Laurens N. Ruben

Wm. R. Kenan Jr. Professor, Emeritus
Work Address: Room 138 Department of Biology, Reed College, Portland, OR 97202-8199
Home Address: 3108 SE Crytstal Springs Blvd., Portland, OR. 97202
Telephone: Work=(503) 777-7276; Home=(503) 771-6948; Fax: (503) 777-7773;
e-mail: laurens_ruben@.reed.edu

 


SELECTED ORIGINAL CONTRIBUTIONS
OF THE LABORATORY
TO IMMUNOLOGIC ISSUES.


The contributions are organized into the following categories:

MACROPHAGE
FUNCTION
T CELL
FUNCTIONS
B CELL
FUNCTIONS
ANAMNESIS
HAPTEIN-SPECIFIC
TOLERANCE
APOPTOSIS
METAMORPHOSIS


MACROPHAGE FUNCTION:
1. Phagocyte blockade will inhibit responses to heterologous RBC’s in the newt. Ruben, L.N., Jonnes, H. and Stack, J. In " Aspects of Developmental and Comparative Immunology I. ", Ed. Solomon, B.J., pp.171-178. Pergamon Press, Oxford, U.K. (1981).
2. The newt can be made to respond to soluble antigens, e.g., keyhole limpet hemocyanin, and helper function can be demonstrated with them, when the antigens are bound to particles. Ruben, L.N. and Stack, J. Dev. Comp. Immunol.6:491-498 (1982).
3. The peritoneal macrophages of Xenopus will take up less radio-labelled ovalbulmin and degrade a smaller percent of what they have taken in, than comparable cells of the mouse. Moreover, newt Mø are unable to take any in, unless it is presented adsorbed to particles. Thus, the failure of primitive vertebrates to respond to soluble immunogens is due to a failure of their Mø's to take up the potential immunogen. Gammie, A. and Ruben, L.N. Cell Immunol.100:577-231 (1986).
4. Antigen-competition can be demonstrated in Xenopus, but not the newt, when two species of RBC’s are injected sequentially. The cell type responsible appears to be the macrophage. Ruben, L.N. and Mette, S.A., Cell Immunol. 51:379-389 (1980).

T CELL FUNCTIONS:
CELL-MEDIATED IMMUNITY:
1. Immunologic factors determine the ability of different frog and salamander organ implants to stimulate the growth capacity of regeneration potent salamander limb tissues. Ruben, L.N. Amer. Nat. 94:427-434 (1960).
2. Lymphoreticular cancer development in Xenopus laevis , the South African clawed toad, suppresses normal allograft rejection capacities. Ruben, L.N. Amer. Zool. 11:229-237 (1971).
3. The cyclical destruction and re-growth in vivo of foci of lymphoreticular cancer in Xenopus depends on immunologic maturation for its existence e.g., Ruben, L.N. Dev. Biol. 22: 43-58 (1970).
4. Mature lymphoid tissue implants suppress the development of the allograft response in larvae of Xenopus differentially. Ruben, L.N., Stevens, J.M and Kidder, G.M. J. Morph. 138:457-466 (1972).

