and Methods. At the time
intervals indicates, 20 Vg/ml of retinoic acid in HEM
with 10% CS was added. Retinoic acid remained on cells throughout the interferon production period. Controls
were treated similarly with an equivalent amount of DMSO (1%).
Interferon yield was determined by pooling supernates
from triplicate cultures. Four dilutions of each
interferon sample were assayed in quadruplicate.




44
















80



0
w
60

Li
1-40
z
LL
0


1-20 coi



e 0
0 2 4 6 8

HOURS AFTER NDV ADSORPTION

































Figure 8. Effect of time of addition of retinoic acid after
addition of poly I:C. Monolayer cultures were
treated with poly I:C as described in Materials and
Methods. At the time intervals indicated 20 pg/ml
of retinoic acid in MEM with 10% CS was added.
Retinoic acid remained on cells throughout the
interferon production period. Controls were treated
similarly with and equivalent amount of DMSO (1%).
Interferon yields were determined by pooling supernates
from triplicate cultures. Four dilutions of each
interferon sample were assayed in triplicate. AandO
show the results of two experiments.




46











100




0
w
LL
60z0
L
0 40
z co
=i:20 A
z
01

0 2 4 6 8

HOURS AFTER ADDITION OF POLY I:C



































Figure 9. Kinetics of interferon production by control and retinoic
acid treated cells. Cultures were infected with NDV as described in Materials and Methods. Cultures were then treated for 2 hours with retinoic acid (20 pglml)
in I4EM with 10% CS. Controls were treated with DMSO (1%).
At the times indicated triplicate cultures from both
groups were frozen at -20'C. At the end of the experiment samples were thawed, pooled, dialyzed and assayed
for interferon. Four dilutions of each interferon
sample were assayed in quadruplicate.





48























35



30



025 0-0 Rtinoic Acid
w 0


S20o z
CL 15- u 0
0) 0

S10ow
0 o


0



0 5 10 15 20 25 30 35

Time (Hrs.)





49



ference with the process of release of interferon, since we found the the same ratio of extracellular!intracellular interferon in both control and retinoic acid treated cells (Table 7). Effect of Cycloheximide on Suppression of Interferon Production By

Retinoic Acid.

The suppression of interferon production by retinoic acid could

be due either to a direct effect of the vitamin on an interferon control mechanism or indirectly through induction of another molecule. Vilvek et al. 36 described a paradoxical effect of inhibitors of RNA and protein synthesis on the production of interferon in cultures of rabbit kidney cells stimulated with poly I:C. In the continuous presence of cycloheximide these cells produced 3 to 10 times more interferon than control cultures. On the basis of kinetic studies it was concluded this effect was most likely explained by preferential inhibition (by cycloheximide) of a cellular regulatory protein (repressor) which controls interferon production. 37 To determine if vitamin A induces the synthesis of a regulatory protein, we studied the effect of cycloheximide on suppression of interferon production by retinoic acid.

Preliminary experiments were performed to determine a concentration of cycloheximide which would not inhibit interferon production. Cells were infected with NDV, as previously described. After adsorption of virus for 1 hour at room temperature 1, 5 or 10 vg/ml of cycloheximide in medium was added to L-cell cultures. Controls received medium without cyclohexinide. Twenty-four hours later supernatant fluids were collected, dialysed and assayed for interferon. Table 10 shows that 10 and

5 Pg/ml of cycloheximide inhibited interferon production while 1 pg/tul did not.




50







TABLE 10

Effect of Cycloheximide on Interferon Production



Cycloheximide % Inhibition
(pg/ml) of Interferon Yield


1 0

5 72

10 99





51



Therefore, 1 vg/mi of cyclobeximide was employed in the following experiment. Cell cultures were infected with NDV. After adsorption of the virus, cultures were washed and refed with 20 pg/ml of retinoic acid in medium with or without cycloheximide. Controls received 1% DMSO in medium with or without cycloheximide. Twentyfour hours later, supernatant fluids were collected, dialysed and assayed for interferon. Table 11 shows that the suppressive effect of retinoic acid on interferon production was completely reversed by cycloheximide. This data suggests that retinoic acid acts indirectly through production of a protein which inhibits interferon production. Kinetics of Interferon Action in the Presence of Retinoic Acid.

