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Circulating thymic hormones: Laboratory quantitation and clinical significance
References 1. Coben, M. H., P. B. Chretien, D. C. Ihle, B. E. Fossicek, R. Makuch, P. A. Bunn , A. V. Johnston, S. E. Shackney, M. J. Mathews, S. O. Lipson, P. E. Kenady, and J. D. Minns. 1979. Thymosin fraction 5 and intensive combination chemotherapy prolonging the survival of patients with small cell lung cancer. J.A.M.A. 241:1813-1815. 2. Dardenne, M., J. M. Pleau, N. K. Man, and J. F. Bach. 1977. Structural study of circulating thymic factor : A peptide isolated from pig serum. I. Isolation and purification. J. Bioi. Chern. 252:8040-8044. 3. Goldstein, A. L., F. D. Slater, and A. White. 1966. Preparation, assay and partial purification of a thymic lymphocytopoietic factor (thymosin) , Proc, Natl. Acad. Sci. USA 56: 10101017. 4. Goldstein, A. L., T. L. K. Low, G. B. Thurman, M. Zatz, N. R. Hall, J. E. McClure, S-K. Hu, and R. S. Schulor. 1980. Thymosins and other hormonal-like factors of the thymus
5. 6.
7.
8.
gland. In E. Mihich (ed.), Immunological aspects of cancer therapeutics, John Wiley and Sons , New York. In press. Goldstein, G. 1975. The isolation of thymopoietin (Thymin). Ann. N.Y. Acad. Sci. 249:177 . Hooper, J. A., M. C. McDaniel, G. B. Thurman, G. H. Cohen, R. S. Schulof, and A. L. Goldstein. 1975. Purification and properties of bovine thymosin. Ann . N.Y. Acad. Sci. 249: 125-144. Low, T. L. K., G. B. Thurman, M. McAdoo, J. McClure, J. L. Rossio, P. H. Naylor, and A. L. Goldstein. 1979. The chemistry and biology of thymosin. I. Isolation, characterization and biological activities of thymosin (XI and polypeptide 131 from calf thymus. J. Biol, Chern. 254:981986. Low, T. L. K., S·K. Hu, and A. L. Goldstein. 1981. Complete amino acid sequence of bovine thymosin p.: A thymic hormone that induces terminal deoxynuc1eotidyl transferase activity in thymocyte populations.
Proc. Natl. Acad, Sci. USA. In press. 9. McClure, J. E., and A. L. Goldstein. 1980. Changes with age in blood levels of thymosina, as measured by rad ioimmunoassay. Proc. 4th Int. Congo Immunol. (Paris) 17:226A . 10. Wetzel, R ., H. L. Heyneker, D. V. Goeddel, P. Jhurani, J. Shapiro, R. Crea, T. L. K. Low, G. B. Thurman, J. E. McClure, and A. L. Goldstein. 1980. Production of biologically active N"-desacetyl thymosin a l in Escherichia coli through expression of a chemically synthesized gene. Bicchem. In press. II. White, A., and A. L. Goldstein. 1975. The endocrine role of the thymus and its hormone, thymosin, in the regulation of the growth and maturation of host immunological competence. Adv. Metab. Disord. 8:359-374.
12. Yaklr, Y., A. I. Kook, and N. Trainin. 1978. Enrichment of in vitro and in vivo immunologic activity of purified fractions of calf thymic hormone. J. Exp. Med. 148:71-83 .
Circulating Thymic Hormones: Laboratory Quantitation and Clinical Significance Jean-Francois Bach MireiUe Dardenne HopitaJ Necker 161, Rue de Sevres 75730 Paris Cedex 15
The thymus gland plays a key role in immunity. The thymus-derived cells (T cells) exert multiple functions: they kill specifically the nucleated cells against which they have been sensitized; they produce the lymphokines that mediate delayed-type hypersensitivity reactions; and, most importantly, they regulate all immune responses whether performed by Bcells (antibody production) or T cells (cell-mediated immunity). It is now known that these diverse functions correspond, at least in part, to physically distinct T-cell subsets, which have been characterized by alloantigens or, more recently, by xenoantigens (using monoclonal anti-Tcell antibodies). The development of these multiple T-cell lineages operates in several phases from the stem cell to mature, competent T cells. Thus, it is possible to schematically characterize several
steps in T-cell differentiation (Table 1) as hematologists do for other blood cell lineages. The difference here is that differentiation phases are not characterized by morphological features (as for erythrocytes or granulocytes) but by antigenic markers
and functions. Several factors control the sequential transformation of T cells. The thymic epithelium plays a central and apparently unique role in the first phases. At its direct contact the prothymocyte (whether or not it is
Table I Stages of T-Cell Differentiation Antigenic markers
Mouse
Man (OKT sertes")
Hemopoietic stem cell Lymphoid stem cell Prothymocyte
Weak Thy-!
OKT6
Immature cortical thymocyte
Lyt-l or 123, Thy-l
OKT4, 6, 8, 10
"Post-thymic" precursor thymocyte (former T1 cell)
Lyt-l and 123, Thy-l
OKTJ,4,8
Helper/inducer
Lyt-l , Thy-!
OKT3,4
Cytotoxic/suppressor
Lyt-2, 3, Thy-!
OKT3,8
Mature T cells (former T2 cells)
·P. C. Kung et al, (18).