HELPER FUNCTION:
1. The capacity of responsivity to heterologous SRBC relates to spleen lymphocytic development. Its cytodynamics in Xenopus laevis are similar to those in mammals . Kidder, G.M., Ruben, L.N. and Stevens, J.M. J. Embry. Exp. Morph. 29:78-85 (1973).
2. Carrier dependent and specific helper function can be demonstrated in a "lower" vertebrate e.g. the newt, Notophthalmus viridescens. Ruben, L.N., Van der Hoven, A. and Dutton, R.W. Cell Immunol. 6:300-314 (1973).
3. Carrier dependent and specific helper function can also be demonstrated in the goldfish, Carassius auratus.. Ruben, L.N., Warr, G.W., Decker, J.M. and Marchalonis, J.J. Cell Immunol. 31:266-283 (1977).
4. Helper function in Xenopus is thymus-related. Gruenewald, D. and Ruben, L.N., Immunol. 38: 191-194 (1979).
5. T cell stimulating lectins e.g. Con A , but not wheat germ agglutinin, can substitute for helper function in Xenopus. Clothier, R.H., James, H.S., Ruben, L.N. and Balls, M. Immunol. 52:703-709 (1984).
6. Human IL-2 will substitute for helper function in Xenopus. Its capacity to do this will last for three hours prior to antigen challenge. Since rIL-2 plus carrier priming fail to further enhance an anti-hapten response, they are likely to be acting on the same cellular population which is maximumized by one or the other protocol. Ruben, L.N. Immunol. Letters. 13:227-230 (1986).
7. Human IL-1, particulate Con A , but not soluble Con A or soluble or particulate WGA or human IL-2, will substitute for carrier primed helper function in the newt. While anti-human IL-2 receptor antibody will bind specifically to freshly biopsied spleen cells, Il-2 will not compete with this binding. The shared lectin specificity suggests that T-like cells are responsible for helper function in this animal which has an "immature" thymus. The lymphokine and Con A data suggest that while human IL-2 is unable to affect the cells of this species, Con A and human IL-1 can, perhaps through the stimulation of autologous IL-2 production. Ruben, L.N., Beadling, C., Langeberg, L, Shiigi, S. and Selden, N. Thymus 11: 213-220 (1988).
8. The Xenopus spleen cells which are PHA activatable with regard to IL-2 receptor density and which bind rIL-2, are related to helper T cell function in cytotoxic and humoral immune responses, since it is removed by N-CH3-N-Nitrosourea (NMU) injection. NMU is selectively lymphotoxic in the toad and specifically removes these two functions. Panning spleen cells with a mAb to Xenopus IgM showed that those cells which bear constitutive IL-2 receptors and bind rIL-2 are equally abundant in the T and B cells populations of freshly biopsied Xenopus spleens. Langeberg, L., Ruben, L.N., Clothier, R.H., and Shiigi, S. Immunol. Letters 16: 43-48 (1987).
9. A monoclonal mouse anti-human IL-2 receptor antibody (anti-p55) will bind receptors on the surface of immunocytes of Xenopus that are only slightly larger than 55kDa. Ruben, L.N., Langeberg, L., Malley, A., Clothier, R.H., Lee, R.O. and Shiigi, S. Immunol. Letters 24:117-126 (1990).
10. Norepinephrine (NE) will stimulate helper function and suppressor function in adult Xenopus. Low dosage immunization will cause the release of NE in the spleen that will affect helper function positively and transiently, while high dosage immunization will affect NE release that is sustained and will stimulate suppressor function. The effect will be prolonged as long as antigen is around. Clothier, R.H., Ruben, L.N., Johnson, R.O., Parker, K., Greenhalgh, L., Ooi, E.E., Sovak, M. and Balls, M. Internat. J. Neurosci. 62: 123-140 (1992).
11. Mortality in developing larvae, particularly during the metamorphic period, can be manipulated by using IL-2 along with an antigen. Thus, it may be possible to lower the amount of IL-2 used in experimental cancer therapy by adding an antigen in conjunction with it (Ruben, L.N., De Leon R.T., Johnson, R.O. and Clothier, R.H. Interleukin-2-induced mortality during metamorphosis of Xenopus laevis. Immunol. Letts 51:157-161 (1996).

