Interferon Production by Human Cells In Vitro

Transcription

1 APPLIED MICROBIOLOGY, Nov p Copyright 1972 American Society for Microbiology Vol. 24, No. 5 Printed in U.S.A. Interferon Production by Human Cells In Vitro CELSA A. SPINA, R. SHIHMAN CHANG, L. MISHRA, AND H. DEAN GOLDEN Department of Medical Microbiology, University of California, Davis, California Received for publication 7 August 1972 The relative capacity of several types of human cells and tissue to produce interferon was studied. Types of cells and tissue included were fibroblasts from embryos, foreskins, and biopsied skins; amnion cells; peripheral leukocytes; established lymphoid cell lines; established heteroploid cell lines; and chorioamniotic membrane. When Newcastle disease virus was used as the inducer, fibroblasts and amnion cells produced more interferon per 106 cells than leukocytes, lymphoid cells, and heteroploid cells. Only minor variations in interferon-producing capacity were observed among fibroblasts from 36 persons. Culture passage level, cell concentration, and inducer were factors that significantly affected interferon production. One important characteristic of interferon is its host specificity; therefore, exogenous interferon intended for use in man must be produced in human cell cultures. Sources of human cells which can be obtained in large quantities for interferon production are: placentae, embryos, foreskins, skin, peripheral blood leukocytes, and established lines of lymphoid cells and heteroploid epithelium-like cells. These cells are known to release interferon upon proper stimulation (1, 7, 9, 11, 13, 18, 19, 21, 23, 26, 30, 35, 37, 39, 42, 43, 45). Optimal conditions for maximal release of interferon by foreskin fibroblasts, peripheral leukocytes, amniotic membrane, and lymphoid cells in continuous culture have also been described (9, 11, 23, 26, 35, 37, 39, 45). Yet, to the best of our knowledge, there is no publication on a comparative study of the relative efficacy in interferon production by cell types from such a wide variety of sources. If exogenous interferon is to be used in the prophylaxis and treatment of viral diseases in man, a method will need to be found which is economical in bulk production of potent human interferon. Knowledge of the relative capacity of various cells to produce human interferon should contribute toward the development of an economical method of production. MATERIALS AND METHOD Inducers. A strain of Newcastle disease virus (NDV), known to be a potent interferon inducer, was kindly supplied by M. Ho. Pools were prepared by allantoic inoculation of 10-day-old chicken embryos. The virus was stored in samples at below -60 C until needed. Infectivity of two pools were 1 x 107 and 4 x plaque-forming units (PFU) per ml when assayed on chick fibroblast cultures. A sample of Chikungunya virus was supplied by S. Baron. It contained 3 X 107 tissue culture infective doses per ml when tested on primary hamster embryo fibroblasts. Poly I: C (lot K) was obtained from the antiviral program of the National Institutes of Health. Vesicular stomatitis virus (VSV). The Indiana strain of VSV was obtained from Y. C. Zee (Univ. of California, Davis). A large pool was prepared by growing the virus in human fibroblast culture and storing it in small samples at below -60 C. Infectivity of the pool was assayed on human fibroblast culture. Culture medium. Culture medium consisted of 20% fetal calf serum in Eagle minimal essential medium (MEM) supplemented with the nonessential amino acids, penicillin (50 units/ml), and streptomycin (50 ug/ml). Human cell culture. Fibroblasts were harvested from embryos, foreskins, and skin biopsies by trypsinization and propagated as described (16). Amnion cells were processed from placentae delivered by Cesarean section and cultivated by a procedure used routinely in this laboratory (5). Leukocytes were harvested from blood and were grown and established into lines as described (6). Established lines of heteroploid cells used in this study were: the u cell derived from human amnion (28), which was supplied by J. Vilcek; the Chang liver cell (4); and HeLa-S3 obtained from T. T. Puck (29). Interferon induction. In a typical experiment, 0.3 to 0.6 million fibroblast, amnion, or heteroploid cells in 4 ml of medium were added to plastic petri dishes (60 mm in diameter), which were then incubated at 37 C in a humidified, 5% CO2 incubator. When the cells formed a confluent monolayer, two cultures were sacrificed for cell count and two were treated with virus inducer as follows. The spent medium was removed; 4 ml of fresh medium containing approximately 5 PFU of NDV per cell or 0.5 PFU of

