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Immunocompetent Cells in the Blood of Immunized Chickens1
GENTAMICIN AND
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antibiotic gentamicin. J. Infect. Dis. 119: 518527. Waitz, J. A., and M. J. Weinstein, 1969. Recent microbiological studies with gentamicin. J. Infect. Dis. 119: 355-360. Weinstein, M. J., G. H. Wagman, E. M. Oden and J. A. Marquez, 1968. Biological activity of the antibiotic components of the gentamicin complex. J. Bact. 94: 789-790. Weinstein, M. J., G. M. Luedemann, E. M. Oden, G. H. Wagman, J. P. Rosselet, J. A. Marquez, C. T. Coniglio, W. Charney, H. L. Herzog and J. Black, 1963. Gentamicin, a new antibiotic complex from Micromonospora. J. Med. Chem. 6: 463-464. Wersall, J., P. G. Lundquist and B. Bjorkroth, 1969. Ototoxicity of gentamicin. J. Infect. Dis. 119: 410-416. Worcester, W. W., 1965. Californians report results of test on paracolon control. Feedstuffs, 37: 6ff. White, A., 1964. In vitro activity of gentamicin. Antimicrob. Agents and Chemotherapy, 1963. p. 17-19.
Immunocompetent Cells in the Blood of Immunized Chickens1 FRANK SETO
Zoology Department, University of Oklahoma, Norman, Oklahoma 73069 (Received for publication June 17, 1970) INTRODUCTION
A
NTIGEN stimulation initiates the • production of specific antibody that reaches a peak concentration in the blood several days later. Concomittant with the antibody response are characteristic histological changes in the activated lymphoid tissues, the proliferation of lymphocytes and differentiation of plasma cells (Leduc et al., 1955; Urso and Makinodan, 1963; Hanna et al., 1966). Direct correlation between the increase in antibody output and an increase in antibody-forming cells in the activated lymphoid organs of mammals 1 Research aided by the Faculty Research Fund of the University of Oklahoma.
have been clearly demonstrated with the agar plaque technic (Jerne et al., 1963) and the immunocytoadherence test (Biozzi et al., 1967). Adoptive immunity experiments have confirmed that spleen and lymph node of antigen-primed mammals contain cells capable of antibody synthesis in suitable hosts (Mitchison, 1957; Albright et al., 1964). Antigen stimulates immunological stem cells to proliferate and produce (a) populations of antibody-forming cells, and (b) reserves of immunologically activated precursors and memory cells which respond anamnestically to subsequent antigen exposure and are responsible for the persistence of immunological mem-
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Kowalski, L. M., and J. F. Stevens, 1968. Arizona 7 : 1 , 7 , 8 infection in young turkeys. Avian Dis. 12: 317-326. Peluffo, C. A., P. R. Edwards and D. W. Bruner, 1942. A group of coliform bacilli serologically related to the genus Salmonella. J. Infect. Dis. 70: 185-192. Rubenfire, M., H. H. Glass, A. S. Goldstein and A. M. Lerner, 1969. Gentamicin therapy of Paracolobactrum epidural abcess and meningitis. Amer. J. Med. Sci. 257: 191-197. Sadler, W. W., R. Yamamoto, H. E. Adler and G. F. Stewart, 1961. Survey of market poultry for Salmonella infection. Applied Microbiol. 9: 7276. Smith, C. B., P. E. Dans and J. N. Wilfert, 1969. The use of gentamicin in combinations with other antibiotics. J. Infect. Dis. 119: 370-377. Stone, H. H., J. D. Martin, Jr., W. E. Huger and L. Kolb, 1965. Gentamicin sulfate in the treatment of Pseudomonas sepsis in burns. Surg. Gynec. Obstet. 120: 351-352. Waisbren, B. A., 1969. Experiences with the new
PARACOLOBACTRUM
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MATERIALS AND METHODS Materials. Juvenile (3^ to 12 weeks old) and baby White Leghorn were donors and White Leghorn embryonated eggs served as
hosts. These were purchased from a local commercial hatchery. METHODS
The in vivo culture system (Albright et al., 1964), as modified for the allogeneic chicken system, detects precursor cells and memory cells, which will be henceforth referred to as immunocompetent cells (ICC), that respond to antigenic challenge. Donor cells, blood and cell suspensions of other tissues, from antigen stimulated chickens are exposed in vitro to antigen, cultured in chick embryo hosts and assayed for antibody production. Appropriate controls indicate that the antibody formed in host embryos do not result from passive transfer of pre-existing antibody-forming cells. The main antigen, mouse erythrocytes (Mrbc), was obtained by cardiac puncture in equal volume of Alsever's solution, washed in sterile 0.15 M NaCl three times, packed by centrifugation and cell suspensions prepared in saline solution. Sheep erythrocytes (Srbc), obtained commercially (Brown Laboratory, Topeka, Kansas) , was used as the other antigen. Prospective donor White Leghorn chickens were immunized with either Mrbc or Srbc. While blood was obtained from each chicken by cardiac puncture in equal volume of Alsever's solution. After centrifugation most of the plasma was discarded and enough antigen suspension was added to restore the original blood volume. A standard amount (0.1 ml.) of donor blood, with or without 4 X 107 washed Mrbc or Srbc depending on the experimental group, was injected intravenously into the chorioallantoic vessel of 14-day White Leghorn embryo recipients. Donor blood was used soon after collection or stored at ice water temperature no longer than two hours before use. Immediately after the antigen was added to the donor blood and thoroughly
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ory (Vasquez and Makinodan, 1966; Sterzl, 1967). Some of the lymphocytes remain in the lymphoid organs whereas others emigrate and presumably enter the general circulation (Harris et al., 1945; Wissler et al., 1957). It is known that blood leucocytes are involved in homograft rejection, graftversus-host reaction, and delayed hypersensitization (Billingham and Barker, 1969), but it is less well established that they are capable of antibody synthesis (Bach and Hirschhorn, 1963). Recent studies employing isotope incorporation techniques, agar plaque assay, immunocytoadherence test, and radioimmunoelectrophoresis have demonstrated antibodyproducing cells in the blood of mammals shortly after immunization (Hulliger and Sorkin, 1963; Landy et al., 1964; Zaalberg, 1964; Kearny and Halliday, 1964; Chessinei al., 1968). Although less documented, the situation is essentially similar in chickens. Histological observations in conjunction with enumeration of hemolytic plaque-forming cells indicate that cellular changes similar to that reported in mammals occur in the spleen of chickens during the immune response (Abramoff and Brien, 1968; Solomon, 1968). Also antibody-producing cells, demonstrated only by the immunocytoadherence test (Duffus and Allan, 1969) and other immunocompetent cells appear in the blood soon after antigenic stimulation (Seto, 1968). This report describes a kinetic analysis of immunocompetent cells in the blood of chickens, as assessed by the in vivo culture technic.