The above data indicates that retinoic acid induces a regulatory protein. One wonders if this protein represses all inducible cell functions. Interferon is thought to inhibit virus replication by inducing the formation of a new protein, the antiviral protein. 30,31,38 If the retinoic acid induced protein non-specifically represses all inducible cell functions, one would expect it, under conditions of suppression of interferon production, to inhibit induction of the antiviral protein in the presence of retinoic acid.

Monolayer cultures were treated for varying periods of time with

4 or 16 PDD50 units/ml of mouse interferon in MEM with 10% CS containing either 20 Vg/ml of retinoic acid or 1% DMSO. Controls received retinoic acid or DNSO in medium. After interferon treatment, cultures were washed 3 times and immediately infected with about 150 PFU of VSV. Forty-eight hours later cultures were stained, virus plaques enumerated





52






TABLE 11

Effect of Cycloheximide on Suppression of
Interferon Production by Retinoic Acid



Treatment Interferon Yield % Inhibition of
PDD50 units/ml Interferon Yield



Retinoic Acid (20 pg/ml) 2,800 75

Retinoic Acid (20 pg/ml) 11,200 0
& cycloheximide (1 pg/ml) DMSO (1%) & cycloheximide 11,200 0
(1 Pg/ml)

DMSO (1%) 11,200 0





53



and the percentage inhibition of the control plaque number determined. Retinoic acid caused no difference in the rate of development of interferon induced viral resistance when compared to controls (Figure 10). This data indicates that the proposed repressor protein induced by retinoic acid did not inhibit interferon induction of the antiviral protein.


































Figure 10. Kinetics of interferon action in the presence of retinoic
acid. Monolayer cultures were treated for the time
intervals indicated with either 4 or 16 PDD50 units/ml
of interferon in MEM with 10% CS containing either
20 pg/ml of retinoic acid of 1% DMSO. At the indicated
times triplicate cultures were washed 3 times and
infected with about 150 PFU of VSV as described
in Materials and Methods.





55














100



90

A0
800



z 70
0



600


(Y 0
< 50



> 40

z ~PDDUNITS/mI of INTERFERON
0
1-30- 4 16
CORETINOIC ACID (2Ojug/mI) A A
z

--020- DMSO (1%) 0o


0
10




0 2 4 6 8 10 24
TREATMENT WITH INTERFERON & RETINOIC ACID OR DMSO(HRS)





56





Structural Requirements of Vitamin A For Suppression

of Interferon Production and Inhibition of Its Action


Different forms of vitamin A and related compounds dissolved in DMSO were tested to determine their effect on the antiviral activity of interferon. Retinoic acid, retinol, retinal, retinyl acetate, vitamin KI, B-carotene and citronellol were each made 6.7 x 10-5 M in an interferon preparation diluted 1 to 10 in HEM. Controls received an equivalent amount of DMSO (1%). After 24 hours at 37*C, the mixtures were further diluted 1 to 100 in MEM with 5% CS and assayed for residual interferon activity. Table 12 shows that retinoic acid and retinol were similarly effective at inhibiting interferon activity while retinal and retinyl acetate were considerably less inhibitory. This data suggested that the terminal group on the vitamin A molecule was important for the inhibitory effect on interferon activity. This concept is supported by the low level of inhibitory activity of s-carotene on interferon action. s-carotene is a dimer of vitamin A with a ring on both ends of the molecule. Since s-carotene was not very inhibitory for interferon action, it seems that the ring portion of the vitamin A molecule was not of much importance in the inhibition of interferon action. Furthermore, citronellol, an analogue of the side chain of retinol, was not inhibitory, suggesting that the conjugated double bond system was also important in the interaction of vitamin A with the interferon molecule. Another fat soluble vitamin, vitamin K., did not inhibit interferon activity.