SUPPRESSOR FUNCTION:
1. Thymus-dependent suppressor function can be demonstrated in vitro with Xenopus laevis. Ruben, L.N., Mette, S.A, Edwards, B.F. and Cochran, S. Thymus 2:19-25 (1980).
2. Thymic suppressor function is antigen dependent, is only partially antigen-specific and is non-MHC restricted in Xenopus. Ruben, L.N., Beunafe, A. and Seivert, D. Thymus 5:13-18 (1982).
3. While thymus suppressor function is not MHC restricted, there are genetic limits to its capacity. R, L.N., James, H.S., Clothier, R.H. and Balls, M. J.Immunogenetics 11:97-102 (1983).
4. The differential temperature sensitivity of suppressor function to long exposure times of cold may account for immune response capacities of ectotherms during the long cold winters in temperate climes. Ruben, L.N., Clothier, R.H., Buenafe, A., Needham, P., James, H.S. and Balls, M. Thymus 6:143-152 (1984).
5. Suppressor function is compartmentalized in adult Xenopus:inducer suppressor cells are in the thymus, while inducer and effector suppressors are in the spleen. Moreover, both functions are sensitive to cyclophosphamide and are macrophage dependent. Ruben, L.N., Buenafe, A. Oliver, S., Malley, A., Barr, K and Lukas, D. Immunol. 54:65-70 (1985).
6. Lectins, e.g. wheat germ agglutinin, peanut agglutinin and Con A can initiate inducer suppressor function in the Xenopus thymus, but only WGA and PNA can stimulate splenic suppressor inducer function. Ruben, L.N., James, H.S., Clothier, R.H., Barr, K. and Balls, M. Thymus 7:161-167 (1985).
7. Suppressor inducer function of the Xenopus thymus, tested in vitro, is mediated by a soluble factor. Ruben, L.N., Barr, K, Clothier, R.H., Nobis, C. and Balls, M. Dev. Comp. Immunol. 9:811-818 (1985).
8. Larval thymocytes have suppressor function which will affect adult immunized splenocytes. However, during metamorphosis this function is impaired. This impairment is not due to suppressor cells migrating to the spleen, since the spleen is also lacking in effective suppressor function during metamorphosis. While wheat germ agglutinin is an effective stimulator of splenic suppressor function in adults, it fails to stimulate it during the metamorphic period. By using fluorescein-labelled WGA, we were able to show that this lack of capacity of wheat germ agglutinin to stimulate splenic suppressor function during metamorphosis was not due to the absence of binding by the Fl-WGA. Thus, the loss of function by the thymic suppressor cells did appear to occur as a consequence of the cells leaving for the spleen, the adult site of suppressor function. Kamali,D., Ruben, L.N. and Gregg, M., Cell. Diff. 18:225-231 (1986).
9. Corticosteroid regulation of IL-1 and IL-2 appears to be responsible for deficient immune suppressor function in the thymus during metamorphosis. Highet, A. and Ruben, L.N., Immunopharm. 13:149-155 (1987).
10. CyP, but not IL-2 will break the tolerance initiated to single haplotype disparate skin grafts made during metamorphosis that are sustained for at least 100 days. Thus, suppressor function appears to be involved in the maintenance of a tolerance which is susceptible to rIL-2 injection, while it is being established. Horton, J.D., Horton, T.L., Varley, C.A. and Ruben, L.N. Transplant. 47:883-887 (1989).
11. There are two types of suppressor cells with respect their differential sensitivity to CyP. Clothier, R.H., Last, Z., Samauroo, J., Ruben, L.N. and Balls, M. Devel. Comp. Immunol. 13: 159-166 (1989).
12. Xenopus suppressor factors will bind antibodies raised against inducer and effector suppressor factors of mouse in Western blots and in ELISA. Moreover, the inducer factor also binds an anti-IL-10 Ab raised against mouse IL-10. Recently, we have found that a protocol that generates suppressor inducer factors in Xenopus will apparently also stimulate mRNA expression particularly of IL-10, IL-5 and IL-4 genes in Xenopus, using RT-PCR with rat oligoprimers of the cytokines.

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B CELL FUNCTIONS:

RECOGNITION AND BINDING:
1. Antigen-binding receptors of primitive lower vertebrates vary in their capacity to discriminate among different haptens after hapten-specific challenge., Ruben, L.N. and Edwards, B.F. Cell Immunol. 31:437-442 (1977).
2. Alpha and ß adrenergics differentially effect the capacity of immunized B cells to bind antigen. Hodgson, R.M., Clothier, R.H., Ruben, L.N. and Balls, M. Eur. J. Immunol. 8:348-351 (1978).
3. Amphibian antibody and complement-dependent lysis is temperature dependent. Ruben, L.N., Edwards, B.F. and Rising, J. Experientia 33:1522-1523 (1977).
4. Immunologic stress may cause the newt to release a low MW antibody which is normally not produced. Warr, G.W., Ruben, L.N. and Edwards, B.F. Immunol. Letts. 4:99-102 (1982).