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IMMUNOCOMPETENT CELLS
mixed it was injected into host embryos. Injection of a group of hosts took several minutes. Some of the donors were killed and the spleen, thymus, bone marrow and bursa of Fabricius were used to prepare cell suspensions. The organs were individually teased apart in sterile Medium 199 (Baltimore Biological Laboratory) to which was added about 2 % by volume of normal chicken se-
14 12 10 8 6 4
a.
2
UJ
*•
B.
3.5 w
C.
3d
15
O
RESULTS 1
2 3 4 5 6 7 8 9 33d T I M E A F T E R ANTIGEN INJECTION
FIG. 1. Effect of antigen dose on the appearance of immunocompetent cells in the blood of chickens of different ages. The response profiles of 9j-week (A), 3^-week (B), and 3-day (C) chickens are shown. Antigen doses were 10' (A), 10s ( • ) , and iO9 (A) Mrbc in a single injection. The S-day mean hemagglutinin responses of 3^-week and 9iweek donors and 7-day response of 3-day donors are shown as a broken line ( ).
The response profile of ICC in the blood of immunized chickens. The initial experiment was conducted to determine the relative concentration of ICC in the blood at various times after immunization. Donor chickens, 9\ weeks of age, were injected with 107, 108, 109, and 1010 washed Mrbc in 1 ml. of.0.15 M NaCl. Blood samples were obtained at intervals from these chickens and assayed in 4 to 6 host em-
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16
rum, dispersed further by gently flushing the fragments through a syringe with a 22 gauge needle and clumps strained out with stainless steel cloth (200 mesh/inch). The cells were washed once with fresh Medium 199, centrifuged, and the supernatant discarded. The pellet was resuspended in fresh medium and a cell count was made with a hemacytometer. Approximately 4-5 X 106 cells in 0.1 ml. of Medium 199, with or without 4 X 10' Mrbc or Srbc as antigen depending on the experiment, were injected intravenously into embryo hosts. Six days later the embryos were bled and antibody titers of the serum samples were estimated by the Microtiter hemagglutination technic (Cooke Engineering, Alexandria, Va.). Preliminary experiments indicated that antibody production in the in vivo hosts was proportional to the amount of inoculated donor blood and thus provided an indirect quantitative assessment of the relative ICC content of the inoculum. Quantitatively similar titers were were obtained, moreover, whether cyclophosphamide-inactivated or untreated 14-day embryos were used as hosts (Seto, 1970). Host embryos at this age are immunologically immature and generally incapable of rejecting the donor cells (Seto, 1970) or responding by hemagglutinin production to heterologous erythrocytes (Seto and Henderson, 1968).
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TABLE 1.—Relative antibody-forming capacity of cells from different tissues of immunized donors1 • Frequency of £1 ^ ' t , Experimental groups r e 5 „ o n d e r s body titer in Antigen only Blood only Bursa+antigen Thymus+antigen Bone marrow-(-antigen Spleen+antigen Blood+antigen 1
^
l0g2
0/6 0/4 0/13 4/17 3/6 16/16 17/1?
0.0 0.0 0.0 0.5 1.0 8.3 8.8
Assessed with the in vivo assay method
Immunocompetent cells in other tissues of immunized chickens. Other experiments were conducted to detect the presence of ICC in other tissues of 5-week donors immunized 4 days earlier. The results of one experiment are summarized in Table 1. As estimated by antibody titers in assay embryos, the ICC were infrequent in the cells suspensions from thymus, bone marrow and bursa of Fabricius. They were abundant only in the blood and spleen. When blood from 5 control unimmunized donors obtained at 4, 5, 6 and 8 weeks of age were similarly tested, no definite positive responded were found among 118 recipients. Although not shown, similar results were obtained with tissues from donors 3 and 6 Donor age and concentration of ICC in the days after immunization, and with older blood. Other experiments were conducted to donors as well. determine the ICC response profiles of The response profiles of ICC in the more immature birds. Juvenile chickens, blood and spleen were compared. Chickens, 3^ weeks of age were injected intracar- 6-12 weeks of age, were immunized with dially with 107, 108, 109, 1010 washed Mrbc Mrbc, then sacrificed at specified time inin 1 ml. of saline solution and their blood tervals and their blood and spleen assayed. assayed at intervals. As with the 9J week The results are shown in Fig. 2. Immunodonors, the response profiles of donors ex- competent cells were detected in the spleen posed to higher doses were much alike and but not in the blood the first day after imthat of the donor receiving the least dose munization. After the second day they were was reduced (Fig. IB). The time of peak present in both tissues and the response
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bryos per blood sample per donor. The mean 6-day anti-mouse hemagglutinin titers obtained in the in vivo hosts for each donor, sampled on different days after immunization, are summarized in Fig. 1A. The response curves for antigen doses of 10s, 109, and 1010 Mrbc were alike and for the sake of clarity in the figure, that for 1010 Mrbc was omitted. Peak concentration of ICC, as reflected in the in vivo antibody titers, were observed in all donors irrespective of antigen dose, by the 3rd day and remained high for many days. Except for the reduced response with the donor exposed to the lowest dose (10 7 ), differences in levels of response were not apparent with the other doses during the first week but the graded differences in levels of these immunocompetent cells persisting in the blood 33 days after immunization was dose dependent.
ICC concentration was 2 days later. Experiments with 7^-week donors immunized with 107 to 1010 washed Srbc showed response profiles essentially like that of the 3^-week group. When 3-day baby chicks were injected with 10s washed Mrbc, only 2 of 10 birds showed definite ICC profiles although 9 of 10 had respectable 7-day hemagglutinin titers. The response profiles of these two birds were averaged and plotted in Fig. 1C. It is apparent that the capacity to produce ICC as measured by their relative amount and persistence in the blood following immunization increases with the maturity of the donors.
IMMUNOCOMPETENT CELLS
patterns were essentially alike although it appears that a more rapid decline occurs in the spleen.
1 2 TIME
AFTER
3 4 ANTIGEN
5 days INJECTION
FIG. 2. A comparison of the response profiles of immunocompetent cells of blood (0)> and spleen ( • ) of 6 to 12-week old White Leghorn donor chickens. 1-to 4 donors were used in each time interval and the number of assay embryos in the sample is shown next to each point.