3
already committed to the T-cell lineage) is transformed into the immature cortical thymocyte and eventually into the "post-thymic thymocyte." This transformation is associated with the acquisition of antigenic markers, and the building up of the repertoire of anti-self receptors necessary for the cognitive function of T cells. It is not known whether thymic hormones intervene in these first phases of differentiation. Further stages of maturation, which will lead the T cell to the post-thymic thymocyte (the still incompetent cell that leaves the thymus) and to the mature T cell, are under thymic hormone control. Other factors that may participate in the very last stages include antigen stimulations, especially through the production of a lymphokine, and the T-cell growth factor (TCOF), also called Interleukin 2. Note, however, that TCOF may only operate as an amplification circuit, since it is exclusively produced by mature T cells, which, by definition, must have differentiated under thymic control. Several thymic hormones have been described, and it has not yet been determined how many molecules intervene physiologicallyas mediators of the humoral function of the thymus. Crude thymic extracts exert a number of biological activities that may generally be interpreted as the consequence of the maturation or the stimulation of the various T-cell subsets mentioned above. The key question is whether these activities are borne by different molecules. The current view is that there is more than one thymic hormone, but that a given hormone probably acts at several levels on different T-cell lines. The picture is confused by the existence in the thymus of several peptides that are probably not physiologically relevant thymic hormones, but which may possess pharmacological activity. These contaminating peptides obscure the interpretation of the effects of the crude thymic extracts that have been used clinicallyuntil now. Several welldefined peptides have been characterized: Thymosins a I, a 7, and {31, and {3. (20); thymopoietin and its h
4
smaller analogue, TP-S, which is also biologically active (1S); thymic humoral factor (24); and the serum thymic factor, facteur thymique serique (FTS) (8). There is no homology in the published amino acid sequences, but there is still room for some degree of chemical relationship other than the splitting of precursor hormones (e.g., cleavage factor, carrier molecule). Clinical trials have been initiated by using the main preparations cited previously. Preliminary results are encouraging, but it is too early to draw any definitive conclusion on the therapeutic efficacy of any of the preparations used so far. No sign of toxicity was detected, however, and correction of T-cell abnormalities (assessed on markers, and functions in vitro or in vivo) were often normalized. Thymic hormones are produced by the thymic epithelium. This has now been directly demonstrated by immunofluorescence using antisera directed at thymosin a I (16) and FrS (22). However, thymic humoral activity may be found in circulating blood. In fact, it is from the serum that FTS was initially isolated (before being chemically isolated and characterized from thymic extracts with identical amino acid analysis). Recently, evidence has been brought in a more indirect fashion that thyme-
poietin and thymosin al also circulate in the blood. Finally, another thymus-dependent serum factor has been described (1), but recent data suggest that such factor is not, as initially thought, a peptide, but merely adenosine. The significance of" the thymic hormone-like activity of" circulating prealbumin (9) is not yet well understood. It is not known definitively whether it is explained by the transport of a small thymic peptide such as FTS, as we have recently suggested on several strong experimental arguments (11). The presence of thymic hormone(s) in the circulation probably has a physiological significance. Adult thymectomy induces several changes that may take place as early as five days after the operation (S), and SUch changes are corrected in vitro by the addition of thymic hormones (6). It is apparent that the T cell that leaves the thymus is not yet completely mature and still depends partly on circulating thymic hormone(s) to terminate its maturation. Thus, in addition to representing a useful means of evaluating the epithelial function of the thymus, the measure, ment of circulating thymic hormones may be interesting to consider as an important parameter in T-cell differentiation. This latter aspect is emphasized by the demonstration in
Editors: Herman Friedman, Mario Escobar, and Noel Rose Editorial Committee: Charles D. Graber. Ph.D.• Medical University of South Carolina; John R. Kateley, Ph.D., Edward W. Sparrow Hospital Assoc.; Bruce S. Rabin, M.D., Ph.D., University of Pittsburgh School of Medicine; Robert F. Ritchie, M.D., Foundation for Blood Research, Maine; John L. Sever, M.D., Ph.D., National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health; Steven Specter, Ph.D., University of South Florida College of Medicine; Roy W. Stevens, Ph.D., New York Health Department Laboratories; Norman Talal, M.D., VA Hospital and University of California Medical Center at San Francisco; Eng M. Tan, M.D., University of Colorado Medical Center; Gabriel Virella, M.D., Ph.D., Medical University of South Carolina. Subscription Rates in U.S. and Canada: one year $46.S0; two years $89.00; three years 5127.S0. All other countries: one year $S3.S0; two years $102.S0; three years $146.S0. Single copies are 52.00 in U.S. and Canada, 52.6S all other countries. Single issues are available in quantity (prices available upon request). All subscriptions are payable in advance. Foreign subscriptions are sent guaranteed air mail. First class postage paid in U.S. and Canada. Address correspondence regarding subscription to: Ctintcal Immunology Newsletter, G. K. Hall & Co., 70 Lincoln St., Boston, Massachusetts 02111. Please include zip code in subscription address. The Clmical Immunology Newsletter is published twice monthly. All rights, including that of translation into other languages, reserved. Photomechanical reproduction (photocopy, microcopy) of this newsletter or parts thereof without special permission of the publisher is prohibited.
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normal serum of several inhibitors that may alter the efficacy of the circulating hormone. In brief, the activity of serum in the rosette assay used for evaluating FTS is very much increased after removal of highmolecular-weight proteins (by mere filtration on Amicon membranes) and still further increased by removal of medium-sized molecules by Sephadex gel filtration (2). More precisely, there are several inhibitors with respective molecular weights of 100,000300,000, 40,000-70,000, and 5,00010,000 daltons. The largest inhibitors are removed before evaluating the serum thymic hormone level, but the smaller ones are not eliminated and interfere with the final evaluation. While this interference may cause difficulties when evaluating central thymic epithelial function, it is interesting to consider, since it might also operate in vivo in the biological effects of circulating thymic hormone. Better appraisal of these inhibitors will be made possible when radioimmunoassays that permit the measurement of the absolute amount of hormones present in the serum are available in addition to the biological assays used presently. It may be relevant to note at this stage that the serum also contains carrier proteins for FrS whose relationship with the inhibitors is not yet clear. In any case, the presence of protein-bound (and possibly inactive) thymic hormone in the serum should be taken into consideration when interpreting serum evaluation of such hormones, by both biological or radiobiological assays. Laboratory Methods for the QuantitatioD of Circulating Thymic Hormones Several methods have been described to measure the levels of circulating thymic hormones. The first of these methods was described in our laboratory in 1972 (4). It is based on the changes induced by thymic hormones on the minority of spleen cells of adult-thymectomized (Tx) mice that form spontaneous rosettes with sheep erythrocytes. The assay consists in detecting the smallest serum concentration that renders