B CELL SUBPOPULATIONS-TI IMMUNOGENIC CARRIERS:

1. Two subpopulations of hapten-specific B cells, which are activated by different carriers, can be demonstrated in Xenopus. Horton, J.D., Edwards, B.F., Ruben, L.N. and Mette, S.A. Dev. Comp. Immunol. 3:621-633 (1979).
2. Two sub-populations of hapten-specific B cells which respond to different carriers can be demonstrated in the newt. Ruben, L.N.and Reischel,G. Dev. Comp. Immunol. 5:513-518 (1981).
3. Responsivity to TNP-Ficoll varies with developmental stage and internal endocrine environmental changes with age. Ruben, L.N., Clothier, R.H., James, H.S. and Balls, M. Cell. Diff. 14:1-5 (1984).
4. T cell-stimulating lectins, e.g. Con A can substitute for the thymus requirement for responses to TNP-Ficoll in Xenopus. Clothier, R.H., Ruben, L.N., James, H.S. and Balls, M. Immunol.52: 483-489 (1984).
5. Murine and human IL-2 can substitute for the thymus requirement in TNP-Ficoll responses in adult Xenopus. Ruben, L.N. Clothier, R.H. Balls, M., Cell. Immunol. 93:229-233 (1985).
6. The TNP-Ficoll response is regulated through two different pathways in Xenopus. The TNP-Ficoll response is T cell-requiring, but this requirement can be substituted for by any protocol which stimulates peripheral T cell activity, including allograft rejection or by optimizing the function of peripheral, but not thymic Mø's. Clothier, R.H., Ruben, L.N., Smart, C. and Balls, M. Dev. Comp. Immunol. 10:577-583 (1986).
7. Responses to TNP-PVP are very similar to those to TNP-Ficoll, i.e. the two will compete for B cells, except that the TNP-PVP is less thymus-dependent.Clothier, R.H., Kandola, L., Mirchandani, M., Ellis, A., Wood, P., Last, Z., Balls, M. and Ruben, L.N. Cell. Diff. 23:213-220 (1989); Clothier, R.H., Quaife, Y., Ali, I., Ruben, L.N. and Balls, M. Herpetopathol. 1:83-88 (1989).
8. Recently, we have found that mouse IL-10 will reduce B cell anti-TNP antibody production when they are exposed early in the immune response to TNP-LPS, but they will increase anti-TNP antibody production, when the exposure to IL-10 is delayed.

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ANAMNESIS:

1. Long term IgM related hapten-specific memory can be initiated in the newt by using TD and TI immunogenic carriers, but TD carriers are required for the second challenge if memory is to be expressed. Only primary responses of the newt are sensitive to Cyclosporine A. Ruben, L.N. Immunol. 48:385-392 (1983).
2. Anamnestic, as well as primary responses to TNP-Ficoll in Xenopus, require T cell activity. This T cell activity appears to support or stimulate the differentiation of the relevant B cell subpopulation from a precursor pool that is responsive to TNP-LPS. Ruben, L.N., Clothier, R.H. and Balls, M. Thymus 8: 341-348 (1986).

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HAPTEN-SPECIFIC TOLERANCE:

1. Hapten-specific tolerance can be stimulated in Xenopus, but not in the newt, Notophthalmus viridescens, by injection of TNBS. This tolerance is restricted to responses that involve TNP conjugated to TI-type 2 carriers in Xenopus. Mette, S.A. and Ruben, L.N. Cell Immunol.53:298-306 (1980).
2. Hapten-specific tolerance in Xenopus is maintained by the stimulation of hapten-specific thymic inducer suppressor cells. Signals provided in vivo by human rIL-2 and Con A can switch hapten-specific tolerance from unresponsivesness to responsiveness in Xenopus. This responsivity is thymus-dependent when TNP-Ficoll, but not, when TNP-PVP is used as the test immunogen. TNP-PVP responses, while activating the same B cells as TNP-Ficoll, are not thymus requiring in Xenopus. Since TNBS will not effect long-term memory responses to TNP-Ficoll, it would appear that the TNP-specific suppressor function this reagant stimulates is acting on the differentiation
of the relevant B cell sub-population which responds to TNP-Ficoll/TNP-PVP. Ruben. L.N., Clothier, R.H., Merchandani, M., Wood, P. and Balls, M. Immunol. 61:235-241 (1987).
3. When a single injection of TNBS is made into premetamorphic, metamorphosing and thiuorea-blocked metamorphosing larvae, no hapten-specific tolerance was initiated. This absence of tolerance correlated with a lack of splenic suppressor function in all three classes of larvae, suggesting that larvae may not have functional in vivo suppressor function and therefore are unable to use this mechanism in establishing self-tolerance for the larval form. This shift from an absence of suppressor regulation of the establishment of tolerance in larval forms to its function in the maintenance of tolerance in the adult, is in agreement with data noted above with single haplotye disparate skin grafts made during metamorphosis, but assayed at least 100 days later. While the establishment of tolerance to the grafts was sensitive to IL-2 injection, its maintenance was insensitive to the reagent. Moreover, it was dependent on suppressor function (Horton, J.D., Horton, T.L., Varley, C.A., and Ruben, L.N. Transplant. 47:883-887 (1989).
4. When TNBS is consistently present (Stage 48 to young adulthood) in the animal's acqueous environment, the tolerance initiated is broad-based. That is, it is effective when TNP is presented on any of the three different classes of carriers used. Moreover, when first applied during metamorphosis, the tolerance is effective with TNP-SRBC and TNP-LPS, but is absent when the TNP is presented on PVP. We suggest that this is because the B1 population required to respond to TNP-PVP, is not differentiated during metamorphosis and therefore is unable to respond to the exposure to TNBS during this period. Interestingly, the capacity to generate tolerance parallels the appearrance of suppressor function in the only secondary lymphoid organ in Xenopus, the spleen, which also develops in the last stages of metamorphosis. No capacity for tolerance, as a consequence of exposure to TNBS, is possible during premetamorphic life, when no splenic suppressor function exists. On the other hand, thymic suppression may function in T cell negative selection, since the thymus during these stages, and only in these stages, possesses both inducer and effector suppressor functions.
5. A theory of cancer resisitance in amphibians was developed that suggested that their resisitance may be related to their diminished capacity for altered-self tolerance (RUBEN, L.N., CLOTHIER, R.H. and BALLS, M. Is the evolutionary increase in epitope-specific tolerance within the vertebrates related to cancer susceptibility ? Herpetopathol.2:99-104 (1995).