maximal production of ICC during the first week that tend to obscure any antigen dosage effect. Later as the production levels off, the antigen dosage effect becomes apparent in the graded levels of immunocompetent cells persisting several weeks later. With sufficiently low antigen dose, a slower response with reduced ICC level is induced that rapidly declines. The blood ICC response of 3-day chicks was weak and brief and probably reflects the immature state of the immune system. The response of 3^-week chickens was considerably greater but that of the 9£week chickens was very rapid, and persisted at high levels. The capacity to produce ICC, as estimated from the peak blood levels, increases over 200-fold during the first month after hatching and an additional 4-fold by the second month, which is comparable in magnitude to the growth of the hemagglutinin-forming potential reported in the chicken earlier (Seto and Henderson, 1968). It has been suspected for some time and later confirmed that precursors of antibody-forming cells circulate in the blood (Gowan and Uhr, 1968; Linna et al., 1968; Davies et al., 1968) and antibodyproducing cells may occur in the blood. It is not readily evident from the literature (Hullinger and Sorkin, 1963; Kearny and Halliday, 1965; Landy et al, 1964; Halasa, 1968; Hiramoto et al., 1968; Duffus and Allan, 1969) or from our results whether these precursor and antibody-producing cells originate from stem cells in the blood or in extravascular sites. There is much indirect evidence, in addition to that presented here however, that suggest their formation in lymphoid tissues in mammals (Leduc et al., 1955; Wissler et al., 1957; Hanna et al., 1966) and in chickens (Wolfe et al., 1950; Abramoff and Brien, 1968; Keily and Abramoff, 1969). Immunocompetent cells occur abundantly in the
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DISCUSSION In our experience immunocompetent cells (ICC) that respond with specific hemagglutinin formation to heterologous erythrocytes are normally absent in the blood but may occur infrequently in the spleen of young unimmunized chickens. Following antigenic stimulation with mammalian erythrocytes, however, they appear in the blood within 2 days and reach peak concentration by the 3rd to 5th day. This period of rise to peak concentration coincides with the logarithmic phase of the hemagglutinin response (Seto and Henderson, 1968). Whether the ICC persisted or declined in the blood was related to the antigen dose and the maturity of the donor. Despite a hundred-fold difference in the amount of antigen administered (108 to 1010), these doses elicited quantitatively similar ICC response profiles during the first week. Apparently moderate to high doses stimulate
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F. SETO
1967) but may include hemocytoblasts as well in chickens (Duffus and Allan, 1969). The ICC include cells that behave much like the proposed memory cells which have been presumed to be small lymphocytes (Gowan and Uhr, 1966; Burnet, 1968). The situation is not as simple in practice, as it now appears that 2 or 3 different cell types—thymus derived antigen-reactive cell, bone marrow-originated antibodyprecursor cell, and the antigen-processing macrophage—are necessary in some way for the immune response (Fishman, 1961; Mosier, 1967; Groves et al., 1970). The identity of the ICC is less clear but functionally it behaves much like the antigensensitive unit, a synergistic combination of antigen-reactive cells and antibody precursor cell (Shearer et al., 1968; Makinodan and Price, 1970). Experiments are in progress to elucidate the nature of the ICC in the blood of chickens. SUMMARY
A modified in vivo culture technic was used in a kinetic study of immonocompetent cells (ICC) in the blood and other tissues of immunized chickens. Cells from antigen-stimulated donors were mixed in vitro with antigen, transferred to 14-day chick embryo hosts, and assayed for antibody production. Of the tissues tested, ICC occurred abundantly in blood and in the spleen but were infrequent in the thymus, bone marrow and bursa of Fabricius. The relative concentration of ICC in the blood varied according to (1) the time after antigen injection, (2) the amount of antigen administered, and (3) the maturity of the donor chickens. Within two days after immunization ICC appeared in the blood, reached a peak concentration on the 3rd or 4th day and then declined. In juvenile birds (3^- and 9hweek old) the ICC response profiles were essentially alike for moderate to high anti-
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spleen of chickens, but are infrequent in the thymus and bone marrow as reported in mammals (Claman et al., 1966; Davies et al., 1967), and also in the bursa of Fabricius. This latter fact does not negate the importance of the bursa in the development of the immune function (Good and Papermaster, 1964; Weber and Weidanz, 1969) and might be expected in current models. Following antigenic stimulation in mammals the antibody-producing cells appear in the blood for a short duration, reach peak concentration on the 3rd and 4th day and then rapidly decline in numbers, whereas in chickens the ICC response profiles described here are more conspicuous and of longer duration. The differences in the pattern could be attributable in part to species differences, antigen dose, age, and previous antigen exposure. It is also conceivable that the type of immunological cell involved may result in different response profiles. The direct correlation between the increase in antibody output and the increase in antibody-forming cells detected by both agar plaque technic and the immunocytoadherence test (Jerne et al., 1963; Biozzi et al., 1967) indicates that the cell type reported in mammalian blood is the antigen activated, terminally differentiated antibody-producing cells (Z or P cells), that have spilled into the blood stream; whereas the cell types investigated here include mainly immunologically activated antigen-sensitive cells (Y or PC 2 ) responsible for immunological memory (Sterzl, 1967; Vasquez and Makinodan, 1966). As to the nature of the blood-borne immunocompetent cells, those involved in cellular immune responses are known to be lymphocytes (Billingham and Barker, 1969) and the antibody-producing types, identified by several technics, are mainly lymphocytes and plasma cells in mammals (Leduc et al., 1955; Urso and Makinodan, 1963; Jerne et al., 1963; Biozzi et al.,
IMMUNOCOMPETENT CELLS
gen doses and perceptibly reduced at the low dose tested. The response profile of 3day chicks was weak and brief, that of 9£week chickens was rapid, high and prolonged, and that of 3^-week chickens was intermediate. Although the ICC has not been identified as to cell type, it behaves much like the immunological memory cells. ACKNOWLEDGMENTS
REFERENCES Abramoff, P., and N. B. Brien, 1968. Studies of the chicken immune response. I. Correlation of the cellular and humoral immune response. J. Immunol. 100: 1204-1209. Albright, J. F., T. Makinodan and E. E. Capalbo, 1964. Factors regulating antibody production by spleen cells cultured in vivo. Proc. 9th Congr. Intern. Blood Transf. Mexico, pp. 301308. Bach, F., and K. Hirschhorn, 1963. Y-gl°bul>n production by human lymphocytes in vitro. Exp. Cell. Res. 32: 592-595. Billingham, R. F., and C. F. Barker, 1969. Recent developments in transplantation immunity Part I. Plastic and Reconstr. Surg. 43: 559-568. Biozzi, G., C. Stiffel and D. Mouton. 1967. A study of antibody-containing cells in the course of immunization. In Immunity, Cancer and Chemotherapy (ed. by E. Mihich), pp. 113130, Academic Press. Burnet, F. M., 1968. Evolution of the immune process in vertebrates. Nature, 218: 426^130. Claman, H. N., E. A. Chaperon and R. F. Triplett, 1966. Immunocompetence of transferred thymus-marrow cell combinations. J. Immunol. 97: 828-832. Chessin, L. N., P. R. Glade, R. Asofsky, P. C. Baker, R. Reisfeld and W. Terry, 1968. Studies on human peripheral blood lymphocytes in vitro. V. Biosynthesis of immunoglobulins. J. Immunol. 101: 458-468. Davies, A. J. S., E. Leuchars, V. Wallis, R. Marchant and E. V. Elliott, 1967. The failure of thymus-derived cells to produce antibody. Transplantation, S: 222-231. Davies, A. J. S., H. Festenstein, E. Leuchars, V. J.
