EFFECT OF TRIIODOTHYRONINE ON CELLS AND ON THEIRRESPONSE TO INFECTION BY POLIOVIRUSES'

WILLIAM H. MURPHY AND CORA BULLIS

Department of Bacteriology, The University of Michigan, Ann Arbor, Michigan

Received for publication October 5, 1961

ABSTRACTMURPHY, W. H. (The University of Michigan,

Ann Arbor) AND CORA BULLIS. Effect of tri-iodothyronine on cells and on their response toinfection by polioviruses. J. Bacteriol. 83:641-648. 1962.-An analysis was made of the effectof triiodothyronine (T3) at physiological (1 Ag/ml) and maximal subliminal toxic levels (35,ug/ml) on HeLa-S3, HeLa-Gey, Chang-liver,and Maben cells, and on their response to infec-tion by cytopathic and submoderate (noncyto-pathic) mutants of type 2 poliovirus. Assays ofcell response to T3 alone, or in combination withthe mutants of poliovirus, were made by con-ventional monolayer cell culture techniques, bystudy of the effect of T3 on plating efficiency ofcells, and by study of its influence on colonies ofcell variants. Cellular response to liminal dosesof T3 was characterized by agglutination of cellsand thickening of the cell membrane. Compactcolonies of Chang-liver and Maben cells were themost sensitive to maximal subliminal amounts ofT3. T3 in combination with cytopathic or sub-moderate (noncytopathic) mutants of poliovirusslightly increased the rate of destruction of cellssusceptible to virus, but did not influence yieldof virus from cell cultures. T3 at physiological orsubliminal concentrations did not induce cyto-pathic response of cell cultures latently infectedby submoderate poliovirus.

The balance of hormones in the living hostregulates resistance to infection as well as otheraspects of the physiopathology of disease (Longand Shewell, 1955; Lurie and Ninos, 1956;Murphy, Wiens, and Watson, 1958). The thyroidhormones were studied particularly in this regardbecause of their systemic effects and influence

' These studies were taken from the thesis sub-mitted to the University of Michigan by CoraBullis in partial fulfillment of the requirements forthe M.S. degree.

on metabolism at the cellular level. Investigationsof the effect in vitro of thyroid hormones on cellsin culture were reported for established cellstrains (Lieberman and Ove, 1959; Leslie andSinclair, 1959; Halevy and Avivi, 1958; VonHaam and Cappel, 1940). Eaton and Scala (1957)analyzed the effect of thyroid hormones on theresponse of fibroblasts from chicken embryos toinfection by influenza virus. The biphasic effectof thyroxine on replication of influenza virus wasmanifested by an increased yield of virus duringthe first 24 hr after infection, followed by suppres-sion of virus multiplication. Although the effectsof a variety of hormones on the replication ofviruses in cell cultures were reported (Kilbourne,1957; Likar and Wilson, 1959; Stewart, 1960),attempts to induce with steroid hormones acytopathic response of HeLa cells to infection byhepatitis viruses thought to be present in clinicalmaterials (Franklin, 1960) were not successful.Because the delicate balance between cytopathicand noncytopathic response of cell cultures toinfection by submoderate poliovirus (Murphyand Armstrong, 1959; Murphy and Landau,in press) could be exploited, a study was made ofthe influence of L-3,3', 5-triiodothyronine (T3)on cellular response to infection. T3 was usedbecause it is more active than thyroxine (Barker,1956; Michel and Pitt-Rivers, 1957) on cellmetabolism and membrane permeability(Lehninger and Ray, 1957; Tapley and Cooper,1956).

MATERIALS AND METHODS

Cell strains and media. The origin of the strainsof HeLa-S3, HeLa-Gey, Chang-liver, and Mabencells was reported previously (Murphy andWisner, 1962). The growth medium used forthe propagation of cells consisted of Eaglemedium (80%) and human serum (20%). Culturefluids were adjusted with 1.4% NaHCO3 solutionto a pH of 7.4 to 7.6 and maintained at this levelby addition of a feeding solution (Murphy and

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Wisner, 1962). The maintenance inedium (Mur-phy and Wisner, 1962) used to sustain the cellsconsisted of: Hanks balanced salt solution (BSS),72.5 ml; 5% lactalbumin hydrolysate, 10 ml;1 % yeast extract, 10 ml; 4.4% NaHCO3 solu-tion, 2.0 ml; 10% glucose solution, 2.5 ml; 1ml of a solution containing 0.128 g of sodiumpyruvate and 0.226 g of sodium acetate per 100ml of BSS; and calf serum, 3.0 ml. None of thecell culture additives contained antibiotics.