57



T X'L!,' 12



Structural l'equL-emcuEs of Vitamin A L'or SuDoresslon

of Interferon Production and Inhibition of Its Action




% SUPPRESSION
OFINTERFERON

COMPOUND TESTED ANTIVIRAL PRODUCTION
ACTIVITY

CH3 CH3 CH3 COOH

63 68
6e 3 RETINOIC ACID
CH3

CH3 CH3 CH3

(: CH201-1
CH
rW3
3 78 47
CH3 RETINOL

CH3 CH3 CH3 CHO

6H 20 68
R E Tl N A L
CH3
0


a H3 H3 H3 1
CH3 "I 1 1 1 1 -O-C21-15 7 40
RETINAL ACETATE
CH3

0 CH, CH3 CH, CH3
1 19
CH3
CH3 VITAMINKI
0 CH3 CH3
CH3 CH3 CH3 24 57
& CH3 CH,3 CH, CH3
CH3 .8-CA90TENE

CH-5 CHA
CH20H
1-11 0 0
CH3 CITRONELLOL


DMSO 0 0
(CONTROL)





58



The same compounds were tested for their effect on interferon production. NDV was adsorbed to L-929 cells for 1 hour at room temperature. Cell cultures were then washed and received either retinoic acid, retinol, retinal, retinyl acetate, vitamin K1, 0-carotene or citronellol at 6.7 x 10-5 M in MEM with 10% CS. Ten percent CS was employed because vitamin A does not inhibit interferon activity under these conditions. In contrast to the marked dependence on the form of vitamin A required for inhibition of interferon action, all forms of vitamin A tested suppressed interferon production (Table 12). This observation suggested that the ring portion of the vitamin A molecule was of primary importance for the suppression of interferon production. That s-carotene, a dimer of vitamin A with a ring group at each end of the molecule, was suppressive for interferon production is consistent with this idea. Also, the lack of a suppression by citronellol, an analogue of the side chain of retinol, supports this concept. Vitamin K1 was also not very suppressive.









DISCUSSION


As we previously reported for human interferon, 32 retinoic acid was shown to inhibit the action of mouse interferon. 39 The inhibition of antiviral activity,which was observed when retinoic acid was mixed with interferon and immediately assayed for interferon activity, apparently resulted from an interaction of retinoic acid with the interferon molecule. This conclusion is supported by the observation that treatment of interferon with retinoic acid and incubation in the absence of cells resulted in a loss of interferon activity when subsequently measured after sufficient dilution to reduce the concentration of retinoic acid to a level which had no effect in the assay. In addition, the inhibitory effect of retinoic acid on interferon activity was prevented by calf serum or bovine serum albumin.

The loss of interferon activity following addition of retinoic

acid was characterized by a dependence on time and temperature. Temperatures above 25C were required for a pronounced inhibitory effect.

Recent reports have shown that human interferon has hydrophobic

binding sites. 40,41,42 These reports have indicated that there is no hydrophobic interaction between mouse interferon with w-carboxypentyl
41
agarose and only slight retention of mouse interferon on albumin immobilized on agarose. 42 Our data is consistent with the concept of a hydrophobic interaction between retinoic acid and interferon. Davey et al. 41 reported that the binding of human interferon to hydrophobic hydrocarbon arms covalently linked to Sepharose was critically dependent on the hydyophilic head group of the hydrocarbon. We have also observed a critical de :edence on the character of the terminal





59





60



group of vitamin A for the inhibitory effect on interferon. Forms of vitamin A with carboxy or hydroxy group.- were effective at inhibiting interferon while vitamin A forms with carbonyl or acetate ester groups were essentially ineffective. Considering the high affinity hydrophobic binding sites on bovine serum albumin, 43 the calf serum dependent prevention of the inhibitory effect of retinoic acid onl interferon probably results from competitive binding of the retinoic acid to albumin and other serum proteins. Bovine serum albumin can be substituted for calf serum in preventing the effect of retinoic acid on interferon.

We have assumed that serum or bovine serum albumin interacts with vitamin A to prevent its binding to interferon. It is possible, of course, that serum interacts with interferon to prevent its binding to vitamin A. However, considering the high affinity that albumin has for fatty acids 43and the low affinity for mouse interferon, 42 it is more likely that serum interacts with vitamin A to prevent vitamin A from binding to int-erferon.