Wallis and M. J. Doenhoff, 1968. The origin of some mouse peripheral blood lymphocytes. Exptl. Hematol. 17: 12. Duffus, W. P. H., and D. Allan, 1969. A study of the ontogeny of specific immune responsiveness amongst circulating leucocytes in the chicken. Immunology, 16: 337-347. Fishman, M. J., 1961. Antibody formation in vitro. J. Exp. Med. 114: 837-856. Good, R. A., and B. W. Papermaster, 1964. Ontogeny and phylogeny of adaptive immunity. Adv. Immunol. 4 : 1-115. Gowan, J. C , and J. W. Uhr, 1966. The carriage of immunological memory by small lymphocytes in the rat. J. Exp. Med. 124: 1017-1030. Groves, D. L., W. E. Lever and T. Makinodan, 1970. A model for the interaction of cell types in the generation of hemolytic plaque-forming cells. J. Immunol. 104: 148-165. Halasa, J., 1968. Plaque forming cells in the spleen, lymph and blood. Folia Microbiol. 13: 253-258. Hanna, M. G., D. C. Swartzendruber and C. C. Congdon, 1966. Morphologic changes in spleen lymphatic tissue during antibody production. Exptl. Molec. Pathol. (Sup. 3 ) : 75-87. Harris, T. M., E. Grimm, E. Mertens and W. E. Ehrich, 1945. The role of the lymphocyte in antibody formation. J. Exp. Med. 8 1 : 73-83. Hiramoto, R. N., N. M. Hamlin and H. L. Harris, 1968. The detection of antibody-containing cells in the blood stream during the IgM cellular antibody response. J. Immunol. 100: 637640. Hulliger, L., and E. Sorkin, 1963. Synthesis of antibodies by blood leucocytes of the rabbit. Nature, 198: 299. Jerne, N. K., A. A. Nordin and C. Henry, 1963. The agar plaque technique for recognizing antibody-producing cells. In Cell Bound Antibodies (ed. B. Amos and H. Koprowski) pp. 109-125, Wistar Inst. Press, Philadelphia. Kearny, R., and W. J. Halliday, 1965. Enumeration of antibody-forming cells in the peripheral blood of immunized rabbits. J. Immunol. 95: 109-112. Keily, S. D., and P. Abramoff, 1969. Studies of the chicken immune response. III. Cellular and humoral antibody production in the splenectomized chicken. J. Immunol. 102: 1058-1063. Landy, M., R. P. Sanderson, M. T. Berstein and A. L. Jackson, 1964. Antibody production by leucocytes in peripheral blood. Nature, 204: 1320-1321. Leduc, E. H., A. H. Coons and J. M. Connolly,
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The author wishes to express appreciation to Dr. T. Makinodan and Dr. J. F. Albright for their encouragement and valuable suggestions during the writing of this paper.
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at A Olt.
Seto, F., 1970. Antibody production in chick embryo hosts by allogeneic donor cells. Proc. Oklahoma Acad. Sci. 50: 45-48. Seto, F., and W. G. Henderson, 1968. Natural and immune hemagglutinin forming capacity of immature chickens. J. Exp. Zool. 169: 501-512. Shearer, G. M., G. Cudkowicz, M. St. J. Connell and R. L. Priore, 1968. Cellular differentiation of the immune system of mice. I. Separate splenic antigen-sensitive units for different antisheep antibody-forming cells. J. Exp. Med. 128: 437-457. Solomon, J. B., 1968. Ontogeny of cells producing
haemolytic antibody or immunocyte-adherence to sheep erythrocytes in chickens. Immunology, 14: 611-619. Sterzl, J., 1967. The effect of immunosuppressive drugs at various stages of differentiation of immunologically competent cells. In Immunity, Cancer and Chemotherapy, (ed. E. Mihich), pp. 71-99, Academic Press, N.Y. Urso, P., and T. Makinodan, 1963. The roles of cellular division and maturation in the formation of precipitating antibody. J. Immunol. 90: 897-907. Vasquez, J. J., and T. Makinodan, 1966. Cytokinetic events following antigenic stimulation. Fed. Proceed. 25: 1727-1733. Weber, W. T., and W. P. Weidanz, 1969. Prolonged bursal lymphocyte depletion and suppression of antibody formation following irradiation of the bursa of Fabricius. J. Immunol. 103: 537-543. Wissler, R. W., F. W. Fitch, M. F. La Via and C. H. Gunderson, 1957. The cellular basis for antibody formation. J. Cell. Comp. Physiol. 50 (sup. 1): 265-301. Wolfe, H. R., S. Norton, E. Springer, M. Goodman and C. A. Herrick, 1950. Precipitin production in chickens. V. The effect of splenectomy on antibody formation. J. Immunol. 64: 179-184. Zaalberg, O. B., 1964. A simple method for detecting single, antibody-forming cells. Nature, 212: 1231.
NEWS AND NOTES (Continued from page 1661) Street, Lancaster, Pennsylvania 17604. Bound: Vol. 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,31,32,33 and 34. Unbound: Vol. 38, No. 4; Vol. 39, No. 3 and S; Vol. 41, No. 2; Vol. 42, No. 2, 3, 4 and 5; and Vol. 44, No. 2.
Science at the University of Idaho were combined in a new Department of Animal Industries. Dr. A. M. Mullins, formerly Professor of Animal Science at Louisiana State University has been named Head of the new Department.
A.I.N. FELLOW At the annual banquet of the American Institute of Nutrition, held at the Shelburne Hotel, on April 15, Dr. H. M. Scott was one of three members appointed Fellows. The citation read: "Harold M. Scott—for over 40 years of dedicated service in undergraduate and graudate teaching and for meritorious research in IDAHO NOTES avian physiology and nutrition. His contribution On July 1, the Departments of Animal Science, to experimental avian nutrition, related to the dePoultry Science, and the production phases of Dairy velopment of high energy rations for broilers and (Continued on page 1727)
HUBBARD NOTES Lowell R. Blass, Hubbard Farms, Walpole, New Hampshire, received an award for outstanding design and sales messages in a trade publication campaign in the annual competition of the Advertising Club of North Jersey.