T3, obtained from Smith, Kline, and French,or Mann Research Laboratories, was dissolvedin 20-mg amounts in 1.3 ml of carbonate-free0.05 N NaOH solution. T3 preparations were

autoclaved at 121 C for 15 min and stored at4 C until used; fresh solutions were made weekly.

Effect of TS on morphology of cells in monolayerculture. Growth medium was decanted frommonolayer cultures (Murphy and Wisner, 1962),the cells washed three times with the bufferedsalt solution (GKN) described by Merchant,Kahn, and Murphy (1960), and graded con-

centrations of T3 in maintenance medium added.Five replicate culture tubes were used for eachconcentration of T3: 0.1, 1.0, 10, 20, 30, 40, 50,and 60 /Ag/ml; five control tubes containedmaintenance medium free of T3. Cell cultureswere incubated at 37 C, examined daily for 7days, and the rate of change in cell morphologyrecorded.

Effect of TS on colonies of cells. The procedureused to plate cells was previously described(Murphy and Wisner, 1962). Cell colonies were

washed three times with GKN, and dilutions ofT3 in maintenance medium were added. Fivereplicate cultures were employed for each dose ofT3; five control cultures contained maintenance

medium without T3. After addition of T3,cultures were incubated at 37 C for 8 days. Arecord was kept of the rate of destruction by T3of compact and diffuse cell colonies (Murphyand Wisner, 1962).

Effect of TS on plating efficiency of cells. Cellswere plated as described above. T3 at concentra-tions of 1 or 35 ,ug/ml was added in growthmedium to each test series of five culture vessels;five cultures containing growth medium aloneserved as controls. After 10 days, cell colonieswere classified, counted, and plating efficiencvof cells calculated.

Polioviruses. The origin (Murphy and Arm-strong, 1959) and characteristics of the cytopathicand submoderate mutants of type 2 polioviruswere reported (Murphy and Wisner, 1962).

Effect of T3 on the morphological response ofcells in monolayer culture to infection by polio-viruses. For each part of each experiment foreach cell strain, five cultures were used: main-tenance medium (cell control); submoderatepoliovirus (control); cytopathic poliovirus (con-trol); 1 ,ug/ml of T3 (control); 35 ,ug/ml of T3(control); 1 ,ug/ml of T3 plus submoderatepoliovirus; and 35 ,ug/ml of T3 plus cytopathicpoliovirus. Cultures were incubated for 7 to14 days at 37 C and examined daily to detectchanges.

Effect of T3 on response of colonies of cells toinfection by polioviruses. Each of the virus strainswas used in the same concentrations and com-

binations with T3 as listed above for monolayercultures. The number of surviving compact anddiffuse cell colonies was enumerated during an

8-day period of observation. Routine procedureswere used (Murphy and Wisner, 1962) to titrate

TABLE 1. Morphological response of cells in monolayer culture to triiodothyronine (TS)

Cell strainConcn of T3* (pg/ml)

0 0.1 1.0 10 20 30 40 50 60

HeLa-S3.......... 0 O O O ot Ot 2+ 3+HeLa-Gey ........ 0 0 0 0 0 0 0 2+ 4+Chang-liver ....... 0 0 0 0 0 0 1+ 2+ 3+Maben........... . 0 0 0 0 0 0 1+ 3+ 4+

* Five culture tubes were used at each concentration of T3; experiments were repeated three timesfor each cell strain. Morphological changes were scored as follows: 0 = cells unchanged compared tocontrols; 1+ = rounding and distortion of the shape of ca. 25% of the cells; 2+ = rounding and dis-tortion of all cells, with detachment of ca. 50% of the cell sheet; 3+ = detachment of ca. 75% of thecell monolayer; 4+ = death and detachment of all cells.

t Cultures were more acid than controls (in each cell strain).

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TRIIODOTHYRONINE AND POLIOVIRUS INFECTION

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FIG. 1. Normal HeLa-Gey cells (magnification 100 X, hematoxylin and eosin stain).FIG. 2. HeLa-Gey cells 48 hr after exposure to 60 ,g/ml of triiodothyronine (T3). Although contraction

and agglutination of cells occurred with all strains, it was most pronounced with the HeLa-Gey cells (magnifi-cation 100 X; hematoxylin and eosin stain). Rate of response was a function of the time of exposure anddose of T3 used.

FIG. 3. Normal Maben cells (magnification 400 X; hematoxylin and eosin stain).FIG. 4. Cytoplasmic contraction and thickening typical of cellular response to toxic doses of TS (Maben

cells 48 hr after exposure to 0 ,ug/ml of T8, magnification 400 X; hematoxylin-eosin stain).