2 736 SPINA ET AL. APPL. MICROBIOL. Chikungunya virus per cell was added; the inoculated culture was incubated at 37 C, and the culture fluid was removed after 48 hr. The fluid was acidified with HCl to ph 2, kept at 0 C for 5 days, and then neutralized with NaOH. The preparation was then stored at 0 C for assay. Those that could not be assayed within 4 weeks were stored at below -60 C and assayed within 6 months. When poly I: C was used as the inducer, the procedure was modified as follows. The spent medium was removed, and 3.6 ml of fresh culture medium containing 2 mg of diethylaminoethyl-dextran (8) was added. After 2 hr at 37 C, 80 ug of poly I: C was added in 0.4 ml of medium. After incubation of the culture at 37 C for 24 hr, the fluid was removed and stored at 0 C for subsequent assay. For induction of leukocytes, cell concentration was adjusted to 2 million cells in 1.5 ml of medium in an 18 by 150 mm culture tube. A 0.5-ml amount of NDV (20 million PFU) was added to the culture; the tube was gassed with 5% CO2 in air and closed with a rubber stopper. After 48 hr at 37 C, the culture was processed for interferon assay as described for fibroblast cultures. The procedures adopted in this study were based on findings published by others (11, 26, 37, 39, 45). We emphasize simplicity. Because preliminary studies with NDV in amnion and fibroblast cells showed that an equal amount of interferon was released with 1, 6, or 10 PFU of NDV per cell, multiplicity of 1 to 10 PFU per cell was used. For NDV and Chikungunya virus, culture fluid was routinely harvested for assay 40 to 48 hr after infection; preliminary studies showed no difference between fluid collected at the 24th or 48th hours. After 20 to 24 hr of incubation with poly I: C, fluid was collected. Reference interferon. A large quantity of interferon was produced in human fibroblast cultures by the procedure described in the preceding section and stored in small samples at below -60 C. It was included in all assays to serve as an internal reference standard. Its average potency was Human interferon reference reagent (69-19) was obtained from the Mill Hill laboratory in London. It was reconstituted in 20% fetal calf serum in MEM at a dilution of 1: 10 and stored in 1-ml samples at below -60 C until use. Interferon assay system. The 50% plaque reduction test was used exclusively (25). In our earlier work dealing with the screening of various cell types, seven lines of human embryonic or foreskin fibroblasts were used as indicator cells. Later, to standardize our procedure, only one line of human embryonic cells was used. VSV was used as the challenge virus. Calculations. Interferon unitage was expressed as the negative logarithm of the highest dilution which produced 50% reduction in plaque number. To account for variation in sensitivity between assays, the titer was adjusted by adding or subtracting the difference between the titer of our internal interferon standard in each assay and 3.5, which was the average value for the standard in 16 consecutive assays. Total fluid volume was accounted for by adding 0.6. Ultimately, the titer was adjusted per 106 cells. RESULTS Variability of interferon assays. Difficulties associated with assays have been reviewed by Finter (10). Although results of interferon assays are known to be quite variable, no data concerning this problem have been published. To provide readers with some basis for evaluating results to be presented, we compiled the following unselected data on the variability of the assay in our laboratory. (i) Observed values for 25 samples of the internal reference interferon standard in 22 consecutive assays were in the range of 2.9 to 4.2, with a mean value of Reproducibility of the assay is not good. (ii) Variation in sensitivity of each assay affected a test preparation in the same way as it affected the reference standard. When the activity of the standard was high, activity of the test preparation was also high. Twelve preparations were compared in two separate assays; the average difference of the values observed was 0.6 i 0.4. This was reduced to if observed values were corrected for variation in sensitivity of the assay according to the calculations described above. (iii) Within