spleen rosette-forming cells (RFC) sensitive to anti-theta serum (A BS) or to azathioprine (Az), the well-known immunosuppressive agent that happens to inhibit, at low concentration, rosette formation in normal mice and to lose this property, as AB S does in Tx mice (3). Results are expressed in serum dilutions (the higher the active dilution, the higher the concentration of the hormone) with the reservation linked to the serum inhibitors previously mentioned, which may influence overall biological activity. Sera are used in the tests after ultrafiltration on Amicon membranes (molecular weight cutoff: 50,(00), but small inhibitors are still present. In selected cases, the relationship of the detected activity to FrS may be verified by passing the serum on an immunoadsorbent prepared with an antibody raised against synthetic FrS. The strict thymus dependency of the activity detected by the rosette assay is demonstrated by its total absence in the sera of nude or Tx mice and of patients with Di George syndrome, and its reappearance in all individuals after thymus grafting. A similar method was described in 1977 by Twomey et al. (26). Serum is incubated with nude-mouse spleen cells, and the increase in (} -positive cells is measured using a cytotoxic assay with trypan blue. The sensitivity of the test is increased by adding ubiquitin at subliminal concentration, a protein known to induce nonspecifically T- (and B-) cell markers. As in the rosette assay, serum ultrafiltrates are used rather than total serum, because of the presence of serum inhibitors. Thymopoietin shows activity in the test. Interestingly, FrS, whose effects are inhibited by ubiquitin (17), is not active in the test. Results are expressed in the nanogram-equivalent of thymopoietin, which does not take full account of molecules other than thymopoietin that could be active in the test, and of the interfering molecules (e.g., carrier proteins, inhibitors). As for the rosette assay, the specificity of the Twomey assay is assessed by its negativity in the sera of
nude and Tx mice, and in sera from patients with Di George syndrome. Radioimmunoassays (RIA) have been described for three well-defined thymic peptides: thymosin a I (Goldstein, unpublished), FrS (23), and thymopoietin (14). Such RIAs have not yet been successfully applied to the evaluation of serum samples for FrS and thymopoietin, essentially because of unsolved interferences ' with serum proteins. More conclusive results have been reported for thymesin al (21), but, unlike the two bioassays previously described, unequivocal demonstration of the total absence of RIA-positive material in the serum of Tx animals or humans as compared to normal individuals has not yet been accomplished. Serum Level of Thymic Hormones in Experimental Models and in Human Diseases The age dependency of FrS serum level has been extensively studied in the mouse. FrS is already present at adult level at birth. Studies in Tx pregnant mothers have shown that FrS is first detectable on the fourteenth day of pregnancy. FrS level remains stable until 5-7 months of age and then progressively declines to become insignificant after 12 months. This age-dependent decline, which parallels that of thymus weight, is premature in autoimmune mice (NZB, (NZB x NZW) FI' MRLI/l, SWAN). A recent study has shown that the disappearance of FTS from the serum in aging mice is due to the simultaneous decline in ITS production and appearance in the serum of FrS inhibitors; grafting an old thymus in young Tx mice induces the appearance of an FrS activity superior to that present in the old mice from which the thymus graft is taken. Similar experiments in NZB mice indicate that in these mice FTS loss is not due to peripheral destruction, but to a deficit in thymic secretion. Evaluation of serum FrS levels has also provided interesting information on thymus physiology. Multiple thymus grafts are associated with increased FrS level grossly propor-
5
tional to the number of thymic lobes grafted, which confirms the absence of thymic-hormone-induced feedback regulation of the thymus, which was suspected from macroscopic studies of the thymus (25). The age dependency of FrS level has also been observed in man. The level is stable until the age of 15-20 years, followed by a progressive decline. The data shown on Figure I, which we initially reported for FrS (7), have been confirmed with the rosette assay by other investigators (12-13), as well as with the Twomey assay (19), with similar kinetics. In contrast to these data, the level of thymosin 0'1 seems to drop earlier than that of FTS, with a decline starting as soon as 10 years of age (Goldstein, unpublished). The serum level of thymic hormones has been evaluated in a number of diseases, using either our rosette assay or the Twomey assay. FrS level is low in human as in murine lupus, although the decrease in FrS level is not a constant feature of lupus patients and is only detectable in young patients (before agematched controls lose their own FTS production). In myasthenia gravis, thymic hormone level is normal in young patients but tends to be higher than normal in older ones. In mycosis fungoides, the level is high, and it has
been verified, using the immunoadsorbent previously mentioned. that such increased FTS activity was absorbed out by the anti-FTS immunoadsorbent. In immunodeficiency syndromes, FTS level is low in thymic aplasia and normal in sex-linked agammaglobulinemia. Interestingly, the FTS level is often low in severe combined immunodeficiencies and tends to increase after bone marrow grafting, parallel to the appearance of thymic shadow. These data indicate that the humoral function of the thymic epithelium may be stimulated by the contact with the colonizing stem cells (absent before bone marrow transplantation). Lastly, note that we have observed epithelial thymoma with high levels of fTS and a high percentage (980/0) of peripheral T cells in one 80-year-old patient, which indicates that in humans there are thymomas secreting inappropriate amounts of thymic hormones, similar to the carcinogen-induced epithelial thymoma in the mouse (10). In conclusion, the dosage of serum thymic hormone is now available for clinical use. Techniques used today are still essentially biologic. Currently, the most frequently used rosette assay has now been employed successfully by several independent groups (4,9, 13, 17), but it is not yet easily available for clinical practice.
serum FTS
1/64
•
1/32
•• ••
.::
1/16
II
••• ••
1/8
•
•
•
••
II: ••
.. •
1/4 ~
1/2 0-2 2-5 5-20
..
•
::. •• ·n..
-:.
•
• ••• ••••
•••
• ·1
....:. ...
• •
•• •••• •• "i·
•
.•.•
.\
30-40 50-60 40-50 ~60 20·30
age (years)
Fig. J. Age dependency of serum FTS levels in man, as evaluated by the rosette assay.