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MURPHY AND BULLIS

TABLE 2. Effect of triiodothyronine (TS) on coloniesof cell variants

Per cent loss* ofcell colonies

Cell strains Agents employed

Compact Diffuse

HeLa-S3 Cell control 0 4± 4 0 ± 11T3, 1/Ag/ml 5 4 6 0 4 12T3, 35,ug/ml 23 i 16 0± 7

HeLa-Gey Cell control 0 i 22 0 4 11T3, 1 ug/ml 10 i 16 6 ± 13T3, 35 ug/ml 7 i 18 15 A 17

Chang-liver Cell control 0 i 15 0 i 15T3,1 ug/ml 0± 10 29 4 7T3, 35,pg/ml 1 ± 12 26 + 22

Maben Cell control 0 ±4 15 0 ±4 9T3 1 jug/ml 21 ± llt 13 ± 8T3, 35,g/ml 23 4 lOt 22 + 12

* Number of surviving colonies compared withthe initial number.

t P value of <0.05 by Student's t-test of com-bined results of three separate experiments.

viruses (Reed and Muench, 1938) and prepareLeighton tube cultures.

RESULTS

Effect of TS on cells in monolayer culture. Todetermine the liminal dose of T3 for the cellstrains, graded amounts were added to culturesin tubes and compared with cultures containingmaintenance medium. All cell strains responded(Table 1) similarly to T3. The maximal subtoxic(subliminal) dose was approximately 35 ,g/ml.The Chang and Maben cell strains appeared tobe more sensitive to toxicity of T3 than thestrains of HeLa cells. The metabolism of cellsin tubes that received toxic doses of the hormonewas inhibited. When a range of 0.1 to 10 ,ug/mlof T3 was used, the cells of each strain producedslightly more acid than the controls. Thesefindings coincided with published biochemicalstudies; viz., T3 at physiological levels (10-4 M)increased the metabolic activity of cells in culture(Leslie and Sinclair, 1959; Halevy and Avivi,1958; Von Haam and Cappel, 1940). The toxicityof T3 for cells, although it varied slightly fromone strain to another, was characterized byagglutination of cells (Fig. 1 and 2) and thickening

TABLE 3. Effect of triiodothyronine (T3) on platingefficiency of cells and distribution of variants

Per cent decrease inplating efficiency of cells

Cell strains Agents employed

Compact Diffuse

HeLa-S3 Cell control 0 ± 17 0 ± 19T3, 1,ug/ml 0± 14 4 ± 13T3, 35,ug/ml 10 ±-5 8 ± 6

HeLa-Gey Cell control 0 ± 5 0 ± 7T3,Ijug/ml 3 ± 2 5 ± 5T3, 35 ug/ml 1 ± 1 4 ± 3

Chang-liver Cell control 0 ± 6 0 ± 2T3, 1 ug/ml 3 + 4 0± 10T3, 35,ug/ml 31 ± 4* 0 ± 15

Maben Cell control 0 ± 18 0 ± 6T3, 1,ug/ml 5 ± 11 0± 12T3, 35,ug/ml 11 ± 16 0± 6

* P value of <0.05 by Student's t-test of com-bined results of three separate experiments.

of the cytoplasmic membrane (Fig. 3 and 4).The agglutinative effect occurred in all cellstrains but was most pronounced with the HeLa-Gey line. Contractive processes that appearedto be related causally to the thickening of cellmembranes were most marked in Chang andMaben cells. The majority of cells that de-generated exhibited the usual fibroblastic ap-pearance commonly seen when cells are exposedto toxic substances.

Effect of TS on colonies of cells. T3 was addedto colonies of cells to confirm the subjectiveobservations recorded in Table 1, and to obtainmore precise data on the dose-response of cellsto the drug. Assays were repeated three times;not less than 4,500 colonies were scored for eachcell strain. A concentration of T3 of 1 or 35,ug/mlhad a significant toxic effect only on compactcolonies of the Maben strain (Table 2).

