lymphocytes. H. & E. x 57. FIG. 2.-Spleen. Male orchidectomized mouse. Well-developed white pulp with densely packed lymphocytes round the arterioles.
Br. J. exp. Path. (1980) 61, 590
EFFECT OF SEX HORMONES ON THE RESPONSE OF MICE TO INFECTION WITH TOXOPLASMA GONDII C. KITTAS AND L. HENRY Froqn the Departntent of P'athology, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX Received for publication July 14, 1980
Summary.-Groups of male and female mice were gonadectomized and some implanted with a pellet of hexoestrol. Half of the animals in each group, including controls, were infected with a low-virulence strain of Toxoplasma gondii. The animals were killed 6 weeks after infection. Gonadectomy increased the relative thymic weight in both sexes, but more so in the male, and the paracortical area of the lymph node was enlarged, as was the thymus-dependent area of the spleen. Hexoestrol administration induced almost complete thymic atrophy and partial involution of the peripheral lymphoid tissues. Greater resistance to toxoplasmic infection was found in the gonadectomized mice than in controls of both sexes, possibly due to a better cell-mediated immunity in the gonadectomized animals. Overwhelming toxoplasmosis with increased mortality was found in the hexoestroltreated mice after infection, probably resulting from a depression of cell-mediated immunity caused by hexoestrol. It is postulated that the cellular immune response is of greater importance than the formation of antibody in resistance to toxoplasmic infection. The role of the thymolymphatic-sex hormonal interrelation on the age-sex differences seen in the incidence of toxoplasmic lymphadenopathy is also discussed.
NUMEROUS MAMAIALIAN SPECIES, including man, show sex differences in the immune response. Several observations suggest a superior immunological capability of the female in terms of both humoral and cellular immunity. Thus, female superiority in antibody formation has been shown following immunization with various antigens (Stern and Davidsohn, 1955; Kenny and Gray, 1971) and a more rapid rejection of both skin allografts (Graff, Lappe and Snell, 1969) and tumours (Snell, 1958) by females has also been reported. These differences may be under genetic control (Steinberg, Pincus and Talal, 1971; Vasilakis, Kunz and Gill, 1974), but sex hormones may also have a direct effect (Eidinger and Garrett, 1972). An age-sex relationship has been noted among patients with reactive lymphadenopathy following infection with Toxoplasma gondii (Beverley et al., 1976). Henry and Beverley (1976) have shown sex
differences in the histological appearances of the lymph node post-capillary venules in control mice and mice infected with T. gondii. These differences have been shown to be under sex hormonal influence (Kittas and Henry, 1979a, b). The present study concerns the effect of gonadectomy and oestrogen administration on the lymphoid organs of the mouse and their response to infection with T. gondii. MATERIALS AND METHODS Animals. Male and female mice were used of a white, non-inbred, laboratory strain, 11 weeks old. Twelve male and 12 female animals were controls, while 24 mice of each sex were subjected to gonadectomy. Half of the gonadectomized mice received an implant of hexoestrol. Eight male and 8 female mice were sham-operated. Half of the animals of each group were infected with Toxoplasma gondii, the remainder being left uninfected. Operations and oestrogen administration. Orchidectomy and oophorectomy were per-
591
SEX HORMONES AND TOXOPLASMA GONlDI)
formned under anaesthetic through a suprapubic incision. The ovaries with the greater part of the tuterus were excised en bloc. The testis and the epididymis were removed after ligation of the spermatic cords. Sham operations were performed through an identical incision. Immediately after gonadectomy, half of the male and female mice received an s.c. implant of a pellet containing 12 mg hexoestrol (Capomatic Tablets S4B, Boots Co. Ltd, Nottingham, England) through a small incision behind the left ear. Inoculations. A cyst-forming, low-virulence strain of Toxoplasma gondii (Beverley strain), first isolated from an apparently healthy rabbit, was used. This has been maintained by s.c. passage of brain emulsion in mice at 3-4monthly intervals. All mice were inoculated at 15 weeks of age. The inoculum was prepared from the brains of mice 9 weeks after infection. Thirty toxoplasma cysts, given as a dilute brain emulsion, were inoculated s.c. Examinations. Six weeks after inoculation all the mice were weighed, anaesthetized and exsanguinated via the axillary vessels. The spleen and thymus were removed free of fibrofatty tissue and weighed before fixation on an electronic balance. One half of the brain of each mouse was emulsified with isotonic sterile saline and exainined as a wet, unstained filmr for the presence of toxoplasma cysts. Using a cystcounting system the total number of cysts per inouse brain was estimated. The remainder of the brain, the lymph nodes, adrenals and speciinens from the luings, heart and liver were fixed in 40o formol saline. The tissues were processed routinely to paraffin, sections cut at 4 ,um and stained with haematoxylin-eosin and methylgreen-pyronin stains. Antibody tests. Titres of toxoplasma antibodies were assessed by a modification of the dye-test described by Sabin and Feldman (1948). Histometric technique. A planimetric analysis of sections by a point-counting system was performed in order to measure the volume percent-