6
We hope that in the near future R...lA methodology will be generalized. In the meantime, the biologic assays will have provided important information on thymic epithelial function in healthy and diseased individuals. In addition. even when valid RIA procedures are available, these biologic assays will still have to be studied in order to appreciate the resulting effects of the various components, (including the carriers and inhibitors) that participate in circulating thYmic hormone activity. The significance of' the various hormones evaluated by RIA methods and their relevance to thymus physiology and pathology must also be studied critically. References I. Astaldl, A., G. C. B. Astaldl, P. T. A. Scbellekens, and V. P. ElJsvoogel. 1976. Thymic factor in human sera demonstrable by a cyclic AMP assay. Nature 260:7I3-71S. 2. Bacb, J. F., M. A. Bacb, D. Blanot. E. Brlcu, J. Cbarrclre, M. Dardenne. C. Fournier, and J. M. Pleau. 1978. Thymic serum factor (FrS). Bull. lost. Pasteur 76:32S-398. 3. Bach, J. F., and M. Dardenne. 1972. Antigen recognition by T lymphocytes. II. Similar effects of azathioprine, ALS and antitheta serum on rosette-forming lymphocytes in normal and neonatally thymectomized mice. Cell Immuno!. 3:11-21. 4. Bacb, J. F., and M. Dardenne. 1972_ Thymus dependency of rosetteforming cells. Evidence of a circulating thymic hormone. Transplant. Proc, 4:34S-3S0. S. Bach, J. F., M. Dardenne, and A. J. S. Davies. 1971. Early effect of adult thymectomy. Nature 231:110-111. 6. Bacb, J. F., M. Dardenne, A. L• Goldstein, and A. Wblte. 1971. Appearance of T-cell markers in bone marrow rosette-forming cells after incubation with purified thymosin, a thymic hormone. Proc. Natl, Acad, Sci. USA 68:2734-2738. 7. Bacb, J. F., M. Dardenne, M. Paplemlk. A. Barols, P. Levasseur. and H. Le Brigand. 1972. Evidence for a serum factor produced by the human thymus. Lancet 2:IOS6-10S8. 8. Bacb, J. F., M. Dardenne, J. M. paeau, and J. Rosa. 1977. Biochemical characterization of a serum thymic hormone. Nature 266:SS-57. 9. Burton, P., S. Iden, K. Mltcbell, and A. White. 1978. Thymic hormonelikerestoration by human prealbumin or azathioprine sensitivity of spleen
cells from thymectomized mice. Proc. Natl. Acad. Sci. USA 75:823826. 10. Dardenne, M., M. Papiernik, J. F. Bach, and O. Stutman. 1974. Studies on thymus products. III. Epithelial origin of the thymic hormone. Immunology 27:299-304. 11. Dardenne, M., J. M. Pleau, and J. F. Bach. 1980. Evidence of the presence in normal serum of a carrier of the serum thymic factor (FrS). Eur. J. Immunol. 10:83-86. 12. Fabris, N. 1980. Serum thymic factor determination in different human pathologies. Int. J. Immunopharmacol. 2(3):157. 13. Garaci, E., R. Ronchetti, V. Del Gobbio, G. Tramutoli, C. RinaldiGaraci, and C. Imperato. 1978. Decreased serum thymic factor activity in asthmatic children. J. Allergy Clin. Immunol. 62:357-362. 14. Goldstein, G. 1976. Radioimmunoassay for thymopoietin. J. Immunol. 117:690-692. 15. Goldstein, G., M. P. Scheid, E. A. Boyse, D. H. Schlesinger, and J. Van Wauwe. 1979. A synthetic pentapeptide with biological activity characteristic of the thymic hormone thymopoietin. Science 204:1309-1312. 16. Hirokawa, K., and K. Saitoh. 1980. Heterogeneity of thymic epithelial
17.
18.
19.
20.
21.
cells, revealed by localization of thymosin a I and various hydrolytic enzymes in human thymus. 4th Int. Congo Immunol. (Paris), abstr. 3.3.14. Iwata, T., G. S. Incefy, and R. A. Good. 1979. Interaction between thymopoietin and facteur thymique serique in the rosette inhibition assay. Biochem. Biophys, Res. Commun. 88:1419-1427. Kung, P. C., et al. 1979. Monoclonal antibodies defining distinctive human T-cell surface antigens. Science 206: 347-349. Lewis, V. M., J. J. Twomey, P. N. Bealmear, G. Goldstein, and R. A. Good. 1978. Age, thymic involution and circulating thymic hormone activity. J. Clin. Endocrinol. Metab. 47:145-150. Low, T. L. K., G. B. Thurman, M. McAdoo, J. McClure, J. L. Rossio, P. H. Naylor, and A. L. Goldstein. 1979. The chemistry and biology of thymosin. I. Isolation, purification and biological activities of thymosin Q, and polypeptide f3, from calf thymus. J. BioI. Chern. 254:981-984. McClure, J. E., and A. L. Goldstein. 1980. Changes with age in blood levels of thymosin Q. as measured by radioimmunoassay. 4th Int. Congo Immunol. (Paris), abstr. 17.2.26.
Letters to the Editor
Case Report and Self-Assessment
To the Editor:
This section was prepared byG. Virella, M.D., Ph.D., Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, South Carolina. Similar questions and answers are solicited from readers.
I question the appropriateness of devoting a page and a half of the Newsletter to the correspondence between Drs. Pitts and Virella. The Newsletter is reasonably expensive and your subscribers deserve better. Thomas W. Green, M.D., Ph.D. Medical Director Berkeley Biologicals Berkeley, California 94710
Editor's Reply: The Newsletter is a new venture, and we appreciate hearing from our readers. Since we view the Newsletter as a forum for the exchange of information and ideas among immunologists, we had hoped to encourage more discussion in this section. We invite Dr. Green and all our other readers to continue to send suggestions and to contribute items for publication.
Case Report In 1969, the patient, a 65-yearold Caucasian male, began to complain of intermittent painful episodes in the epigastrium. Two years later, he developed symptoms of pyloric stenosis. At that time, routine electrophoresis showed a slight increase in alpha 2 globulins. A 6 x 4 em juxtapyloric tumor was surgically removed; no obvious lymphadenopathies were seen. The histological examination of the tumor was compatible with lymphatic lymphoma. The patient was discharged without additional therapy. Three years later, the patient developed gastrointestinal symptoms (nausea, vomiting, diarrhea) with hepatosplenomegaly. A liver biopsy
22. Monier, J. C., M. Dardenne, J. M. Pleau, D. Schmitt, P. Deschaux, and J. F. Bach. Characterization of FrS in the thymus. I. Fixation of anti-F'I'S antibodies on thymic reticulo-epithelial cells. Clin. Exp. Immunol. In press. 23. Pleau, J. M., D. Pasques, and J. F. Bach. 1978. Dosage radioimmunologique du facteur thymique serique (FrS) In Radioimmunoassay and related procedures in medicine, vol. 2. International Atomic Energy Agency, Vienna. 24. Trainin, N., M. Small, D. Zipori, T. Umiel, A. I. Kook, and V. Rotter. 1975. Characteristics of TAF, a thymic hormone, p. 117. In D. W. Van Bekkum (ed.), The biological activity of thymic hormones. Kooyker Scientific Publications, Rotterdam. 25. Tubiana, N., and M. Dardenne. 1979. Neonatal thymus grafts. I. Studies on the regulation of the level of circulating thymic factor (FrS). Immunology 36:207-213. 26. Twomey, J. J., G. Goldstein, V. M. Lewis, P. M. Bealmear, and R. A. Good. 1977. Bioassay determinations of thymopoietin and thymic hormone levels in human plasma. Proc. Natl. Acad. Sci. USA 6:2541-2545.
showed portal space infiltration by differentiated mononucleated lymphocytes. Proteinuria was 20.5 mg/dl, The patient was treated supportively and discharged. In 1979, the patient complained of weakness, exertion dyspnea, easy fatigue, and loss of weight. He showed pallor of skin and mucosae, hepatosplenomegaly, and right-sided pleural effusion. Hemoglobin was 8.S g/dl. White blood cell counts varied between 3,000 and 5,000 mm' with normal differential count. A bone marrow aspirate showed pancytopenia with relative increase of reticulocytes and lymphocytes. A gastrointestinal x-ray examination showed widespread mucosal infiltration with multiple constrictions and dilations of the jejunal loops. Urinary proteins were 35 mg/dl. The patient was treated with a combination of vinblastine, triethylene phosphoramide, rufochromomycin, procarbazine, and prednisone. At the end of two months
7
5. 6.