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19S5. Studies on antibody production. II. The primary and secondary responses in the popliteal lymph node of the rabbit. J. Exp. Med. 102: 61-71. Linna, T. J., T. Brenning and E. Hemmingsson, 1968. Lymphoid cell migration and the germinal centers. Exptl. Hematol. 17 : 4. Makinodan, T., and G. B. Price, 1970. Radiation effects in immune response: Its significance to transplantation. Transplantation (in press). Mitchison, N. A., 19S7. Adoptive transfer of immune reactions by cells. J. Cell. Comp. Physiol. 50(sup. 1 ) : 247-264. Mosier, D. E., 1967. A requirement for two cell types for antibody formation in vitro. Science, 158: 1573-1575. Seto, F., 1968. Immunocytes in the peripheral blood of immunized chickens. Amer. Zool. 8 :
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antibiotic gentamicin. J. Infect. Dis. 119: 518527. Waitz, J. A., and M. J. Weinstein, 1969. Recent microbiological studies with gentamicin. J. Infect. Dis. 119: 355-360. Weinstein, M. J., G. H. Wagman, E. M. Oden and J. A. Marquez, 1968. Biological activity of the antibiotic components of the gentamicin complex. J. Bact. 94: 789-790. Weinstein, M. J., G. M. Luedemann, E. M. Oden, G. H. Wagman, J. P. Rosselet, J. A. Marquez, C. T. Coniglio, W. Charney, H. L. Herzog and J. Black, 1963. Gentamicin, a new antibiotic complex from Micromonospora. J. Med. Chem. 6: 463-464. Wersall, J., P. G. Lundquist and B. Bjorkroth, 1969. Ototoxicity of gentamicin. J. Infect. Dis. 119: 410-416. Worcester, W. W., 1965. Californians report results of test on paracolon control. Feedstuffs, 37: 6ff. White, A., 1964. In vitro activity of gentamicin. Antimicrob. Agents and Chemotherapy, 1963. p. 17-19.
Immunocompetent Cells in the Blood of Immunized Chickens1 FRANK SETO
Zoology Department, University of Oklahoma, Norman, Oklahoma 73069 (Received for publication June 17, 1970) INTRODUCTION
A
NTIGEN stimulation initiates the • production of specific antibody that reaches a peak concentration in the blood several days later. Concomittant with the antibody response are characteristic histological changes in the activated lymphoid tissues, the proliferation of lymphocytes and differentiation of plasma cells (Leduc et al., 1955; Urso and Makinodan, 1963; Hanna et al., 1966). Direct correlation between the increase in antibody output and an increase in antibody-forming cells in the activated lymphoid organs of mammals 1 Research aided by the Faculty Research Fund of the University of Oklahoma.
have been clearly demonstrated with the agar plaque technic (Jerne et al., 1963) and the immunocytoadherence test (Biozzi et al., 1967). Adoptive immunity experiments have confirmed that spleen and lymph node of antigen-primed mammals contain cells capable of antibody synthesis in suitable hosts (Mitchison, 1957; Albright et al., 1964). Antigen stimulates immunological stem cells to proliferate and produce (a) populations of antibody-forming cells, and (b) reserves of immunologically activated precursors and memory cells which respond anamnestically to subsequent antigen exposure and are responsible for the persistence of immunological mem-
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Kowalski, L. M., and J. F. Stevens, 1968. Arizona 7 : 1 , 7 , 8 infection in young turkeys. Avian Dis. 12: 317-326. Peluffo, C. A., P. R. Edwards and D. W. Bruner, 1942. A group of coliform bacilli serologically related to the genus Salmonella. J. Infect. Dis. 70: 185-192. Rubenfire, M., H. H. Glass, A. S. Goldstein and A. M. Lerner, 1969. Gentamicin therapy of Paracolobactrum epidural abcess and meningitis. Amer. J. Med. Sci. 257: 191-197. Sadler, W. W., R. Yamamoto, H. E. Adler and G. F. Stewart, 1961. Survey of market poultry for Salmonella infection. Applied Microbiol. 9: 7276. Smith, C. B., P. E. Dans and J. N. Wilfert, 1969. The use of gentamicin in combinations with other antibiotics. J. Infect. Dis. 119: 370-377. Stone, H. H., J. D. Martin, Jr., W. E. Huger and L. Kolb, 1965. Gentamicin sulfate in the treatment of Pseudomonas sepsis in burns. Surg. Gynec. Obstet. 120: 351-352. Waisbren, B. A., 1969. Experiences with the new
PARACOLOBACTRUM
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MATERIALS AND METHODS Materials. Juvenile (3^ to 12 weeks old) and baby White Leghorn were donors and White Leghorn embryonated eggs served as
hosts. These were purchased from a local commercial hatchery. METHODS
The in vivo culture system (Albright et al., 1964), as modified for the allogeneic chicken system, detects precursor cells and memory cells, which will be henceforth referred to as immunocompetent cells (ICC), that respond to antigenic challenge. Donor cells, blood and cell suspensions of other tissues, from antigen stimulated chickens are exposed in vitro to antigen, cultured in chick embryo hosts and assayed for antibody production. Appropriate controls indicate that the antibody formed in host embryos do not result from passive transfer of pre-existing antibody-forming cells. The main antigen, mouse erythrocytes (Mrbc), was obtained by cardiac puncture in equal volume of Alsever's solution, washed in sterile 0.15 M NaCl three times, packed by centrifugation and cell suspensions prepared in saline solution. Sheep erythrocytes (Srbc), obtained commercially (Brown Laboratory, Topeka, Kansas) , was used as the other antigen. Prospective donor White Leghorn chickens were immunized with either Mrbc or Srbc. While blood was obtained from each chicken by cardiac puncture in equal volume of Alsever's solution. After centrifugation most of the plasma was discarded and enough antigen suspension was added to restore the original blood volume. A standard amount (0.1 ml.) of donor blood, with or without 4 X 107 washed Mrbc or Srbc depending on the experimental group, was injected intravenously into the chorioallantoic vessel of 14-day White Leghorn embryo recipients. Donor blood was used soon after collection or stored at ice water temperature no longer than two hours before use. Immediately after the antigen was added to the donor blood and thoroughly
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ory (Vasquez and Makinodan, 1966; Sterzl, 1967). Some of the lymphocytes remain in the lymphoid organs whereas others emigrate and presumably enter the general circulation (Harris et al., 1945; Wissler et al., 1957). It is known that blood leucocytes are involved in homograft rejection, graftversus-host reaction, and delayed hypersensitization (Billingham and Barker, 1969), but it is less well established that they are capable of antibody synthesis (Bach and Hirschhorn, 1963). Recent studies employing isotope incorporation techniques, agar plaque assay, immunocytoadherence test, and radioimmunoelectrophoresis have demonstrated antibodyproducing cells in the blood of mammals shortly after immunization (Hulliger and Sorkin, 1963; Landy et al., 1964; Zaalberg, 1964; Kearny and Halliday, 1964; Chessinei al., 1968). Although less documented, the situation is essentially similar in chickens. Histological observations in conjunction with enumeration of hemolytic plaque-forming cells indicate that cellular changes similar to that reported in mammals occur in the spleen of chickens during the immune response (Abramoff and Brien, 1968; Solomon, 1968). Also antibody-producing cells, demonstrated only by the immunocytoadherence test (Duffus and Allan, 1969) and other immunocompetent cells appear in the blood soon after antigenic stimulation (Seto, 1968). This report describes a kinetic analysis of immunocompetent cells in the blood of chickens, as assessed by the in vivo culture technic.