the same assay, however, results were less variable. When four replicate samples of the standard were tested in one assay, values of 3.6, 3.5, 3.6, and 3.7 were obtained. In a second assay, three replicate samples gave titers of 3.5, 3.2, and 3.4. (iv) When two replicate cultures were induced and assayed for interferon activity in parallel, differences in results were relatively small. Ten pairs of cultures were so tested, and differences in activity for each pair were found to vary from 0 to 0.5 (average = 0.2 i 0.1). (v) In 10 assays, the potency of the internal reference standard was compared to that of the World Health Organization interferon standard. The average value for the internal standard was 3.3 ± 0.2, and the average titer for the Mill Hill standard was 4.0 ± 0.3. (vi) It was previously reported that embryonic cells are less sensitive than neonatal cells to the action of interferon (33). When our internal reference standard was tested on 7 lines of neonatal (foreskin) fibroblasts in 22 assays, the average titer obtained was 3.5 ± 0.3. When tested on 12 lines of embryonic fibroblasts in 20 assays, the internal standard gave an average value of 3.4 i 0.4. In our assay system, embryonic and neonatal fibroblasts show no difference in sensitivity to interferon. Interferon production by various ceil cultures. Various types of human cell cultures

3 VOL. 24, 1972 INTERFERON PRODUCTION BY HUMAN CELLS IN VITRO 737 were tested for their ability to produce interferon. NDV was used as the inducer, and titer was expressed as adjusted units per 106 cells. (i) Embryonic fibroblasts: 11 lines of embryonic fibroblasts produced an average value of 3.7 i 0.2 units per 106 cells (range, 3.3 to 4.1). (ii) Neonatal foreskin fibroblasts: five lines of foreskin fibroblasts released interferon within a range of 2.5 to 4.0. The average titer was (iii) Children's skin fibroblasts: fibroblasts from four children 3, 8, 12, and 15 years of age yielded 3.6, 3.3, 2.9, and 3.8 units of interferon, respectively. (iv) Adult skin fibroblasts: 16 specimens of skin fibroblasts from normal persons 18 to 92 years of age gave an average interferon titer of (range 3.1 to 3.9). (v) Amnion cells: 11 cultures from 9 placentae released from 2.8 to 4.1 units of interferon with a mean titer of (vi) Leukocytes: leukocytes from 7 individuals were tested as primary suspension cultures. Results varied from 2.1 to 2.9 interferon units, giving an average value of (vii) Established lymphoid cell lines: three lines of low-passaged (<5) lymphoid cells yielded values of 2.5, 2.3, and 2.1 (average = 2.3); four lines of high-passaged (> 50) cells released 2.2, 2.3, 2.1, and 2.3 adjusted units (average = 2.2). (viii) Heteroploid cell lines: only 2.5, 2.3, and 2.7 units of interferon were released, respectively, by the Chang liver, u, and HeLa-S3 cells. Interferon production by intact amniotic membrane. Fournier and associates reported that intact amniotic membrane released a significant quantity of interferon upon induction (11). Because intact membrane in culture medium has a distinct advantage over cell culture in its simplicity, we performed three experiments to determine its capacity to release interferon as compared to amnion cell culture. Amniotic membrane was stripped, washed, drained, weighed, and cut into portions of approximately 1 g. Several portions were induced with NDV as described in Table 1. Other portions were processed for maximal yield of cells for culture in petri dishes and induced with NDV as described in Materials and Methods. For placenta no. 255 induced under condition A, a total of 4.4 adjusted interferon units in 40 ml was obtained in 4 days from 1 g of tissue. When the cells were grown as monolayers, and the cultures were induced on day 4, a total of 3.6 units of 20 ml was collected. For placenta no. 256, also under condition A, 20 ml containing 4.0 units of interferon was collected in the first 2 days from 1 g of tissue. When this tissue was processed for cultures and induced on day 11, 16 ml containing 3.1 units of interferon was obtained. When the membrane was induced under condition B (108 PFU of NDV per g of tissue), interferon production was slightly greater than under condition A. Intact amniotic membranes appeared superior to cell cultures for interferon production. Factors affecting interferon production by fibroblasts. Because fibroblasts are better interferon producers than leukocytes and heteroploid cells, and