If retinoic acid exerts its inhibitory effect by binding to hydrophobic regions on interferon molecules, it would seem this binding must be of relatively high affinity as compared with calf serum since the molar concentration of protein in serum should be considerably higher than the molar concentration of interferon. Since mouse interferon seems to have hydrophobic regions, this data suggested that mouse interferon might be purified by hydrophobic chromatography. Unfortunately, there are no means by which to couple the ring portion of the vitamin A molecule to a solid matrix. Coupling by the ring, portion would seem





61




essential since the side chain and hydrophilic end of the vitamin A molecule appears to be most important in the interaction with the interferon molecule. In fact, interferon did not bind to retinoic acid coupled by its carboxy group to Sepharose. Recently, however, mouse interferon has been greatly purified by hydrophobic chromatography. 4

We have also shown a suppressive effect of vitamin A on interferon production. 45Our in vitro suppression is in agreement with a report of an in vivo suppression of interferon production by vitamin A. 46 Based on our previous findings the first possible explanation was that the suppressive effect might result from inactivation of interferon by vitamin A after it was produced. This was eliminated by several lines of evidence: removal of retinoic acid after 2 hours did not cause any less suppression than its continued presence throughout the interferon production period (24 hours); interferon production was suppressed in a calf serum concentration (10%) in which 20 iig/ml of retinoic acid does not inactivate interferon activity; and a form of vitamin A (retinal) which was very effective at suppressing interferon production was ineffective at inactivating interferon activity. The enhanced suppression of interferon production observed in 3% CS, however, might be accounted for by inactivation of interferon by retinoic acid.

A second possible explanation was that the suppressed production resulted from non-specific toxicity. This possibility was eliminated since in 10% CS the concentration of vitamin A employed was non-toxic as measured by: cell number; cell viability as determined by trypan blue dye exclusion; plaque forming ability of VSV; 3H-uridine incorporation; and the ability of cells to proliferate in vitamin, A.




62




We have found that retinoic acid treatment reduced the amount of interferon recovered from lysed NDV infected cells and that the ratio of extracellular/intracellular interferon is apparently 6 in both control and retinoic acid treated cultures 24 hours after NDV infection. This data indicates that retinoic acid does not impede the release of interferon from the synthesizing cells.

Unimpaired protein synthesis is, of course, a prerequisite to

maximal interferon production. Known inhibitors of protein synthesis, such as puromycin and p-fluorophenylalanine, are suppressive throughout the period of interferon production. 47 If retinoic acid acted by inhibition of protein synthesis it should also be active throughout the cycle of interferon production. Contrary to this, we have shown that addition of retinoic acid 8 hours after interferon induction, the time at which interferon is first detectable, did not suppress the final yield of interferon. Furthermore, it seems unlikely that vitamin A acted by inhibition of protein synthesis since cells can proliferate in 20 hg/ml of retinoic acid and VSV and SFV can grow normally in vitamin A treated cells.

This work also demonstrates that vitamin A suppressed an early event in the production of interferon. The earliest event in the production cycle of interferon isthought to be the generation of the
35
inducer, double stranded RNA. However, retinoic acid inhibited

interferon production in resonse to SFV without inhibiting the yield of virus. Since double stranded RNA is a by product of virus replication and virus replication was not inhibited, it seems improbable that retinoic acid interfered with the generation of the inducer.





63



Furthermore, retinoic acid suppressed poly I:C induced interferon production. Since poiy I:C is a synthetic double stranded RNA molecule, the interferon inducer had been formed prior to interaction with the cell and would, therefore, preclude interference with generation of the inducer.

Specific m-RNA synthesis must also be an early event in the production of interferon. There are several reports concerning the effects of adding actinomycin D at different times after induction of interferon synthesis and these have shown that interferon messenger RNA synthesis is completed within a few hours following virus infection. 49,50,51 Although total RNA synthesis was not altered in our system, the kinetics of the suppressive effect of vitamin A on interferon production are consistent with the suppression of interferon messenger RNA synthesis since the maximum effect occurred about 2 hours after adsorption of NIW or addition of poly T:C and the kinetics of the effect are very similar to those seen with actinomycin D. 49If only a few messenger RNA species were suppressed, one would not detect a difference in total RNA. This would be an important difference between the two systems (actinomycin D vs. vitamin A), since actinomycin D caused a marked inhibition of total RNIA synthesis while vitamin A did not.