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IMMUNOCOMPETENT CELLS
mixed it was injected into host embryos. Injection of a group of hosts took several minutes. Some of the donors were killed and the spleen, thymus, bone marrow and bursa of Fabricius were used to prepare cell suspensions. The organs were individually teased apart in sterile Medium 199 (Baltimore Biological Laboratory) to which was added about 2 % by volume of normal chicken se-
14 12 10 8 6 4
a.
2
UJ
*•
B.
3.5 w
C.
3d
15
O
RESULTS 1
2 3 4 5 6 7 8 9 33d T I M E A F T E R ANTIGEN INJECTION
FIG. 1. Effect of antigen dose on the appearance of immunocompetent cells in the blood of chickens of different ages. The response profiles of 9j-week (A), 3^-week (B), and 3-day (C) chickens are shown. Antigen doses were 10' (A), 10s ( • ) , and iO9 (A) Mrbc in a single injection. The S-day mean hemagglutinin responses of 3^-week and 9iweek donors and 7-day response of 3-day donors are shown as a broken line ( ).
The response profile of ICC in the blood of immunized chickens. The initial experiment was conducted to determine the relative concentration of ICC in the blood at various times after immunization. Donor chickens, 9\ weeks of age, were injected with 107, 108, 109, and 1010 washed Mrbc in 1 ml. of.0.15 M NaCl. Blood samples were obtained at intervals from these chickens and assayed in 4 to 6 host em-
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16
rum, dispersed further by gently flushing the fragments through a syringe with a 22 gauge needle and clumps strained out with stainless steel cloth (200 mesh/inch). The cells were washed once with fresh Medium 199, centrifuged, and the supernatant discarded. The pellet was resuspended in fresh medium and a cell count was made with a hemacytometer. Approximately 4-5 X 106 cells in 0.1 ml. of Medium 199, with or without 4 X 10' Mrbc or Srbc as antigen depending on the experiment, were injected intravenously into embryo hosts. Six days later the embryos were bled and antibody titers of the serum samples were estimated by the Microtiter hemagglutination technic (Cooke Engineering, Alexandria, Va.). Preliminary experiments indicated that antibody production in the in vivo hosts was proportional to the amount of inoculated donor blood and thus provided an indirect quantitative assessment of the relative ICC content of the inoculum. Quantitatively similar titers were were obtained, moreover, whether cyclophosphamide-inactivated or untreated 14-day embryos were used as hosts (Seto, 1970). Host embryos at this age are immunologically immature and generally incapable of rejecting the donor cells (Seto, 1970) or responding by hemagglutinin production to heterologous erythrocytes (Seto and Henderson, 1968).
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TABLE 1.—Relative antibody-forming capacity of cells from different tissues of immunized donors1 • Frequency of £1 ^ ' t , Experimental groups r e 5 „ o n d e r s body titer in Antigen only Blood only Bursa+antigen Thymus+antigen Bone marrow-(-antigen Spleen+antigen Blood+antigen 1
^
l0g2
0/6 0/4 0/13 4/17 3/6 16/16 17/1?
0.0 0.0 0.0 0.5 1.0 8.3 8.8
Assessed with the in vivo assay method
Immunocompetent cells in other tissues of immunized chickens. Other experiments were conducted to detect the presence of ICC in other tissues of 5-week donors immunized 4 days earlier. The results of one experiment are summarized in Table 1. As estimated by antibody titers in assay embryos, the ICC were infrequent in the cells suspensions from thymus, bone marrow and bursa of Fabricius. They were abundant only in the blood and spleen. When blood from 5 control unimmunized donors obtained at 4, 5, 6 and 8 weeks of age were similarly tested, no definite positive responded were found among 118 recipients. Although not shown, similar results were obtained with tissues from donors 3 and 6 Donor age and concentration of ICC in the days after immunization, and with older blood. Other experiments were conducted to donors as well. determine the ICC response profiles of The response profiles of ICC in the more immature birds. Juvenile chickens, blood and spleen were compared. Chickens, 3^ weeks of age were injected intracar- 6-12 weeks of age, were immunized with dially with 107, 108, 109, 1010 washed Mrbc Mrbc, then sacrificed at specified time inin 1 ml. of saline solution and their blood tervals and their blood and spleen assayed. assayed at intervals. As with the 9J week The results are shown in Fig. 2. Immunodonors, the response profiles of donors ex- competent cells were detected in the spleen posed to higher doses were much alike and but not in the blood the first day after imthat of the donor receiving the least dose munization. After the second day they were was reduced (Fig. IB). The time of peak present in both tissues and the response
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bryos per blood sample per donor. The mean 6-day anti-mouse hemagglutinin titers obtained in the in vivo hosts for each donor, sampled on different days after immunization, are summarized in Fig. 1A. The response curves for antigen doses of 10s, 109, and 1010 Mrbc were alike and for the sake of clarity in the figure, that for 1010 Mrbc was omitted. Peak concentration of ICC, as reflected in the in vivo antibody titers, were observed in all donors irrespective of antigen dose, by the 3rd day and remained high for many days. Except for the reduced response with the donor exposed to the lowest dose (10 7 ), differences in levels of response were not apparent with the other doses during the first week but the graded differences in levels of these immunocompetent cells persisting in the blood 33 days after immunization was dose dependent.
ICC concentration was 2 days later. Experiments with 7^-week donors immunized with 107 to 1010 washed Srbc showed response profiles essentially like that of the 3^-week group. When 3-day baby chicks were injected with 10s washed Mrbc, only 2 of 10 birds showed definite ICC profiles although 9 of 10 had respectable 7-day hemagglutinin titers. The response profiles of these two birds were averaged and plotted in Fig. 1C. It is apparent that the capacity to produce ICC as measured by their relative amount and persistence in the blood following immunization increases with the maturity of the donors.
IMMUNOCOMPETENT CELLS
patterns were essentially alike although it appears that a more rapid decline occurs in the spleen.
1 2 TIME
AFTER
3 4 ANTIGEN
5 days INJECTION
FIG. 2. A comparison of the response profiles of immunocompetent cells of blood (0)> and spleen ( • ) of 6 to 12-week old White Leghorn donor chickens. 1-to 4 donors were used in each time interval and the number of assay embryos in the sample is shown next to each point.