7.
8.
gland. In E. Mihich (ed.), Immunological aspects of cancer therapeutics, John Wiley and Sons , New York. In press. Goldstein, G. 1975. The isolation of thymopoietin (Thymin). Ann. N.Y. Acad. Sci. 249:177 . Hooper, J. A., M. C. McDaniel, G. B. Thurman, G. H. Cohen, R. S. Schulof, and A. L. Goldstein. 1975. Purification and properties of bovine thymosin. Ann . N.Y. Acad. Sci. 249: 125-144. Low, T. L. K., G. B. Thurman, M. McAdoo, J. McClure, J. L. Rossio, P. H. Naylor, and A. L. Goldstein. 1979. The chemistry and biology of thymosin. I. Isolation, characterization and biological activities of thymosin (XI and polypeptide 131 from calf thymus. J. Biol, Chern. 254:981986. Low, T. L. K., S·K. Hu, and A. L. Goldstein. 1981. Complete amino acid sequence of bovine thymosin p.: A thymic hormone that induces terminal deoxynuc1eotidyl transferase activity in thymocyte populations.
Proc. Natl. Acad, Sci. USA. In press. 9. McClure, J. E., and A. L. Goldstein. 1980. Changes with age in blood levels of thymosina, as measured by rad ioimmunoassay. Proc. 4th Int. Congo Immunol. (Paris) 17:226A . 10. Wetzel, R ., H. L. Heyneker, D. V. Goeddel, P. Jhurani, J. Shapiro, R. Crea, T. L. K. Low, G. B. Thurman, J. E. McClure, and A. L. Goldstein. 1980. Production of biologically active N"-desacetyl thymosin a l in Escherichia coli through expression of a chemically synthesized gene. Bicchem. In press. II. White, A., and A. L. Goldstein. 1975. The endocrine role of the thymus and its hormone, thymosin, in the regulation of the growth and maturation of host immunological competence. Adv. Metab. Disord. 8:359-374.
12. Yaklr, Y., A. I. Kook, and N. Trainin. 1978. Enrichment of in vitro and in vivo immunologic activity of purified fractions of calf thymic hormone. J. Exp. Med. 148:71-83 .
Circulating Thymic Hormones: Laboratory Quantitation and Clinical Significance Jean-Francois Bach MireiUe Dardenne HopitaJ Necker 161, Rue de Sevres 75730 Paris Cedex 15
The thymus gland plays a key role in immunity. The thymus-derived cells (T cells) exert multiple functions: they kill specifically the nucleated cells against which they have been sensitized; they produce the lymphokines that mediate delayed-type hypersensitivity reactions; and, most importantly, they regulate all immune responses whether performed by Bcells (antibody production) or T cells (cell-mediated immunity). It is now known that these diverse functions correspond, at least in part, to physically distinct T-cell subsets, which have been characterized by alloantigens or, more recently, by xenoantigens (using monoclonal anti-Tcell antibodies). The development of these multiple T-cell lineages operates in several phases from the stem cell to mature, competent T cells. Thus, it is possible to schematically characterize several
steps in T-cell differentiation (Table 1) as hematologists do for other blood cell lineages. The difference here is that differentiation phases are not characterized by morphological features (as for erythrocytes or granulocytes) but by antigenic markers
and functions. Several factors control the sequential transformation of T cells. The thymic epithelium plays a central and apparently unique role in the first phases. At its direct contact the prothymocyte (whether or not it is
Table I Stages of T-Cell Differentiation Antigenic markers
Mouse
Man (OKT sertes")
Hemopoietic stem cell Lymphoid stem cell Prothymocyte
Weak Thy-!
OKT6
Immature cortical thymocyte
Lyt-l or 123, Thy-l
OKT4, 6, 8, 10
"Post-thymic" precursor thymocyte (former T1 cell)
Lyt-l and 123, Thy-l
OKTJ,4,8
Helper/inducer
Lyt-l , Thy-!
OKT3,4
Cytotoxic/suppressor
Lyt-2, 3, Thy-!
OKT3,8
Mature T cells (former T2 cells)
·P. C. Kung et al, (18).