Vi1cek and Ng 52have postulated that interferon synthesis in

rabbit kidney cells is controlled by a cellular repressor. Their conclusi-on -is based on the increased yield of interferon in the continued presence of low concentrations of cycloheximide. This data -indicated a preferential inhibition of the repressor of interferon production by cyclobexitide. If rabbit Cultures induced to synthesize interferon





64



and exposed to cycloheximide for 4 hours were then treated with actinomycirn D and the cycloheximide block reversed, an even larger increase in interferon yield resulted, termed superinduction. It was virtually impossible that further synthesis of interferon m-RNA could have occurred after treatment with actinomycin D (5 jig/ml). Actinomycin D, therefore. inhibited a cellular function which otherwise would have prematurely terminated the translation of interferon m-RNA. Hence, the repressor apparently acted at the post-transcriptional level. We have observed that interferon production by L-cells is resistant to

1 jig/mil of cvcloheximide. This finding is not surprising since interferon is induced by double stranded RNA which itself inhibits protein synthesis. 53 When cell cultures were treated with this concentration of cycloheximide and with retinoic acid (20 jig/ml) there was no suppression of interferon production by the vitamin, whereas vitamin A treatment alone caused a 75% inhibition of interferon yield. Although other interpretations are possible, one explanation of the data is that retinoic acid indirectly suppressed interferon production through induction of a new protein. The synthesis of this protein must, therefore, be more sensitive to cvcloheximide than is interferon synthesis. If retinoic acid had only increased the speed or amount of production of a protein already present or normally induced, one would have expected cycloheximide alone to have increased

the yield of interferon as compared to the control. Whether this postulated repressor acts at the transcriptional or post-transcriptional level is not presently known. However. the kinetics of the suppressive effect of retinoic acid suggests that transcription of the m-RNA for





65




interferon is suppressed. This idea is based on the finding that addition of retinoic acid at 8 hours after interferon induction did not suppress interferon production. Even considering a possible 2 hour requirement for the maximum suppressive effect of retinoic acid, this would be only 10 hours into the interferon production cycle. Ten hours post induction is well before the bulk of the interferon has been synthesized. Since interferon release into the culture fluid very closely follows translation of the interferon messenger RNA, 54this data indicates that translation is not suppressed.

Interferon acts through the induction of a new protein, the antiviral protein. 30,31,38 The postulated vitamin A induced repressor

is probably somewhat specific in its action since retinoic acid did not interfere with the generation of the antiviral state in cells treated with interferon. Furthermore, vitamin A did not suppress SFV replication which also requires new protein synthesis. It is tempting to speculate

the vitamin A may induce a regulatory protein which specifically suppresses induction of certain messenger RNAs, one of which is the interferon messenger RNA.

Numerous studies of the action of vitamin A have yielded a wealth of information. For example, vitamin A can: Stimulate growth of chick heart fibroblasts; 55alter differentiation of epithelial ,cells; suppress induction and growth of tumors in response to Chemical carcinc- ris; 6, and alter gl1ycoprotein synthesis. 27,28 Although all of these studies indicate a role for vitamin A in gene expression, none of them have shown a specific site of action. An





66




intracellular retinoic acid binding protein has recently been discovered 56,57 and has led to speculation that retinoic acid may bind to this protein and act directly at the gene level, similar to a steroid-steroid receptor complex. Our data suggests that the site of action of vitamin A is at the gene level since it appears that interferon production is suppressed at the transcriptional level by a vitamin A induced protein. Interestingly, superinduction of interferon has only been reported in primary (normal) cell cultures. 52,58,59 Superinduction by cycloheximide and actinomycin D might, therefore, be thought of as resulting from a block in a control mechanism for interferon which is operational in normal cells. We have not observed superinduction in L-cells (transformed cells) which implies they either do not have or do not express the normal regulatory mechanism for interferon production. If this is the case, L-cells, once induced, produce a maximal amount of interferon whereas normal cells do not. The suppression of interferon production by vitamin A might then be interpreted as a restoration of regulation of interferon production with the 10 to 25% of interferon which is not suppressed representing the aMOUnt produced by normal cells. The vitamin A treated cell would, in a sense, be normal and the cycloheximide block of the vitamin A induced suppression of interferon production could be interpreted as superinduction. In other words, vitamin A might

restore control of the production of a r_ gulatory protein for interferon production. Supportive of this hypothesis is the observation that L-cells, since they are transformed, have lost control of proliferation and vitamin A can apparently restore control of their





67



proliferation. 9 Hence, we wish to propose that vitamin A controls, or is necessary for regulation o inducible. gene expression and that control of the production of interferon is one manifestation of this potential. The system might, thereforeprovide a model for the study of the action of vitamin A at the molecular level.