age of the lymph nodes occupied by cortex, paracortex and medulla. A 21mm disc containing a graticule with 100 line intersections was used (G14, Graticules Ltd, England), a network of regularly spaced points being superimposed upon the sections. A particular component in the tissue samnple will be directly proportional to the relative numbers of line intersections that overlie the component in the sections (Hennig and Meyer-Arendt, 1963). Several random sections fromn each block containing the whole lymph node were so examnined. Statistics. The body and relative organ weights, cysts per mouse brain, and percentage of the 3 lymph-node compartments were presented as the arithmetic mean + s.e. mean. The t test was used for the deterinination of statistical significance. RESULTS
The results in the sham-operated mice were similar to those in the control animals. None of the mice receiving hexoestrol alone died, but all showed a mild wasting illness. A mortality of 50%0 was seen after infection of the hexoestrol-treated mice. Further mice were used to restore the numbers. Body and organ weight The body weight and relative weight of thymus and spleen are shown in Table I. Male control mice were consistently heavier than females. However, females had a significantly heavier thymus (P< 0-001). Gonadectomy induced a slight increase in body weight and a significant increase in spleen and thymus relative weights. The rise in thymus relative
TABLE I.-Mean body weight and relative* weights of the thymnus and spleen of male and female mice Spleein relative weiglht Thlymus relativ-e weiglht Body weiglt (g) r-
Group Control Not infecte(d Infected Gonadectomy Not infected Infected Gonadectomy +
r-
A
--\
Male
Female
167-3 + 2-9
371-1 + 10-1 716-2 + 5-7
374-3+6-1 889-4+ 11-7
154-7+4-8 182-1 + 5-9
418 5+6-3 431-9+7-9 868-8 + 19-9 799-9 ± 12-4
Male
Female
Male
Female
42-7+0-7 37-4 + 0-6
37-8+0-6 32 + 0 4
984+3 118-9 + 3
45+0-5
39-9+0-5 33-5 + 0-2
181-7+4-9 173-5+ 7-2
38-7 + 0-6
Hexoestrol Not infected 25-8 + 0 9 21-3 + 0 3 Infected 22-8 + 0-6 20 7 + 0 4 * Relative weight (mg/100 g body wvt). + S.e. mean.
139-6+±41
439-7 + 6-6 507-6 + 18-7
515-9 + 11-2 523-6 + 15-9
592
C. KITTAS AND L. HENRY
1
2
-
|
s-
FIG. 1.-Thymus. Male orchidectomized mouse. The cortex is widened and densely packed with lymphocytes. H. & E. x 57. FIG. 2.-Spleen. Male orchidectomized mouse. Well-developed white pulp with densely packed lymphocytes round the arterioles. H. & E. x 57.
593
SEX HORMONES AND TOXOPLASMA GONDII
TABLE II.-Mean percentage of the areas occupied by the cortex, paracortex and medulla in the lymph nodes of male and female mice Cortex
Medulla
Paracortex C-
Control Not infected Infected Gonadectomy Not infected Infected + S.e. mean.
Male
Female
Male
Male
Female
25+2 25+0-9
21+0-7 27+3
50+2-7 54-5+2 34+4 34++3 3
25+1 41+3
27+2 28+1
22+0-7 26+3
56+1-5 47+3
17+0-8 25+2
weight was considerably greater in male than in female gonadectomized mice. As a result, the thymus of males was heavier than that of females after gonadectomy (P < 001). Hexoestrol administration induced a progressive loss of weight, the weight of male and female controls being twice as much as that of hexoestroltreated animals. The thymus was almost completely atrophic after hexoestrol. Infected animals were invariably less heavv than the non-infected ones. Significantly heavier spleens were found after infection of control and gonadectomized mice of both sexes but only a small change was found when infection followed hexoestrol administration. Effect of gonadectomy and hexoestrol administration on the histology of the lymphoid
organs Gonadectomy induced a widening of the thymic lobules with a cortex more densely packed by lymphocytes (Fig. 1). These changes were more prominent in male than female mice. The spleen in both sexes showed a higher proportion of white to red pulp because of an increase in the lymphocytic population mainly round the central arterioles (Fig. 2). The percentage occupied by each of the 3 lymph-node compartments is shown in Table II. The paracortex was slightly more prominent in female than male controls and increased after gonadectomy in both sexes. The lymphocytes in the paracortical area, mainly round the post-capillary venules, were more densely packed in gonadectomized than control mice (Fig. 3). The in-
57+2 53+3-5
Female 24-5+2 39+3 20+2 21+2
crease of the paracortical area after gonadectomy was slightly higher in male than female mice although neither value was statistically significant. The area occupied by medulla was decreased in both sexes by gonadectomy although this only achieved significance in the male (P < 0 001). Hexoestrol administration induced severe thymic atrophy in both sexes. Remnants of thymic tissue could only be detected by removing the entire upper mediastinum and cutting serial sections of the whole blocks. The completely involuted thymus was entirely depleted of its normal cortical lymphocytic content. The main surviving cells were the reticular cells of the medulla with some medullary lymphocytes and occasional Hassall's corpuscles (Fig. 4). The spleens of male and female mice after hexoestrol administration showed a decreased proportion of white to red pulp (Fig. 5). A relative increase in the number of megakaryocytes was also found. The lymph nodes of the gonadectomized hexoestrol-treated animals were small and showed varying degrees of involution. Thus several atrophic lymph nodes showed distortion of their normal architecture with an increased proportion of fibro-histiocytic elements, a few lymphocytes and occasional plasma cells (Fig. 6). By contrast some lymph nodes were small but their morphology was normal. Because of this discrepancy in the histological appearances no attempt was made to evaluate the percentage distribution of cortex, paracortex and medulla in the lymph nodes of hexoestrol-treated animals.