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APOPTOSIS

1. We find that the in vivo thymic apoptotic levels are very low during metamorphosis and therefore are not likely to be responsible for the depressed immune activity seen at this time. Moreover, in vivo apoptosis doesn’t appear to be regulated by the internal glucocorticoid levels in the plasma (Ruben, L.N., Ahamadi, P., Buchholz, D., Johnson, R.O., Clothier, R.H. and Shiigi, S. Apoptosis in the thymus of developing Xenopus laevis. Dev. Comp. Immunol. 18:343-352 (1994); Grant, P., Clothier, R.H., Johnson, R.O. and Ruben, L.N. Lumphocyte apoptosis in Xenopus laevis, the South African clawed toad, during metamorphosis. (Dev. Comp. Immunol. In Press).
2. Few, if any, new antigens are being expressed during premetamorphic development. As the animal grows, however, only modest measures may be needed to diminish anti-self reactivity to preserve the integrity of self. A relationship between apoptosis and the expression of new antigens is supported by our findings that during metamorphosis in vitro thymic apoptotic rates are extremely high in comparison and that if one freezes the animals in metamorphosis by blocking their development using thiourea to reduce the production of thyroxine, the hormone responsible for driving metamorphosis and the multitude of new differentiations that result, the thymic apoptotic rate drops from 55-80% to near zero (∞7-9%).(Ruben, L.N., Ahamadi, P., Buchholz, D., Johnson, R.O., Clothier, R.H. and Shiigi, S. Apoptosis in the thymus of developing Xenopus laevis. Dev. Comp. Immunol. 18:343-352 (1994). Thus, apoptosis responsible for clonal deletion in the thymus, is driven by antigenicity. Since the peaks in apoptotic rates do not correspond to the glucocorticoid levels in the animals, it would appear that while corticosterone is responsible for regulating T cell functions (Highet, A. and Ruben, L.N. Immunopharm. 13:149-155 (1987), it does affect apoptosis in the thymuses as it does in mammals. Peripheral tolerance to TNP, in developing Xenopus, is suppressor T cell dependent, as shown by prior treatment with cyclophosphamide (Ruben, L.N., Goodman, A.R., Johnson, R.O., Kaleeba, J.A.R. and Clothier, R.H. Devel. Comp. Immunol.19:405-415 (1995).
3. Adult thymocytes of Xenopus , like those of mammals, will have enhanced apoptosis following in vitro exposure to the glucocorticoid, dexamethasone, PHA (phytahemagglutinin), as a lectin, and TNBS (trinitrobenzene sulfonic acid) as an antigen (Ruben, L.N., Buchholz, D., Ahmadi, P.,Johnson, R.O., Clothier, R.H. and Shiigi, S. Apoptosis in the thymus of adult Xenopus laevis. Dev. Comp. Immunol.18: 231-238 (1994).
4. In situ thymic and splenic apoptosis has been described over a 24 hour experimental period in adult Xenopus after injection of a T cell (PHA) and B (LPS) cell specific lectins. The medulla of the thymus and the red pulp area of the spleen are primarily involved in the apoptosis which peaks around 12 hours after injection (Grant, P., Clothier, R.H., Johnson, R.O., Schott, S., and Ruben, L.N. The kinetics and distribution of T and B cell mitogen-stimulated apoptosis in vivo. Immunol. Letters 47:227-231 (1995).
5. A Fas-like molecule which can initiate apoptosis is present on the surface of a variety of developing and adult tissues, including the thymus and spleen, of Xenopus (Mangurian, C., Johnson, R.O., MacMahan, R., Shiigi, S., Clothier, R.H. and Ruben, L.N. Expression of a Fas-like apoptotic molecule on larval and adult cells of Xenopus laevis. (In press,Immunol. Letts.).
6. Adrenoceptor ligation will strongly enhance or reduce concurrent apoptosis stimulated by either the calcium ionophore, A23187 or the phorbol diester, PMA, in accordance with the class of receptor stimulated and the length of time of exposure to either the alpha-2, clonodine, or beta, isoproterenol, agonist used. Apoptosis induced by dexamethasone was resistant to modulation by both adrenergics (Haberfeld, M., Johnson, R.O., Ruben, L.N., Clothier, R.H. and Shiigi, S. Adrenoceptor modulation of apoptosis in splenocytes of Xenopus laevis in vitro. (In press, NeuroImmunoModulation).

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METAMORPHOSIS, A DEVELOPMENTAL PERIOD WITH AN UNUSUAL ENDOCRINE ENVIRONMENT; REGULATION BY LYMPHOKINES/NEURO-ENDOCRINE SECRETIONS:

(Some references which fall into specific categories above have been reproduced for this category below)