because fibroblasts have greater growth potential than amnion cells, we chose fibroblasts for the evaluation of factors that may affect interferon production. The following factors were investigated (all interferon titers were again expressed as adjusted units per 106 cells). (i) Inducers: A comparison of two strains of NDV (Ho's and Hertz) in stimulating interferon production was made. At a multiplicity of 6 PFU per cell, both strains stimulated the release of similar amounts of interferon from fibroblasts and amnion cultures. The strain supplied by Ho was used for all studies included in this report. The relative effectiveness of NDV, Chikungunya virus, and poly I: C in inducing inter- TABLE 1. Placentaa Condition' Interferon production by amniotic membrane Interferon" produced on day A B C A <3.2 <3.2 B < 3.2 C < A B a Amounts of 21 and 28 g of membrane were stripped, respectively, from placenta 255 and 256; 4.5 and 4.0 million cells were harvested from 1 g of membrane from placenta 255 and 256, respectively. b Condition A, 1 g of membrane was suspended in 10 ml of medium and inoculated with 107 PFU of NDV; fluid was replaced with fresh medium every 24 hr. Condition B, as A except that 108 PFU of NDV was used. Condition C, as A except that the membrane was suspended in 2 ml of medium. All specimens of one experiment were tested in one assay. cadjusted interferon units released by 1 g of tissue; expressed as the negative logarithm of the highest dilution which gave 50% reduction in VSV plaque number.

4 738 SPINA ET AL. APPL. MICROBIOL. feron production was tested on nine lines of skin fibroblasts. Average amounts of interferon released per 106 cells were 3.6 ± 0.3, , and for NDV, Chikungunya virus, and poly I: C, respectively. (ii) Culture passage level: Since human fibroblasts can undergo numerous cell divisions sequentially and thereby yield an astronomical number of cells for production purpose (17), it would be important to determine whether the capacity to release interferon changes upon in vitro passage. In this study, the passage level is determined by Hayflick's procedure (16); one passage is therefore equivalent to one cell doubling. Results are shown in Table 2. In nine lines of embryonic fibroblasts, early passaged cells (7th and earlier) with an average interferon titer of 3.3 ± 0.2 appeared to be poorer producers than the corresponding later passaged cells TABLE 2. Interferon production by NDV-treated embryonic fibroblasts at different passage level Passage Interferon' Line level units/106 cells 2a a < < a a The 7th and 31st passage levels of line 2; 7th and 30th of line 10; and 2nd and 13th of line 35 were induced and assayed simultaneously. 'Expressed as the log of the adjusted titer of interferon activity. Average titer of early-passaged cells (7th and earlier) = ; average titer of high-passaged cells (13th and over) = ; P < (13th and over) which gave an average titer of 3.9 ± 0.2. (iii) Age of culture and cell concentration: Since fibroblasts are actively growing cells until they become confluent, replicate cultures tested at varying intervals after preparation can vary in cell concentration and in the proportion of actively dividing cells. To determine the relative importance of these two factors, the following experiment was performed. Eighteen petri dishes were inoculated with approximately 0.3 x 101 cells per dish. On day 1 to 6, two cultures were sacrificed for cell count and one culture was induced with NDV. Culture medium was changed on day 3 and 5 for cultures not treated with NDV. All preparations were assayed for interferon activity in a single assay. Results of two experiments are shown in Table 3. There was a definite, progressive increase from day 1 to day 4 in the amount of interferon released by a culture, but there was also a corresponding increase in cell concentration. When expressed on the basis of interferon released by a million cells, there appeared to be little difference in the capacity TABLE 3. Interferon production by fibroblast cultures of varying culture age Cells PsaeCulture Itefrn lvl (day) per cul- 106, cells Line Passae age (log) Per Per clueture Foreskin no Embryo no a Interferon units expressed as the log of the adjusted titer of interferon activity. TABLE 4. Differences in interferon production due to differences in sex Female Male Interferona No. Interferon" ± i 0.4 a Log of the average adjusted titer of interferon activity per 106 cells. 'P = 0.2.