3
already committed to the T-cell lineage) is transformed into the immature cortical thymocyte and eventually into the "post-thymic thymocyte." This transformation is associated with the acquisition of antigenic markers, and the building up of the repertoire of anti-self receptors necessary for the cognitive function of T cells. It is not known whether thymic hormones intervene in these first phases of differentiation. Further stages of maturation, which will lead the T cell to the post-thymic thymocyte (the still incompetent cell that leaves the thymus) and to the mature T cell, are under thymic hormone control. Other factors that may participate in the very last stages include antigen stimulations, especially through the production of a lymphokine, and the T-cell growth factor (TCOF), also called Interleukin 2. Note, however, that TCOF may only operate as an amplification circuit, since it is exclusively produced by mature T cells, which, by definition, must have differentiated under thymic control. Several thymic hormones have been described, and it has not yet been determined how many molecules intervene physiologicallyas mediators of the humoral function of the thymus. Crude thymic extracts exert a number of biological activities that may generally be interpreted as the consequence of the maturation or the stimulation of the various T-cell subsets mentioned above. The key question is whether these activities are borne by different molecules. The current view is that there is more than one thymic hormone, but that a given hormone probably acts at several levels on different T-cell lines. The picture is confused by the existence in the thymus of several peptides that are probably not physiologically relevant thymic hormones, but which may possess pharmacological activity. These contaminating peptides obscure the interpretation of the effects of the crude thymic extracts that have been used clinicallyuntil now. Several welldefined peptides have been characterized: Thymosins a I, a 7, and {31, and {3. (20); thymopoietin and its h
4
smaller analogue, TP-S, which is also biologically active (1S); thymic humoral factor (24); and the serum thymic factor, facteur thymique serique (FTS) (8). There is no homology in the published amino acid sequences, but there is still room for some degree of chemical relationship other than the splitting of precursor hormones (e.g., cleavage factor, carrier molecule). Clinical trials have been initiated by using the main preparations cited previously. Preliminary results are encouraging, but it is too early to draw any definitive conclusion on the therapeutic efficacy of any of the preparations used so far. No sign of toxicity was detected, however, and correction of T-cell abnormalities (assessed on markers, and functions in vitro or in vivo) were often normalized. Thymic hormones are produced by the thymic epithelium. This has now been directly demonstrated by immunofluorescence using antisera directed at thymosin a I (16) and FrS (22). However, thymic humoral activity may be found in circulating blood. In fact, it is from the serum that FTS was initially isolated (before being chemically isolated and characterized from thymic extracts with identical amino acid analysis). Recently, evidence has been brought in a more indirect fashion that thyme-
poietin and thymosin al also circulate in the blood. Finally, another thymus-dependent serum factor has been described (1), but recent data suggest that such factor is not, as initially thought, a peptide, but merely adenosine. The significance of" the thymic hormone-like activity of" circulating prealbumin (9) is not yet well understood. It is not known definitively whether it is explained by the transport of a small thymic peptide such as FTS, as we have recently suggested on several strong experimental arguments (11). The presence of thymic hormone(s) in the circulation probably has a physiological significance. Adult thymectomy induces several changes that may take place as early as five days after the operation (S), and SUch changes are corrected in vitro by the addition of thymic hormones (6). It is apparent that the T cell that leaves the thymus is not yet completely mature and still depends partly on circulating thymic hormone(s) to terminate its maturation. Thus, in addition to representing a useful means of evaluating the epithelial function of the thymus, the measure, ment of circulating thymic hormones may be interesting to consider as an important parameter in T-cell differentiation. This latter aspect is emphasized by the demonstration in
Editors: Herman Friedman, Mario Escobar, and Noel Rose Editorial Committee: Charles D. Graber. Ph.D.• Medical University of South Carolina; John R. Kateley, Ph.D., Edward W. Sparrow Hospital Assoc.; Bruce S. Rabin, M.D., Ph.D., University of Pittsburgh School of Medicine; Robert F. Ritchie, M.D., Foundation for Blood Research, Maine; John L. Sever, M.D., Ph.D., National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health; Steven Specter, Ph.D., University of South Florida College of Medicine; Roy W. Stevens, Ph.D., New York Health Department Laboratories; Norman Talal, M.D., VA Hospital and University of California Medical Center at San Francisco; Eng M. Tan, M.D., University of Colorado Medical Center; Gabriel Virella, M.D., Ph.D., Medical University of South Carolina. Subscription Rates in U.S. and Canada: one year $46.S0; two years $89.00; three years 5127.S0. All other countries: one year $S3.S0; two years $102.S0; three years $146.S0. Single copies are 52.00 in U.S. and Canada, 52.6S all other countries. Single issues are available in quantity (prices available upon request). All subscriptions are payable in advance. Foreign subscriptions are sent guaranteed air mail. First class postage paid in U.S. and Canada. Address correspondence regarding subscription to: Ctintcal Immunology Newsletter, G. K. Hall & Co., 70 Lincoln St., Boston, Massachusetts 02111. Please include zip code in subscription address. The Clmical Immunology Newsletter is published twice monthly. All rights, including that of translation into other languages, reserved. Photomechanical reproduction (photocopy, microcopy) of this newsletter or parts thereof without special permission of the publisher is prohibited.
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normal serum of several inhibitors that may alter the efficacy of the circulating hormone. In brief, the activity of serum in the rosette assay used for evaluating FTS is very much increased after removal of highmolecular-weight proteins (by mere filtration on Amicon membranes) and still further increased by removal of medium-sized molecules by Sephadex gel filtration (2). More precisely, there are several inhibitors with respective molecular weights of 100,000300,000, 40,000-70,000, and 5,00010,000 daltons. The largest inhibitors are removed before evaluating the serum thymic hormone level, but the smaller ones are not eliminated and interfere with the final evaluation. While this interference may cause difficulties when evaluating central thymic epithelial function, it is interesting to consider, since it might also operate in vivo in the biological effects of circulating thymic hormone. Better appraisal of these inhibitors will be made possible when radioimmunoassays that permit the measurement of the absolute amount of hormones present in the serum are available in addition to the biological assays used presently. It may be relevant to note at this stage that the serum also contains carrier proteins for FrS whose relationship with the inhibitors is not yet clear. In any case, the presence of protein-bound (and possibly inactive) thymic hormone in the serum should be taken into consideration when interpreting serum evaluation of such hormones, by both biological or radiobiological assays. Laboratory Methods for the QuantitatioD of Circulating Thymic Hormones Several methods have been described to measure the levels of circulating thymic hormones. The first of these methods was described in our laboratory in 1972 (4). It is based on the changes induced by thymic hormones on the minority of spleen cells of adult-thymectomized (Tx) mice that form spontaneous rosettes with sheep erythrocytes. The assay consists in detecting the smallest serum concentration that renders