Effect of T3 on plating efficiency of cells. Ex-periments were devised to test whether T3exerted a significant effect on the plating efficiencyof cells. The design of the analysis made itpossible to assess whether the hormone exerted adifferential effect that gave rise to each of thetwo colony types. The techniques outlined abovewere used for these experiments. All experimentswere repeated three times; not less than 3,000

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TABLE 4. Effect of triiodothyronine (T3) on the replication of polioviruses by cells in monolayer culture

Yield (log TcIDs0/ml) of cytopathic poliovirus* Yield (log TCID50/ml) of submoderate poliovirus

Cell strainsVirus control 1Virus plus Virus plus 35 Virus control Virus plus I Virus plus 35VIjg/ml of T3 VAg/ml of T3 Vg/ml of T3 pAg/ml of T3

HeLa-S3........ 8.10 8.0 7.91 4.35 3.63 4.0HeLa-Gey........ 7.74 7.96 8.40 6.11 6.19 6.0Chang-liver........ 8.32 7.64 8.18 3.62 4.19 4.33Maben........ 7.24 7.42 7.30 5.65 5.75 6.09

* Data presented are the composite results from two experiments. Each tube of each test series re-ceived initially 102 plaque-forming units of virus. Differences in the yields of viruses from cell strainswere not statistically significant.

TABLE 5. Effect of triiodothyronine (TS) on morphological response of cells in monolayer culture to infectionby submoderate and cytopathic polioviruses

Agents added*

Cell strains -______-______ ______-___________________T3 (l)t T3 (35) NCPt NCP + T3 NCP + T3 MEF MEF+ T3 MEF + T3 Cell control

(1) (35) (1) (35)

HeLa-S3 ..... 0 0 0 0 0 4+ 4+ 4+ 0HeLa-Gey . 0 0 4+ 4+ 4+ 4+ 4+ 4+ 0Chang-liver. 0 0 0 0 0 4+ 4+ 4+ 0Maben ....... 0 0 2+ 2+ 2+ 4+ 4+ 4+ 0

* Three experiments were done with each cell strain. Cellular response was scored as: 0 = no morpho-logical change compared to controls; 1+ = cellular contraction of ca. 25% of the cells of the monolayer;2+ = contraction, rounding, and detachment of ca. 50% of the cells in the monolayer; 3+ = destructionof ca. 75% of the cells in the monolayer; 4+ = death and detachment of all cells.

t T3 concentrations are given in ,ug/ml.$ Submoderate (NCP) and cytopathic (MEF) variants of type 2 poliovirus were used. Each tube

received an inoculum of 102 plaque-forming units of virus.

colonies of each strain were counted. T3 de-creased the plating efficiency of only the compactcell variants of the Chang strain (Table 3).

Effect of T3 on replication of polioviruses bycells in monolayer culture. The production ofvirus by cells was studied to determine whetherT3 influenced the yield of either the cytopathicor submoderate (noncytopathic) mutants ofpoliovirus. Virus specimens for titration consistedof pooled supernatant fluids from each set oftube cultures exposed to each test virus andgraded amounts of T3. For each cell strain, theyield of virus from cultures exposed to 1 or 35,ug/ml of T3 was compared with untreated con-trols containing virus without T3. The pooledspecimens from each part of each experimentfor each cell line were titrated separately. Eachexperiment was repeated three times. T3 didnot significantly change the yield of viruses frominfected cell cultures (Table 4).

Effect of T3 on morphological response of cellsin monolayer culture to polioviruses. Morphologicalstudies of the effect on each cell strain of thetwo viruses alone and in combination with T3were carried out in parallel in an attempt toprovide evidence for the induction of submoderatevirus cytopathic effect.The results (Table 5) showed that HeLa-S3

and Chang cells remained resistant to thecytopathicity of submoderate poliovirus (Murphyand Armstrong, 1959). In one experiment, anattempt was made to overcome the resistanceof Chang cells to infection by submoderatepoliovirus by increasing 100-fold the virusinoculum. At the higher concentration, approxi-mately 20%O of the monolayer was destroyed;however, the effect of virus and T3 was similarto that of virus alone. The morphological ap-pearance of cells infected with polioviruses in

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combination with T3 was the same as that forvirus alone.

Effect of T3 on response of colonies of cells toinfection by polioviruses. Colonies of cells were

used to quantitate the combined cytopathiceffect of test viruses and T3 and to test whetherT3 changed the rate of destruction of cell colonies.Experimental design made it possible to detect

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FIG. 5. Effect of triiodothyronine (TS) on rate ofdestruction of colonies of HeLa-SS and HeLa-Geycells by noncytopathic (NCP) and cytopathic (MEF)mutants of type 2 poliovirus. NCP alone, 0; NCPplus TS, A; MEF alone, *; MEF plus 72, A.

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FIG. 6. Effect of triiodothyronine (TS) on rate ofdestruction of colonies of Chang-liver and Mabencells by noncytopathic (NCP) and cytopathic (MEF)mutants of type 2 poliovirus. NCP alone, 0; NCPplus TS, 0; MEF alone, A; MEF plus TS, A.