maximal production of ICC during the first week that tend to obscure any antigen dosage effect. Later as the production levels off, the antigen dosage effect becomes apparent in the graded levels of immunocompetent cells persisting several weeks later. With sufficiently low antigen dose, a slower response with reduced ICC level is induced that rapidly declines. The blood ICC response of 3-day chicks was weak and brief and probably reflects the immature state of the immune system. The response of 3^-week chickens was considerably greater but that of the 9£week chickens was very rapid, and persisted at high levels. The capacity to produce ICC, as estimated from the peak blood levels, increases over 200-fold during the first month after hatching and an additional 4-fold by the second month, which is comparable in magnitude to the growth of the hemagglutinin-forming potential reported in the chicken earlier (Seto and Henderson, 1968). It has been suspected for some time and later confirmed that precursors of antibody-forming cells circulate in the blood (Gowan and Uhr, 1968; Linna et al., 1968; Davies et al., 1968) and antibodyproducing cells may occur in the blood. It is not readily evident from the literature (Hullinger and Sorkin, 1963; Kearny and Halliday, 1965; Landy et al, 1964; Halasa, 1968; Hiramoto et al., 1968; Duffus and Allan, 1969) or from our results whether these precursor and antibody-producing cells originate from stem cells in the blood or in extravascular sites. There is much indirect evidence, in addition to that presented here however, that suggest their formation in lymphoid tissues in mammals (Leduc et al., 1955; Wissler et al., 1957; Hanna et al., 1966) and in chickens (Wolfe et al., 1950; Abramoff and Brien, 1968; Keily and Abramoff, 1969). Immunocompetent cells occur abundantly in the
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DISCUSSION In our experience immunocompetent cells (ICC) that respond with specific hemagglutinin formation to heterologous erythrocytes are normally absent in the blood but may occur infrequently in the spleen of young unimmunized chickens. Following antigenic stimulation with mammalian erythrocytes, however, they appear in the blood within 2 days and reach peak concentration by the 3rd to 5th day. This period of rise to peak concentration coincides with the logarithmic phase of the hemagglutinin response (Seto and Henderson, 1968). Whether the ICC persisted or declined in the blood was related to the antigen dose and the maturity of the donor. Despite a hundred-fold difference in the amount of antigen administered (108 to 1010), these doses elicited quantitatively similar ICC response profiles during the first week. Apparently moderate to high doses stimulate
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F. SETO
1967) but may include hemocytoblasts as well in chickens (Duffus and Allan, 1969). The ICC include cells that behave much like the proposed memory cells which have been presumed to be small lymphocytes (Gowan and Uhr, 1966; Burnet, 1968). The situation is not as simple in practice, as it now appears that 2 or 3 different cell types—thymus derived antigen-reactive cell, bone marrow-originated antibodyprecursor cell, and the antigen-processing macrophage—are necessary in some way for the immune response (Fishman, 1961; Mosier, 1967; Groves et al., 1970). The identity of the ICC is less clear but functionally it behaves much like the antigensensitive unit, a synergistic combination of antigen-reactive cells and antibody precursor cell (Shearer et al., 1968; Makinodan and Price, 1970). Experiments are in progress to elucidate the nature of the ICC in the blood of chickens. SUMMARY
A modified in vivo culture technic was used in a kinetic study of immonocompetent cells (ICC) in the blood and other tissues of immunized chickens. Cells from antigen-stimulated donors were mixed in vitro with antigen, transferred to 14-day chick embryo hosts, and assayed for antibody production. Of the tissues tested, ICC occurred abundantly in blood and in the spleen but were infrequent in the thymus, bone marrow and bursa of Fabricius. The relative concentration of ICC in the blood varied according to (1) the time after antigen injection, (2) the amount of antigen administered, and (3) the maturity of the donor chickens. Within two days after immunization ICC appeared in the blood, reached a peak concentration on the 3rd or 4th day and then declined. In juvenile birds (3^- and 9hweek old) the ICC response profiles were essentially alike for moderate to high anti-
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spleen of chickens, but are infrequent in the thymus and bone marrow as reported in mammals (Claman et al., 1966; Davies et al., 1967), and also in the bursa of Fabricius. This latter fact does not negate the importance of the bursa in the development of the immune function (Good and Papermaster, 1964; Weber and Weidanz, 1969) and might be expected in current models. Following antigenic stimulation in mammals the antibody-producing cells appear in the blood for a short duration, reach peak concentration on the 3rd and 4th day and then rapidly decline in numbers, whereas in chickens the ICC response profiles described here are more conspicuous and of longer duration. The differences in the pattern could be attributable in part to species differences, antigen dose, age, and previous antigen exposure. It is also conceivable that the type of immunological cell involved may result in different response profiles. The direct correlation between the increase in antibody output and the increase in antibody-forming cells detected by both agar plaque technic and the immunocytoadherence test (Jerne et al., 1963; Biozzi et al., 1967) indicates that the cell type reported in mammalian blood is the antigen activated, terminally differentiated antibody-producing cells (Z or P cells), that have spilled into the blood stream; whereas the cell types investigated here include mainly immunologically activated antigen-sensitive cells (Y or PC 2 ) responsible for immunological memory (Sterzl, 1967; Vasquez and Makinodan, 1966). As to the nature of the blood-borne immunocompetent cells, those involved in cellular immune responses are known to be lymphocytes (Billingham and Barker, 1969) and the antibody-producing types, identified by several technics, are mainly lymphocytes and plasma cells in mammals (Leduc et al., 1955; Urso and Makinodan, 1963; Jerne et al., 1963; Biozzi et al.,
IMMUNOCOMPETENT CELLS
gen doses and perceptibly reduced at the low dose tested. The response profile of 3day chicks was weak and brief, that of 9£week chickens was rapid, high and prolonged, and that of 3^-week chickens was intermediate. Although the ICC has not been identified as to cell type, it behaves much like the immunological memory cells. ACKNOWLEDGMENTS
REFERENCES Abramoff, P., and N. B. Brien, 1968. Studies of the chicken immune response. I. Correlation of the cellular and humoral immune response. J. Immunol. 100: 1204-1209. Albright, J. F., T. Makinodan and E. E. Capalbo, 1964. Factors regulating antibody production by spleen cells cultured in vivo. Proc. 9th Congr. Intern. Blood Transf. Mexico, pp. 301308. Bach, F., and K. Hirschhorn, 1963. Y-gl°bul>n production by human lymphocytes in vitro. Exp. Cell. Res. 32: 592-595. Billingham, R. F., and C. F. Barker, 1969. Recent developments in transplantation immunity Part I. Plastic and Reconstr. Surg. 43: 559-568. Biozzi, G., C. Stiffel and D. Mouton. 1967. A study of antibody-containing cells in the course of immunization. In Immunity, Cancer and Chemotherapy (ed. by E. Mihich), pp. 113130, Academic Press. Burnet, F. M., 1968. Evolution of the immune process in vertebrates. Nature, 218: 426^130. Claman, H. N., E. A. Chaperon and R. F. Triplett, 1966. Immunocompetence of transferred thymus-marrow cell combinations. J. Immunol. 97: 828-832. Chessin, L. N., P. R. Glade, R. Asofsky, P. C. Baker, R. Reisfeld and W. Terry, 1968. Studies on human peripheral blood lymphocytes in vitro. V. Biosynthesis of immunoglobulins. J. Immunol. 101: 458-468. Davies, A. J. S., E. Leuchars, V. Wallis, R. Marchant and E. V. Elliott, 1967. The failure of thymus-derived cells to produce antibody. Transplantation, S: 222-231. Davies, A. J. S., H. Festenstein, E. Leuchars, V. J.