spleen rosette-forming cells (RFC) sensitive to anti-theta serum (A BS) or to azathioprine (Az), the well-known immunosuppressive agent that happens to inhibit, at low concentration, rosette formation in normal mice and to lose this property, as AB S does in Tx mice (3). Results are expressed in serum dilutions (the higher the active dilution, the higher the concentration of the hormone) with the reservation linked to the serum inhibitors previously mentioned, which may influence overall biological activity. Sera are used in the tests after ultrafiltration on Amicon membranes (molecular weight cutoff: 50,(00), but small inhibitors are still present. In selected cases, the relationship of the detected activity to FrS may be verified by passing the serum on an immunoadsorbent prepared with an antibody raised against synthetic FrS. The strict thymus dependency of the activity detected by the rosette assay is demonstrated by its total absence in the sera of nude or Tx mice and of patients with Di George syndrome, and its reappearance in all individuals after thymus grafting. A similar method was described in 1977 by Twomey et al. (26). Serum is incubated with nude-mouse spleen cells, and the increase in (} -positive cells is measured using a cytotoxic assay with trypan blue. The sensitivity of the test is increased by adding ubiquitin at subliminal concentration, a protein known to induce nonspecifically T- (and B-) cell markers. As in the rosette assay, serum ultrafiltrates are used rather than total serum, because of the presence of serum inhibitors. Thymopoietin shows activity in the test. Interestingly, FrS, whose effects are inhibited by ubiquitin (17), is not active in the test. Results are expressed in the nanogram-equivalent of thymopoietin, which does not take full account of molecules other than thymopoietin that could be active in the test, and of the interfering molecules (e.g., carrier proteins, inhibitors). As for the rosette assay, the specificity of the Twomey assay is assessed by its negativity in the sera of
nude and Tx mice, and in sera from patients with Di George syndrome. Radioimmunoassays (RIA) have been described for three well-defined thymic peptides: thymosin a I (Goldstein, unpublished), FrS (23), and thymopoietin (14). Such RIAs have not yet been successfully applied to the evaluation of serum samples for FrS and thymopoietin, essentially because of unsolved interferences ' with serum proteins. More conclusive results have been reported for thymesin al (21), but, unlike the two bioassays previously described, unequivocal demonstration of the total absence of RIA-positive material in the serum of Tx animals or humans as compared to normal individuals has not yet been accomplished. Serum Level of Thymic Hormones in Experimental Models and in Human Diseases The age dependency of FrS serum level has been extensively studied in the mouse. FrS is already present at adult level at birth. Studies in Tx pregnant mothers have shown that FrS is first detectable on the fourteenth day of pregnancy. FrS level remains stable until 5-7 months of age and then progressively declines to become insignificant after 12 months. This age-dependent decline, which parallels that of thymus weight, is premature in autoimmune mice (NZB, (NZB x NZW) FI' MRLI/l, SWAN). A recent study has shown that the disappearance of FTS from the serum in aging mice is due to the simultaneous decline in ITS production and appearance in the serum of FrS inhibitors; grafting an old thymus in young Tx mice induces the appearance of an FrS activity superior to that present in the old mice from which the thymus graft is taken. Similar experiments in NZB mice indicate that in these mice FTS loss is not due to peripheral destruction, but to a deficit in thymic secretion. Evaluation of serum FrS levels has also provided interesting information on thymus physiology. Multiple thymus grafts are associated with increased FrS level grossly propor-
5
tional to the number of thymic lobes grafted, which confirms the absence of thymic-hormone-induced feedback regulation of the thymus, which was suspected from macroscopic studies of the thymus (25). The age dependency of FrS level has also been observed in man. The level is stable until the age of 15-20 years, followed by a progressive decline. The data shown on Figure I, which we initially reported for FrS (7), have been confirmed with the rosette assay by other investigators (12-13), as well as with the Twomey assay (19), with similar kinetics. In contrast to these data, the level of thymosin 0'1 seems to drop earlier than that of FTS, with a decline starting as soon as 10 years of age (Goldstein, unpublished). The serum level of thymic hormones has been evaluated in a number of diseases, using either our rosette assay or the Twomey assay. FrS level is low in human as in murine lupus, although the decrease in FrS level is not a constant feature of lupus patients and is only detectable in young patients (before agematched controls lose their own FTS production). In myasthenia gravis, thymic hormone level is normal in young patients but tends to be higher than normal in older ones. In mycosis fungoides, the level is high, and it has
been verified, using the immunoadsorbent previously mentioned. that such increased FTS activity was absorbed out by the anti-FTS immunoadsorbent. In immunodeficiency syndromes, FTS level is low in thymic aplasia and normal in sex-linked agammaglobulinemia. Interestingly, the FTS level is often low in severe combined immunodeficiencies and tends to increase after bone marrow grafting, parallel to the appearance of thymic shadow. These data indicate that the humoral function of the thymic epithelium may be stimulated by the contact with the colonizing stem cells (absent before bone marrow transplantation). Lastly, note that we have observed epithelial thymoma with high levels of fTS and a high percentage (980/0) of peripheral T cells in one 80-year-old patient, which indicates that in humans there are thymomas secreting inappropriate amounts of thymic hormones, similar to the carcinogen-induced epithelial thymoma in the mouse (10). In conclusion, the dosage of serum thymic hormone is now available for clinical use. Techniques used today are still essentially biologic. Currently, the most frequently used rosette assay has now been employed successfully by several independent groups (4,9, 13, 17), but it is not yet easily available for clinical practice.
serum FTS
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•
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•• ••
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1/16
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• •
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.\
30-40 50-60 40-50 ~60 20·30
age (years)
Fig. J. Age dependency of serum FTS levels in man, as evaluated by the rosette assay.
6
We hope that in the near future R...lA methodology will be generalized. In the meantime, the biologic assays will have provided important information on thymic epithelial function in healthy and diseased individuals. In addition. even when valid RIA procedures are available, these biologic assays will still have to be studied in order to appreciate the resulting effects of the various components, (including the carriers and inhibitors) that participate in circulating thYmic hormone activity. The significance of' the various hormones evaluated by RIA methods and their relevance to thymus physiology and pathology must also be studied critically. References I. Astaldl, A., G. C. B. Astaldl, P. T. A. Scbellekens, and V. P. ElJsvoogel. 1976. Thymic factor in human sera demonstrable by a cyclic AMP assay. Nature 260:7I3-71S. 2. Bacb, J. F., M. A. Bacb, D. Blanot. E. Brlcu, J. Cbarrclre, M. Dardenne. C. Fournier, and J. M. Pleau. 1978. Thymic serum factor (FrS). Bull. lost. Pasteur 76:32S-398. 3. Bach, J. F., and M. Dardenne. 1972. Antigen recognition by T lymphocytes. II. Similar effects of azathioprine, ALS and antitheta serum on rosette-forming lymphocytes in normal and neonatally thymectomized mice. Cell Immuno!. 3:11-21. 4. Bacb, J. F., and M. Dardenne. 1972_ Thymus dependency of rosetteforming cells. Evidence of a circulating thymic hormone. Transplant. Proc, 4:34S-3S0. S. Bach, J. F., M. Dardenne, and A. J. S. Davies. 1971. Early effect of adult thymectomy. Nature 231:110-111. 6. Bacb, J. F., M. Dardenne, A. L• Goldstein, and A. Wblte. 1971. Appearance of T-cell markers in bone marrow rosette-forming cells after incubation with purified thymosin, a thymic hormone. Proc. Natl, Acad, Sci. USA 68:2734-2738. 7. Bacb, J. F., M. Dardenne, M. Paplemlk. A. Barols, P. Levasseur. and H. Le Brigand. 1972. Evidence for a serum factor produced by the human thymus. Lancet 2:IOS6-10S8. 8. Bacb, J. F., M. Dardenne, J. M. paeau, and J. Rosa. 1977. Biochemical characterization of a serum thymic hormone. Nature 266:SS-57. 9. Burton, P., S. Iden, K. Mltcbell, and A. White. 1978. Thymic hormonelikerestoration by human prealbumin or azathioprine sensitivity of spleen
cells from thymectomized mice. Proc. Natl. Acad. Sci. USA 75:823826. 10. Dardenne, M., M. Papiernik, J. F. Bach, and O. Stutman. 1974. Studies on thymus products. III. Epithelial origin of the thymic hormone. Immunology 27:299-304. 11. Dardenne, M., J. M. Pleau, and J. F. Bach. 1980. Evidence of the presence in normal serum of a carrier of the serum thymic factor (FrS). Eur. J. Immunol. 10:83-86. 12. Fabris, N. 1980. Serum thymic factor determination in different human pathologies. Int. J. Immunopharmacol. 2(3):157. 13. Garaci, E., R. Ronchetti, V. Del Gobbio, G. Tramutoli, C. RinaldiGaraci, and C. Imperato. 1978. Decreased serum thymic factor activity in asthmatic children. J. Allergy Clin. Immunol. 62:357-362. 14. Goldstein, G. 1976. Radioimmunoassay for thymopoietin. J. Immunol. 117:690-692. 15. Goldstein, G., M. P. Scheid, E. A. Boyse, D. H. Schlesinger, and J. Van Wauwe. 1979. A synthetic pentapeptide with biological activity characteristic of the thymic hormone thymopoietin. Science 204:1309-1312. 16. Hirokawa, K., and K. Saitoh. 1980. Heterogeneity of thymic epithelial
17.