Finally, these results have illustrated that vitamin A can suppress both interferon action and production. The inhibitory effects, however, appear to result from different mechanisms. The inhibition of interferon activity by vitamin A seems to be due to an effect of vitamin A on the interferon molecule, while the suppressive effect on interferon production is clearly due to an effect of vitamin A on the cell which has been induced to make interferon. It is interesting that the different mechanisms of action of vitamin A on interferon action and production apparently occur by different moieties of the vitamin A molecule, 60 Inhibition of interferon action seems to be most dependent on the side chain of the vitamin A molecule. The side chain apparently requires a conjugated double bond system with a hydrophilic terminal group (hydroxy group for retinol and carboxy group for retinoic acid) to inhibit interferon action. In contrast, the ring portion of the vitamin A molecule seems to be most important for the suppression of interferon production.










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11. Wald, C. 1968. The molecular basis of visual excitation.
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23. Levi, A.S. and Wolf, G. 1969. Purification and properties of
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28. DeLuca, L. and Yuspa, S.H. 1974. Altered glycoprotein synthesis
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29. Burke, D.C. 1966. "Mechanisms of interferon production."
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31. Taylor, J. 1965. Studies on the mechanism of action of
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32. Blalock, J.E. and Gifford, G.E. 1974. Effect of Aquasol A,
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33. Lindenmann, J. and Gifford, G.E. 1963. Studies on vaccinia
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34. Viliek, J. and Lowy, D.R. 1967. Interaction of interferon with
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37. Vilcek, J. 1970. Metabolic determinants in the induction of
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38. Baron, S., Buckler, C.E., Friedman, R.M. and McCloskey, R.V.
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40. Davey, M.W., Huang, J.W., Sulkowski, E. and Carter, W.A. 1974.
Hydrophobic interaction of human interferon with concanavalin
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41. Davey, M.W., Huang, J.W., Sulkowski, E. and Carter, W.A. 1975.
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42. Huang, J.W., Davey, M.W., Hejna, C.J., Von Muenchhausen, W.,
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43. Jonas, A. and Weber, G. 1971. Presence of arginine residues at
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44. Davey, M.W., Sulkowski, E. and Carter, W.A. 1976. Purification
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45. Blalock, J.W. and Gifford, G.E. 1976. Suppression of interferon
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46. Tokumaru, T. 1967. The effect of trauma on production of
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47. Buchan, A. and Burke, D.C. 1966. Interferon production in chick
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48. Ho, M. and Armstrong, J.A. 1975. interferon. Ann. Rev.
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49. Wagner, R.R. and Huang, A.S. 1965. Reversible inhibition of
interferon synthesis by puromycin. Evidence for an interferonspecific messenger RNA. Proc. Nat. Acad. Sci. U.S.A. 54:1112.

50. Levy, H.B., Axelrod, D. and Bar)n, S. 1965. Messenger RNA for
interferon production. Proc. Soc. Exp. Biol. Med, 118:384.

51. Ho, M. and Breinig, M.K. 1965. Metabolic determinants of interferon formation. Virology 25:331.

52. Vildek, J. and Ng, M.H. 1971. Post-transcriptional control
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53. Ehrenfeld, E. and Hunt, T. 1971. Double-stranded PNA inhibits
initiation of protein synthesis by reticulocyte lysates. Proc.
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54. Reynolds, F.H., Jr. and Pitha, P. 1974. The induction of interferon
and its messenger RNA in human fibroblasts. Biochem. Biophys.
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55. Lasnitzki, I. 1955. The effect of excess vitamin A on mitosis
in chick heart fibroblasts in vitro. Exp. Cell Res. 8:121.

56. Sani, B.P. and Hill, D.L. 1975. A retinoic acid-binding protein
from chick embryo skin. Cancer Res. 36:404.

57. Ong, D.E., Page. D.L. and Chytil, F. 1975. Retinoic acid-binding
protein: Occurrence in human tumors. Science 190:60.

58. Tan, Y.H., Armstrong, J.A., Ke, Y.H., and Ho, M. 1970.
Regulation of cellular interferon production: Enhancement by
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59. Havell, E.A. and Vilcek, J. 1972, Production of high-titered
interferon in cultures of human diploid cells. Antimicrob.
Agents Chemother. 2:476.