5 VOL. 24, 1972 INTERFERON PRODUCTION BY HUMAN CELLS IN VITRO of 1- to 6-day-old cultures to produce interferon. (iv) Sex of donor: Table 4 shows that in 23 lines of human skin fibroblasts, there is no apparent correlation between interferon production and the donor's sex. (v) Age of donor: There is no consistent relation between the donor's age and the ability of his cells to produce interferon. Results are summarized in Table 5. DISCUSSION The main objectives of this study were: to compare the interferon-producing capacity of various types of human cells that can be obtained in bulk quantities; to search for a person whose cells are superior interferon producers; to compare the effectiveness of three well-known interferon inducers; and to delineate culture conditions that may enhance interferon production. The capacity of various types of human cells to produce interferon upon stimulation with NDV is summarized in Table 6. Fibroblasts and amnion cells are, in general, better interferon producers than leukocytes and heteroploid cells. Although there were minor differences between cell lines in interferon production, we did not encounter a single per- TABLE 5. Agenof Interferon produced by skin fibroblastsa from donors of various ages Agdonr Interferon' units/10 cells treated with (years) NDV Poly I: C < < < < < < < < <3.0 aculture passage levels, 9th to 25th. b Expressed as the log of the adjusted titer of interferon activity. son whose cells were superior producers (arbitrarily defined as one capable of producing 4.5 units of interferon per 106 cells). Differences in interferon-producing capacity of cells from persons with various diseases have been reported (see Table 7). It is not possible, however, to determine from those published data whether their cells were superior interferon producers. Age of cultures has been reported to affect interferon production (10). We have examined this parameter in considerable detail with the fibroblast-ndv system and have obtained the following results. (i) The higher yield of interferon by "aged" cultures could be accounted for by the higher cell number in these "aged" cultures (see Table 3). (ii) In general, embryonic fibroblasts that have undergone less than seven serial cell doublings were poorer producers than those that have undergone more than 15 cell doublings (see Table 2). (iii) The age of donors did not affect interferon production (see Table 5). The relationship between the donor's age and interferon production has been studied by several investigators (2, 3, 14, 31, 34, 36). The results vary between different studies; there is not complete agreement among these studies nor with our own findings. Of the three inducers, NDV and Chikungunya virus were superior to poly I: C in stimulating maximal yield of interferon from skin fibroblasts. In conclusion, the best cell for large-scale production of human interferon is, in our opinion, the embryonic fibroblast. This cell is one of the two best interferon-producing cells and can TABLE Interferon production by various human cells induced with NDV No. of Interferona Cell type lines or produced persons by 106 cells tested Fibroblasts Embryonic" Neonatal foreskin i 0.6 Children's skin ± 0.4 Adult skin ± 0.3 Amnion cells ± 0.4 Leukocytes Peripheral blood ± 0.3 Established lines ± 0.2 Heteroploid cell lines ± 0.2 a Expressed as the log of the adjusted titer of interferon activity. Interferon produced by embryonic fibroblasts versus peripheral leukocytes, P < ; embryonic fibroblasts versus heteroploid lines, P <