1. Helper function can be demonstrated during larval Xenopus development but, it is aberrant during metamorphosis. Ruben, L.N., Welch, J.M. and Jones, R.E. In " Development and Differentiation of Lymphocytes of Vertebrate Lymphocytes ". Ed. Horton, J.D., pp.227-240, No. Holland Biomed. Press, Amsterdam (1980).
2. An internal MLR, leading to the release of thymus replacing factor during metamorphosis, may establish a "bypass" of the usual requirement for helper function in Xenopus. Jones, S.E. and Ruben, L.N. Immunol. 43:741-745 (1981).
3. Inducer and effector suppressor cells are functonally inhibited and become anatomically compartmentalized during metamorphosis of Xenopus. The thymus of pre-metamorphic larvae, unlike that of adult toads, contains both inducer and effector suppressor functions. Kamali, D., Ruben, L.N. and Gregg, M. Cell Diff.18:225-231 (1986).
4. Responsivity to TNP-Ficoll varies with developmental stages and internal endocrine environmental changes with age. Ruben, L.N., Clothier, R.H., James, H.S. and Balls, M. Cell Diff. 14:1-5 (1984).
5. Corticosteroid will inhibit a TD response in an adult lower vertebrate. Ruben, L.N. and Vaughan, M. J. Exp. Zool. 190:229-235 (1974).
6. The number of corticosteroid receptors/cell of splenic lymphocytes during metamorphosis, when serum titers are high, is 10x the level found in young adults. Pre-metamorphic larvae will not bind the hormones.Since corticosteroids will inhibit T cell mitogenesis, helper and suppressor functions, it seems likely that T cell functional deficiencies during this period are mediated by corticosteroids. Marx, M., Ruben, L.N., Nobis, C. and Duffy, D., In " Developmental and Comparative Immunology ", Ed. Cooper, E.L., pp 129-140. Alan R. Liss Inc. Publ., New York (1987).
7. Corticosteroid is responsible for the inhibition of inducer suppressor function during Xenopus metamorphosis. This inhibition can be relieved by injection of metyrapone, human IL-2, Con A or human IL-1in vivo. Thus, this T cell immune deficiency seems to be due to inhibition of Mø secretion of IL-1 and, in turn, too little IL-2, by high endogenous corticosteroid titers
Highet, A. and Ruben, L.N. Immunopharm. 13: 149-155 (1987).
8. Alpha and ß adrenergics effect the capacity of immunized adult Xenopus, but not of newt, B cells to bind antigen differentially. Hodgson, R.M., Clothier, R.H., Ruben, L.N. and Balls, M. Eur. J. Immunol. 8:348-351 (1978).
9. Human IL-2 will substitute for helper function in Xenopus. Its capacity to do this will last for three hours prior to antigen challenge. Ruben, L.N. Immunol. Letts. 13:227-230 (1986).
10. Human IL-2 and Con A can stimulate responses to sub-immunogenic dosages of TNP-Ficoll. Ruben, L.N., Barr, K., Clothier, R.H., Nobis, C. and Balls, M. Dev. Comp. Immunol. 9:811-818 (1985).
11. Murine and human IL-2 can substitute for the thymus requirement in TNP-Ficoll responses in adult Xenopus. Ruben, L.N., Clothier, R.H. and Balls, M. Cell Immunol. 93:229-233 (1985).
12. Signals provided in vivo by human IL-2 and Con A can switch hapten-specific tolerance from unresponsivesness to responsiveness in Xenopus. This release from tolerance is thymus-dependent when TNP-Ficoll, but not when TNP-PVP is used as the test immunogen. TNP-PVP responses, while activating the same B cells as TNP-Ficoll, are not thymus requiring in Xenopus. Ruben. L.N., Clothier, R.H., Mirchandani, M., Wood, P. and Balls, M. Immunol. 61: 235-241 (1987).
13. Xenopus splenocytes display constitutive molecules which bind mouse-anti-human IL-2 receptor antibody specifically. Il-2 will compete with this binding. Moreover, the number of cells able to bind both the anti-receptor antibody and IL-2, as well as the number of receptors/cells can be increased by lectin activiation of the freshly biopsied spleen cells in vitro. Langeberg, L., Ruben, L.N., Malley, A., Shiigi, S. and Beadling, C. Immunol. Letts. 14: 103-110 (1987).
14. The Xenopus spleen cells which are PHA activatable with regard to IL-2 R receptor density and which bind rIL-2 are related to helper T cell function in cytotoxic and humoral immune responses, since it is removed by N-CH3-N-Nitrosourea (NMU) injection. NMU is selectively lymphotoxic in the toad and specifically removes these two functions. Panning spleen cells with a mAb to Xenopus IgM showed that those cells which bear constitutive IL-2 receptors and bind rIL-2 are equally abundant in the T and B cells populations of freshly biopsied Xenopus spleens. Langeberg, L., Ruben, L.N., Clothier, R.H., and Shiigi, S. Immunol. Letters 16: 43-48 (1987).