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A: CELL CONTROL D: NCP-VIRUSB: T3, Igg/ml E: NCP-VIRUS+T3,115g/mlC: T3.35jug/ml F: NCP-VIRUS.+T3.35,ug/mi

I COMPACT COLONIES

0 DIFFUSE COLONIES

FIG. 7. Effect of triiodothyronine (T7) on cell colonies infected with noncytopathic poliovirus (NCP-virus). Any differences noted in the response of cell colonies to NCP-virus were not statistically significant(p >0.05). Since HeLa-Gey cells normally were susceptible to cytopathic effects of NCP-virus, cell destructionoccurred as anticipated (columns D, E, F); T3 did not increase the proportion of Maben cells destroyed byNCP-virus.

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whether the combined effect of T3 and viruseswas manifested by a differential response ofcompact and diffuse colonies to infection. Ex-periments with submoderate and cytopathicpolioviruses were carried out in parallel. Assayswere repeated in triplicate; not less than 3,000colonies were counted per cell strain.

Since the effect of virus-cell interaction of T3at concentrations of 35 and 1 ug/ml was essen-tially the same, only results for T3 at a dose of1 ,ug/ml are recorded (Fig. 5 and 6). Thesefindings show that T3 accelerated only the rateof destruction by cytopathic poliovirus of coloniesof HeLa-S3 cells. T3 did not alter the responseof colonies of cells to infection by submoderatelpoliovirus (Fig. 7). The results of these assays,like those of the preceding experiments, failedto provide evidence for the induction by T3 ofsubmoderate virus cytopathicity.

DISCUSSION

The effects of T3 on lpolpulations of HeLa-S3,HeLa-Gey, Chang-liver, and MAaben cells wereassayed by the conventional use (Merchantet al., 1960) of cells in monolayer culture, byassay of its action on the plating efficiency ofcells, and by study of its influence on coloniesof variants of the wild-type cell p)opulations.T3 at physiological concentration (1 ,ug/ml)failed to influence the morp)hological appearanceof cells. However, the morphological response ofcells in monolayer culture to a liminal (40,ug/ml) toxic dose of T3 was manifested byagglutination of cells and a marked thickeningof the cytoplasmic membrane, which app)earedto result from contraction of the peripheralelements of the cytoplasrn. Wl!hen monolayer eellcultures were employed to assess cellular responseto T3, the results showed that all of the cellstrains reacted similarly. However, by use of a

cell-colony screening technique (Murphy andLandau, 1961) it was possible to show differencesin the response of variants of the wild l)olpula-tions; i.e., comp)act variants of the AMaben strainweere the most sensitive to toxicity of T3. By

al)p)lication of the Marcus and Puck (1959)technique for plating cells, conclusive evidencewas provided that T3 at maximal subliminalconcentration (35 Ag/ml) decreased the platingefficieney of only the compact variants of theChang-liver strain of cells.The possibility of inducing latent virus in-

fection by exposure of cells to T3 was suggestedbv consideration of the mechanisms by which

cellular susceptibility to cytopathic virus infec-tion may be controlled: (i) changes that affectthe cell surface and thus determine whethervirus infectively attaches (McLaren, Holland, andSyverton, 1960), (ii) alterations in the balanceof cell metabolic pathways, (iii) inefficientrecombination between virus nucleic acid andhost genetic elements (Darnell and Sawyer,1960; Brenner, 1959), or (iv) other factors thatgovern cellular response to infection (M\elnicket al., 1957). Because T3 has an effect on themetabolism of cells and on the membranes ofthe cell surface and internal organelles (VonHaam and Cappel, 1940; Emmelot and Brom-bacher, 1957; Lehninger and Ray, 1957; Park,1Ieriwether, and Ilark, 1958; Halevy and Avivi,1958; Leslie and Sinclair, 1959), a study wasmade to determine whether the dual effect ofT3 and submoderate poliovirus induced cyto-pathic response of cells to infection. 'WVhen T3was used at maximal subliminal concentration(35 Ag/ml), it accelerated the rate of destructionof HeLa-S3 cells by cytopathic poliovirus. T3did not change the yield of virus from cells. T3at physiological or subliminal concentrations didinduce cytopathic response of cells normallyresistant to the cytopathic effect of submoderatepoliovirus. Since no attempt was made to testwhether the kinetics of virus replication werealtered by the addition of T3 to cultures, theresults described are not inconsistent with thoseof Eaton and Scala (1957), who reported thatthe addition of thyroid hormones at physiologicallevels to cultures of chicken embryo fibroblastsincreased the yield of influenza virus during theearly stages of v-irus production.

ACKNOWLEDGMENTS

These studies were supported by U. S. PublicHealth Service grant E-2279 and Army ChemicalCorps grant FD-GR-60-5.

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