6 740 SPINA ET AL. APPL. MICROBIOL. Cell type TABLE 7. Interferon production in cells derived from patients with various diseases Interferon production Enhanced Same as control Depressed Leukocytes Chronic myelogenous leu- Chronic lymphocytic leu- Acute lymphocytic leukemia kemia (20, 22)a kemia (short-term) (22) (20, 22) Infectious Down's syndrome (38) Chronic lymphocytic leumononucleosis(12) Fanconi's anemia (38) kemia (long-term) (20, 22) SSPE (38) Acute myelogenous leukemia Blast-cell leukemia (38) (20, 22) Polycythemia vera (15) Uremia (32) Fibroblasts Fanconi's anemia (44) Carcinoma (44) Sarcoma (44) a References cited in parentheses. be obtained in large quantity through serial propagation without decline in interferon-producing capacity. We have estimated that at least 1030 fibroblasts can be obtained from a 3-month-old embryo through serial propagation. Interferon production by the fibroblast can be further boosted by the addition of antimetabolites (27, 40, 41). Of course, the main difficulty with the use of embryonic fibroblasts for the production of biologicals for use in man is the difficulty of excluding oncogenic viruses that may be present in these cells. This difficulty and many other aspects on the use of human cells for human vaccine production have already been discussed (24). ACKNOWLEDGMENTS This investigation was supported by the Antiviral Substances Program (PH ) of the National Institute of Allergy and Infectious Diseases. We are grateful to W. Nelson-Rees (PH ) for some of the cells included in this study. LITERATURE CITED 1. Cantell, K., and K. Paucker Studies on viral interference in 2 lines of HeLa cells. Virology 19: Cantell, K., H. Strander, L. Saxen, and B. Meyer Interferon response of human leukocytes during intrauterine and postnatal life. J. Immunol. 100: Carter, W., K. Hande. B. Essien, E. Prochownik, and M. Koback Comparative production of interferon by human fetal, neonatal, and maternal cells. Infect. Immi 'ity 3: Chang, R. S Continuous subcultivation of epithelium-like cells from normal human tissues. Proc. Soc. Exp. Biol. Med. 87: Chang, R. S Observations on the growth phases of human amnion cell cultures. J. Nat. Cancer Inst. 40: Chang, R. S The loss of growth vitality of human lymphoid cell lines derived from healthy adults. Proc. Soc. Exp. Biol. Med. 135: Chany, C Interferon-like inhibitor of viral multiplication from malignant cells. Virology 13: Diazani. F., S. Baron, C. E. Buckler. and H. Levy Mechanisms of DEAE-dextran enhancement of polynucleotide induction of interferon. Proc. Soc. Exp. Biol. Med. 136: Falcoff, E., R. Falcoff, and C. Chany Mass production of human interferon for therapeutic trials. Med. Appl. Virol., p Finter, N. B Frontiers of biology: interferons, vol. 2, p North Holland Publishing Co., Amsterdam. 11. Fournier, F., E. Falcoff, and C. Chany Demonstration, mass production and characterization of a heavy molecular weight human interferon. J. Immunol. 99: Gergely, L., F. D. T6th, G. Hadhazy, and B. Szabo Enhanced interferon production in vitro by leukocytes from children with infectious mononucleosis. Acta Microbiol. Acad. Sci. Hung. 17: Gresser, I Production of interferon by suspensions of human leucocytes. Proc. Soc. Exp. Biol. Med. 108: Gresser, I.. and D. J. Lang Relationships between viruses and leukocvtes. Progr. Med. Virol. 8: Hadhazy, G., L. Gergely, G. Nagy, and F. D. T6th Comparison of interferon production in vitro by leukocytes from healthy and polycythemic persons. Acta Microbiol. Acad. Sci. Hung. 15: Hayflick, L The limited in vitro lifetime of human diploid cell strains. Exp. Cell. Res. 37: Hayflick, L Cell substrates for human virus vaccine preparation. Nat. Cancer Inst. Monograph 29, p Ho, M., and J. F. Enders Further studies on an inhibitor of viral activity appearing in infected cell cultures and its role in chronic viral infection. Virology 9: Johnson, T. C.. and L. C. McLaren Plaque development and induction of interferon synthesis by RMC poliovirus. J. Bacteriol. 