15. Human IL-1, Con A on sepharose or agarose, but not soluble Con A or soluble or particulate WGA or human IL-2, will substitute for carrier primed helper function in the newt. While anti-human IL-2 receptor antibody will bind specifically to freshly biospsied spleen cells, Il-2 will not compete with this binding. The shared lectin specificity suggests that T-like cells are responsible for helper function in this animal with an " immature " thymus. The lymphokine and Con A data suggest that while human IL-2 is unable to affect the cells of this species, Con A and human IL-1 can initiate the stimulation, perhaps through the stimulation of autologous IL-2 production. Ruben, L.N., Beadling, C., Langeberg, L, Shiigi, S.and Selden, N. Thymus 11: 77-87 (1988).
16. The monoclonal mouse-anti-human IL-2 recedptor antibody that we have used recognizes molecules of the cell surface of Xenopus lymphocytes that specifically bind rIL-2 and are essentially the size as the Tac molecule (p55) Ruben, L.N., Langeberg, L., malley, A., Clothier, R.H., Beadling, C., Lee, R.O. and Shiigi, S. Immunol. Letters 24:117-126 (1990).
17. During metamorphosis, when T cell functions are impaired by a high glucocorticoid titer, T lymphocytes also have reduced capacities for the expression of IL-2 receptors, upon exposure to PHA, and to generate autologous TCGF. Thus, we have suggested that during this developmental period, when adult cells and antigens are being expressed within the larval body, T cell functions are impaired by glucocortioid-imposed anergy and that this is what is responsible for the prevention of immune self-destruction (Ruben, L.N., Scheinman, M. A., Johnson, R.O., Shiigi, S., Clothier, R.H. and Balls, M. Mech. of Develop. 37:167-172 (1992).
18. Mortality in developing larvae, particularly during the metamorphic period, can be manipulated by using IL-2 along with an antigen. Thus, it may be possible to lower the amount of IL-2 used in experimental cancer therapy by adding an antigen in conjunction with it (Ruben, L.N., De Leon R.T., Johnson, R.O. and Clothier, R.H. Interleukin-2-induced mortality during metamorphosis of Xenopus laevis. Immunol. Letts 51:157-161 (1996).
19. In vivo thymic apoptotic levels are very low during metamorphosis and therefore are not likely to be responsible for the depressed immune activity seen at this time. Moreover, in vivo apoptosis doesn’t appear to be regulated by the internal glucocorticoid levels in the plasma (Ruben, L.N., Ahamadi, P., Buchholz, D., Johnson, R.O., Clothier, R.H. and Shiigi, S. Apoptosis in the thymus of developing Xenopus laevis. Dev. Comp. Immunol. 18:343-352 (1994); Grant, P., Clothier, R.H., Johnson, R.O. and Ruben, L.N. Lumphocyte apoptosis in Xenopus laevis, the South African clawed toad, during metamorphosis. (Dev. Comp. Immunol. In Press).
20. Few, if any, new antigens are being expressed during premetamorphic development. As the animal grows, however, only modest measures may be needed to diminish anti-self reactivity to preserve the integrity of self. A relationship between apoptosis and the expression of new antigens is supported by our findings that during metamorphosis in vitro thymic apoptotic rates are extremely high in comparison and that if one freezes the animals in metamorphosis by blocking their development using thiourea to reduce the production of thyroxine, the hormone responsible for driving metamorphosis and the multitude of new differentiations that result, the thymic apoptotic rate drops from 55-80% to near zero (∞7-9%).(Ruben, L.N., Ahamadi, P., Buchholz, D., Johnson, R.O., Clothier, R.H. and Shiigi, S. Apoptosis in the thymus of developing Xenopus laevis. Dev. Comp. Immunol. 18:343-352 (1994). Thus, apoptosis responsible for clonal deletion in the thymus, is driven by antigenicity. Since the peaks in apoptotic rates do not correspond to the glucocorticoid levels in the animals, it would appear that while corticosterone is responsible for regulating T cell functions (Highet, A. and Ruben, L.N. Immunopharm. 13:149-155 (1987), it does affect apoptosis in the thymuses as it does in mammals. Peripheral tolerance to TNP, in developing Xenopus, is suppressor T cell dependent, as shown by prior treatment with cyclophosphamide (Ruben, L.N., Goodman, A.R., Johnson, R.O., Kaleeba, J.A.R. and Clothier, R.H. Devel. Comp. Immunol.19:405-415 (1995).

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