18.
19.
20.
21.
cells, revealed by localization of thymosin a I and various hydrolytic enzymes in human thymus. 4th Int. Congo Immunol. (Paris), abstr. 3.3.14. Iwata, T., G. S. Incefy, and R. A. Good. 1979. Interaction between thymopoietin and facteur thymique serique in the rosette inhibition assay. Biochem. Biophys, Res. Commun. 88:1419-1427. Kung, P. C., et al. 1979. Monoclonal antibodies defining distinctive human T-cell surface antigens. Science 206: 347-349. Lewis, V. M., J. J. Twomey, P. N. Bealmear, G. Goldstein, and R. A. Good. 1978. Age, thymic involution and circulating thymic hormone activity. J. Clin. Endocrinol. Metab. 47:145-150. Low, T. L. K., G. B. Thurman, M. McAdoo, J. McClure, J. L. Rossio, P. H. Naylor, and A. L. Goldstein. 1979. The chemistry and biology of thymosin. I. Isolation, purification and biological activities of thymosin Q, and polypeptide f3, from calf thymus. J. BioI. Chern. 254:981-984. McClure, J. E., and A. L. Goldstein. 1980. Changes with age in blood levels of thymosin Q. as measured by radioimmunoassay. 4th Int. Congo Immunol. (Paris), abstr. 17.2.26.
Letters to the Editor
Case Report and Self-Assessment
To the Editor:
This section was prepared byG. Virella, M.D., Ph.D., Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, South Carolina. Similar questions and answers are solicited from readers.
I question the appropriateness of devoting a page and a half of the Newsletter to the correspondence between Drs. Pitts and Virella. The Newsletter is reasonably expensive and your subscribers deserve better. Thomas W. Green, M.D., Ph.D. Medical Director Berkeley Biologicals Berkeley, California 94710
Editor's Reply: The Newsletter is a new venture, and we appreciate hearing from our readers. Since we view the Newsletter as a forum for the exchange of information and ideas among immunologists, we had hoped to encourage more discussion in this section. We invite Dr. Green and all our other readers to continue to send suggestions and to contribute items for publication.
Case Report In 1969, the patient, a 65-yearold Caucasian male, began to complain of intermittent painful episodes in the epigastrium. Two years later, he developed symptoms of pyloric stenosis. At that time, routine electrophoresis showed a slight increase in alpha 2 globulins. A 6 x 4 em juxtapyloric tumor was surgically removed; no obvious lymphadenopathies were seen. The histological examination of the tumor was compatible with lymphatic lymphoma. The patient was discharged without additional therapy. Three years later, the patient developed gastrointestinal symptoms (nausea, vomiting, diarrhea) with hepatosplenomegaly. A liver biopsy
22. Monier, J. C., M. Dardenne, J. M. Pleau, D. Schmitt, P. Deschaux, and J. F. Bach. Characterization of FrS in the thymus. I. Fixation of anti-F'I'S antibodies on thymic reticulo-epithelial cells. Clin. Exp. Immunol. In press. 23. Pleau, J. M., D. Pasques, and J. F. Bach. 1978. Dosage radioimmunologique du facteur thymique serique (FrS) In Radioimmunoassay and related procedures in medicine, vol. 2. International Atomic Energy Agency, Vienna. 24. Trainin, N., M. Small, D. Zipori, T. Umiel, A. I. Kook, and V. Rotter. 1975. Characteristics of TAF, a thymic hormone, p. 117. In D. W. Van Bekkum (ed.), The biological activity of thymic hormones. Kooyker Scientific Publications, Rotterdam. 25. Tubiana, N., and M. Dardenne. 1979. Neonatal thymus grafts. I. Studies on the regulation of the level of circulating thymic factor (FrS). Immunology 36:207-213. 26. Twomey, J. J., G. Goldstein, V. M. Lewis, P. M. Bealmear, and R. A. Good. 1977. Bioassay determinations of thymopoietin and thymic hormone levels in human plasma. Proc. Natl. Acad. Sci. USA 6:2541-2545.
showed portal space infiltration by differentiated mononucleated lymphocytes. Proteinuria was 20.5 mg/dl, The patient was treated supportively and discharged. In 1979, the patient complained of weakness, exertion dyspnea, easy fatigue, and loss of weight. He showed pallor of skin and mucosae, hepatosplenomegaly, and right-sided pleural effusion. Hemoglobin was 8.S g/dl. White blood cell counts varied between 3,000 and 5,000 mm' with normal differential count. A bone marrow aspirate showed pancytopenia with relative increase of reticulocytes and lymphocytes. A gastrointestinal x-ray examination showed widespread mucosal infiltration with multiple constrictions and dilations of the jejunal loops. Urinary proteins were 35 mg/dl. The patient was treated with a combination of vinblastine, triethylene phosphoramide, rufochromomycin, procarbazine, and prednisone. At the end of two months
7