90: Lee, S. H. S Interferon production by human leukocytes in vitro. Appl. Microbiol. 18: Lee, S. H. S., and R. L. Ozere Production of interferon by human mononuclear leucocytes. Proc. Soc. Exp. Biol. Med. 118: Lee, S. H. S., C. E. Van Rooyen, and R. L. Ozere Additional studies of interferon production by human leukemic leukocytes in vitro. Cancer Res. 29: Marchenko, V. I., A. A. Babayants, and L. N. Pokidysheva Production of human amniotic interferon. Vop. Virusol. 13: Merchant, D. J Cell culture for virus vaccine production. Nat. Cancer Inst. Monograph Merigan, T. C A plaque inhibition assay for human interferon employing human neonate skin fibroblast monolavers and bovine vesicular stomatitis virus p In B. R. Brown and P. R. Glade (ed.), In vitro

7 VOL. 24, 1972 INTERFERON PRODUCTION BY HUMAN CELLS IN VITRO 741 methods in cell-mediated immunity. Academic Press Inc., New York. 26. Merigan, T. C., D. F. Gregory, and J. K. Petralli Physical properties of human interferon prepared in vitro and in vivo. Virology 29: Myers, M. W., and R. M. Friedman Potentiation of human interferon production by superinfection. J. Nat. Cancer Inst. 47: Pohjanpelto, P Two different thermostable varients of poliovirus. Virology 15: Puck, T. T., and H. W. Fisher Genetics of somatic mammalian cells. I. Demonstration of the existence of mutant with different growth requirements in a human cancer cell strain (HeLa). J. Exp. Med. 104: Rabson, A. S., G. T. O'Connor, S. Baron, J. J. Whang, and F. Y. Legallais Morphologic, cytogenic. and virologic studies in vitro of a malignant lymphoma from an African child. Int. J. Cancer 1: Ray, C. G The ontogeny of interferon production by human leukocytes. J. Pediat. 76: Sanders, C. V Suppression of interferon response in lymphocytes from patients with uremia. J. Lab. Clin. Med. 77: Siewers, C. M. F., C. E. John, and D. N. Medearis, Jr Sensitivity of human cell strains to interferon. Proc. Soc. Exp. Biol. Med. 133: Soloviev, V. D., T. A. Bektemirov, N. V. Vorotyntseva, et al Interferon production outside the body by leukocytes of healthy children and children with acute respiratory infection. Vop. Virusol. 14: Soloviev, V. D., L. N. Pokidysheva, N. R. Gutman, V. I. Marchenko, and V. P. Kuznetsov A study of the productional conditions for obtaining human leukocytic interferon. Vop. Virusol. 14: St. Geme, J. W., and D. Horrigan Comparative production of interferon by foreskin fibroblasts from newborn infants and children. J. Lab. Clin. Med. 74: Strander, H Production of interferon in serum-free human leukocyte suspensions. Appl. Microbiol. 18: Strander, H Interferon response of lymphocytes in disorders with decreased resistance to infections. Clin. Exp. Immunol. 6: Strander, H., and K. Cantell Production of interferon by human leukocytes in vitro. Ann. Med. Exp. Fenn. 44: Tan, Y. H., J. A. Armstrong, Y. H. Ke, and M. Ho Regulation of cellular interferon production: enhancement by anti-metabolites. Proc. Nat. Acad. Sci. U.S.A. 67: Vilcek, J., T. G. Rossman, and F. Varacalli Differential effects of actinomycin D and puromycin on the release of interferon induced by double stranded RNA. Nature (London) 222: Wheelock, E. F I.F.-like virus-inhibitor induced in human leucocytes by PHA. Science 149: Wheelock, E. F., and W. A. Silbey Interferon ip human serum during clinical viral infections. Lancet u: Worthington, M., and S. A. Aaronson Interferon system in cells from human tumors and from persons predisposed to cancer. Infect. Immunity 3: Zajac, B. A., W. Henle, and G. Henle Autogenous and virus-induced interferons from lines of lymphoblastoid cells. Cancer Res. 29: Downloaded from on July 12, 2018 by guest