Wallis and M. J. Doenhoff, 1968. The origin of some mouse peripheral blood lymphocytes. Exptl. Hematol. 17: 12. Duffus, W. P. H., and D. Allan, 1969. A study of the ontogeny of specific immune responsiveness amongst circulating leucocytes in the chicken. Immunology, 16: 337-347. Fishman, M. J., 1961. Antibody formation in vitro. J. Exp. Med. 114: 837-856. Good, R. A., and B. W. Papermaster, 1964. Ontogeny and phylogeny of adaptive immunity. Adv. Immunol. 4 : 1-115. Gowan, J. C , and J. W. Uhr, 1966. The carriage of immunological memory by small lymphocytes in the rat. J. Exp. Med. 124: 1017-1030. Groves, D. L., W. E. Lever and T. Makinodan, 1970. A model for the interaction of cell types in the generation of hemolytic plaque-forming cells. J. Immunol. 104: 148-165. Halasa, J., 1968. Plaque forming cells in the spleen, lymph and blood. Folia Microbiol. 13: 253-258. Hanna, M. G., D. C. Swartzendruber and C. C. Congdon, 1966. Morphologic changes in spleen lymphatic tissue during antibody production. Exptl. Molec. Pathol. (Sup. 3 ) : 75-87. Harris, T. M., E. Grimm, E. Mertens and W. E. Ehrich, 1945. The role of the lymphocyte in antibody formation. J. Exp. Med. 8 1 : 73-83. Hiramoto, R. N., N. M. Hamlin and H. L. Harris, 1968. The detection of antibody-containing cells in the blood stream during the IgM cellular antibody response. J. Immunol. 100: 637640. Hulliger, L., and E. Sorkin, 1963. Synthesis of antibodies by blood leucocytes of the rabbit. Nature, 198: 299. Jerne, N. K., A. A. Nordin and C. Henry, 1963. The agar plaque technique for recognizing antibody-producing cells. In Cell Bound Antibodies (ed. B. Amos and H. Koprowski) pp. 109-125, Wistar Inst. Press, Philadelphia. Kearny, R., and W. J. Halliday, 1965. Enumeration of antibody-forming cells in the peripheral blood of immunized rabbits. J. Immunol. 95: 109-112. Keily, S. D., and P. Abramoff, 1969. Studies of the chicken immune response. III. Cellular and humoral antibody production in the splenectomized chicken. J. Immunol. 102: 1058-1063. Landy, M., R. P. Sanderson, M. T. Berstein and A. L. Jackson, 1964. Antibody production by leucocytes in peripheral blood. Nature, 204: 1320-1321. Leduc, E. H., A. H. Coons and J. M. Connolly,
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The author wishes to express appreciation to Dr. T. Makinodan and Dr. J. F. Albright for their encouragement and valuable suggestions during the writing of this paper.
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at A Olt.
Seto, F., 1970. Antibody production in chick embryo hosts by allogeneic donor cells. Proc. Oklahoma Acad. Sci. 50: 45-48. Seto, F., and W. G. Henderson, 1968. Natural and immune hemagglutinin forming capacity of immature chickens. J. Exp. Zool. 169: 501-512. Shearer, G. M., G. Cudkowicz, M. St. J. Connell and R. L. Priore, 1968. Cellular differentiation of the immune system of mice. I. Separate splenic antigen-sensitive units for different antisheep antibody-forming cells. J. Exp. Med. 128: 437-457. Solomon, J. B., 1968. Ontogeny of cells producing
haemolytic antibody or immunocyte-adherence to sheep erythrocytes in chickens. Immunology, 14: 611-619. Sterzl, J., 1967. The effect of immunosuppressive drugs at various stages of differentiation of immunologically competent cells. In Immunity, Cancer and Chemotherapy, (ed. E. Mihich), pp. 71-99, Academic Press, N.Y. Urso, P., and T. Makinodan, 1963. The roles of cellular division and maturation in the formation of precipitating antibody. J. Immunol. 90: 897-907. Vasquez, J. J., and T. Makinodan, 1966. Cytokinetic events following antigenic stimulation. Fed. Proceed. 25: 1727-1733. Weber, W. T., and W. P. Weidanz, 1969. Prolonged bursal lymphocyte depletion and suppression of antibody formation following irradiation of the bursa of Fabricius. J. Immunol. 103: 537-543. Wissler, R. W., F. W. Fitch, M. F. La Via and C. H. Gunderson, 1957. The cellular basis for antibody formation. J. Cell. Comp. Physiol. 50 (sup. 1): 265-301. Wolfe, H. R., S. Norton, E. Springer, M. Goodman and C. A. Herrick, 1950. Precipitin production in chickens. V. The effect of splenectomy on antibody formation. J. Immunol. 64: 179-184. Zaalberg, O. B., 1964. A simple method for detecting single, antibody-forming cells. Nature, 212: 1231.
NEWS AND NOTES (Continued from page 1661) Street, Lancaster, Pennsylvania 17604. Bound: Vol. 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,31,32,33 and 34. Unbound: Vol. 38, No. 4; Vol. 39, No. 3 and S; Vol. 41, No. 2; Vol. 42, No. 2, 3, 4 and 5; and Vol. 44, No. 2.
Science at the University of Idaho were combined in a new Department of Animal Industries. Dr. A. M. Mullins, formerly Professor of Animal Science at Louisiana State University has been named Head of the new Department.
A.I.N. FELLOW At the annual banquet of the American Institute of Nutrition, held at the Shelburne Hotel, on April 15, Dr. H. M. Scott was one of three members appointed Fellows. The citation read: "Harold M. Scott—for over 40 years of dedicated service in undergraduate and graudate teaching and for meritorious research in IDAHO NOTES avian physiology and nutrition. His contribution On July 1, the Departments of Animal Science, to experimental avian nutrition, related to the dePoultry Science, and the production phases of Dairy velopment of high energy rations for broilers and (Continued on page 1727)
HUBBARD NOTES Lowell R. Blass, Hubbard Farms, Walpole, New Hampshire, received an award for outstanding design and sales messages in a trade publication campaign in the annual competition of the Advertising Club of North Jersey.
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19S5. Studies on antibody production. II. The primary and secondary responses in the popliteal lymph node of the rabbit. J. Exp. Med. 102: 61-71. Linna, T. J., T. Brenning and E. Hemmingsson, 1968. Lymphoid cell migration and the germinal centers. Exptl. Hematol. 17 : 4. Makinodan, T., and G. B. Price, 1970. Radiation effects in immune response: Its significance to transplantation. Transplantation (in press). Mitchison, N. A., 19S7. Adoptive transfer of immune reactions by cells. J. Cell. Comp. Physiol. 50(sup. 1 ) : 247-264. Mosier, D. E., 1967. A requirement for two cell types for antibody formation in vitro. Science, 158: 1573-1575. Seto, F., 1968. Immunocytes in the peripheral blood of immunized chickens. Amer. Zool. 8 :