60. Blalock, J.E. and Gifford, G.E. 1976. Comparison of the
suppression of interferon production and inhibition of its action by vitamin A and related compounds. Proc. Soc. Exp.
Biol. Med. Submitted for publication









BIOGRAPH IICAL S1KLTC1I


fan~os Edwin Blalock was born in Madison, Florida, on Scptember 29, 1949. fie, attended the public ;choois in Madison County and graduated from Madison High School in June, 1967. From September, 1967, to June, 1969, he attended North Florida Junior College, Madison, Florida and received an Associate of Arts degree m gpna cum laude in June, 1969. The remainder of his undergraduate work was performed at the University of Florida, Gainesville, Florida where he was awarded a Bachelor of Science degree in December, 1971, with a major in Microbiology. From January, 1972, until the present time he has pursued the degree of Doctor of Philosophy in the Department of Immunology and Medical Microbiology, College of Medicine, University of Florida. During this time his research received First Place among Junior Investigators at the 197.5 meeting of the Southeastern Section of the Society for Experimental Biology and Medicine at Duke University, Durham, North Carolina. In June, 1976, he received the annual Graduate Student Research Award from the University of Florida Chapter of the Society of the Sig-za Xi. lie was supported by N111 Training Grant 5TI AT-0128 and NTH Research Grant AT-10900

during his graduate training.

Following his graduation from the Unive~rsity of Florida in June, 1976, he accepted a position as a postdoctoral fellow in the laboratory of Dr. Samuel Baron, Chairman of the Department of Microbiology at the

Medical Branch o[ the University of Texas at Galveston, Texas.












73





74



As a result of his research while a graduate student at the University

of Ficrida, J.E. Blalock published or was in the process of publishing

at the time of graduation the following papers,


1. Blalock, J.E. and Gifford, G.E. 1974. Effect of Aquasol A, vitamin
A and Tween 80 on vesicular stomatitis virus plaque formation and on interferon action. Arch. ges. Virusforsch. 45:161-164.

2. Blalock, J.E., Tullish, J. and Gifford, G.E. 1975. Effect of Tween
80 and Aquasol A on virus plaque formation. Inf. and Imm.
12:490-494.

3. Blalock, J.E. and Gifford, G.E. 1975. Inhibition of interferon
action by vitamin A. J. Gen. Virol. 29:315-324.

4. Blalock, J.E. and Gifford, G.E. 1976. Suppression of interferon
production by vitamin A. J. Gen. Virol. in press.

5. Blalock, J.E. and Gifford, G.E. 1976. Comparison of the suppression
of interferon production and inhibition of its action by
vitamin A and related compounds. Proc. Soc. Exp. Biol. Med.
submitted for publication.

6. Dion, L.D., Blalock, J.E. and Gifford, G.E. 1976. Vitamin A
induced density dependent inhibition of L-cell proliferation.
J. Natl. Can. Inst. submitted for publication.

7. Blalock, J.E. and Gifford, G.E. Effect of vitamin A and a nonionic
surface active agenu oan vesicular scomaritis virus plaque formation. Abs. Ann. Meeting Amer. Soc. Microbiol., 1973, p. 205.

8. Blalock, J.E. Effect of vitamin A and a nonionic surface active
agent on virus replication and on interferon action. Abs.
Southeastern Sectional Meeting Society for Experimental Biology
and Medicine, 1973, p. 16.

9. Blalock, J.E. Suppression of interferon production by vitamin A.
Abs. Southeastern Sectional Meeting Society for Experimental
Biology and Medicine, 1975.











1 certify that I have read this Study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



16eorge9. Gifford, Ph.D., Chairman Professor of Immunology and Medical Microbiology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Joeph W. Shands, Jr.,MD ofessor of Immunology and Medical Microbiology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertati gi for the degree of Doctor of Philosophy.



L. William Clem, Ph.D. Professor of Immunology and Medical Microbiology



I certify that I have read this Study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Donna It. Duckworth, Ph.D. Associate Professor of Immunology and Medical Microbiology










I certify that I have read this study and that in my opinion it
conforna to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Kenneth Ley, Ph.D., a t
Professor of Veterinary Science


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.



Paul A. Klein, Ph.D., Associate Professor of Pathology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is tully adequate, in scope and quality, as a dissertation for the deg Doctor of Philosophy.



v, Parker A. S6 7M.D/
Professor and Interim Cl irman Immunology and Medical Microbiology


This dissertation was submitted to the Dean of the College of Medicine and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor Philosophy.

June, 1976



Dean, lege of Medicine






Dean, Graduate School