594
C. KITTAS AND L. HENRY
iS~ ~ ~ ~ ~ ~ ~ ~_ 22
FIG. 3. Lymph node. Male orchidectomized mouse. Prominent paracortex with densely packed lymphocytes round the post-capillary venules. H. & E. x 23. FIG. 4. Thymus. Male orchidectomized mouse after hexoestrol administration. Remnants of thymic medullary tissue with complete cortical atrophy. H. & E. x 57.
SEX HORMONES AND TOXOPLASMA GONDII
5
. -
' r*'O NA
FIG. 5. Spleen. Male orchidectomized mouse after hexoestrol administration. Atrophy of the splenic issue, mainly the white pulp. H. & E. x 57. FiG. 6.-Lymph node. Male orchidectomized mouse after hexoestrol administration showing atrophy witli increase(d diffuse histiocytic elements. H. & E. x 154.
595
C. KITTAS AND L. HENRY
596
......
......
....... ..
....
.....
FiG. 7. Brain. Male orchidectomized, hexoestrol-treated mouse after infection. Aggregate of toxoplasma cysts. H. & E. x 360. FIG. 8. Peripheral nerve. Male orchidectomized, hexoestrol-treated mouse after infection. A toxoplasma cyst involving the nerve (arrow). H. & E. x 225.
597
SEX HORMONES AND TOXOPLA4S5MA GONDII
Effect of toxoplasma infection The thymus of the control mice after infection showed only focal areas of cortical lymphoid depletion. No changes were seen in gonadectomized infected animals. The lymph nodes of control and gonadectomized mice after infection showed varying degrees of follicular reaction with germinal centre phagocytosis. Epithelioid histiocytes were also present. The paracortex of control male and female mice was decreased after infection (P < 001 and P < 0*001 respectively), while the medulla was better developed than that of the non-infected controls containing numerous plasma cells. Male and female gonadectomized mice showed more paracortical and less medullary reaction to infection. However, large numbers of plasma cells were again present. The latter were also increased even when infection followed hexoestrol administration. This was the only change occurring in the lymph nodes compared to the non-infected, hexoestroltreated groups. Infection induced a follicular reaction and increased numbers of plasma cells both round the follicles and in relation to the fibrous septa of the spleen of control and gonadectomized mice. After hexoestrol the follicular reaction to infection was poor but the numbers of plasma cells were
TABLE III.-Mean numbers of toxoplasma cysts found in the brains of male and female mice infected with T. gondii and subjected to gonadectomy and administration of hexoestrol
Sex MIale Female
Conitrol andl infect ion 220+2.3 222 + 42
Gonadectomy an(c infection 178± 24 189+ 22
Gonadectomy andl hexoestrol and infection 599+45 598 + 64
+ S.e. mean.
ministration, becoming polymorphonuclear in nature and less dense than in the other infected groups. Although no sex differences were found in the number of cysts in the brains of control mice, these numbers decreased in both sexes when infection followed gonadectomy. This was constant but not statistically significant. By contrast, toxoplasma cysts were numerous in the brains of male and female hexoestrol-treated mice, forming aggregates in many instances (Fig. 7). The numbers of the cysts were significantly above those found in the infected controls (P < 0.001). The heart of the infected mice of all groups showed a similar inflammatory infiltrate but toxoplasma cysts were only found in sections from the hexoestroltreated mice. After infection the lungs and again increased. liver from the control and gonadectomized animals showed only mild focal chronic Other organs No significant changes in the size and inflammatory infiltration with focal areas the morphology of the adrenals were found of parenchymal cell necrosis, mainly of the after gonadectomy and hexoestrol ad- liver. More severe lesions of pneumonia ministration with or without toxoplasma and extensive liver cell necrosis were present when hexoestrol administration infection. Table III illustrates the numbers of was followed by infection. In the latter group, involvement of cysts found in the brains of mice of the peripheral nerves by toxoplasma cysts was various infected groups. The meninges and brains of male and found with only minor, inflammatory female control and gonadectomized mice reaction (Fig. 8). showed a focal, dense inflammatory infiltrate consisting of lymphocytes and a few Serology plasma cells. The infiltrate was more proNo differences were detected in the antinounced in gonadectomized than in con- body titres of the various infected groups, trol mice and its character was altered the average value being 1: 4,000 for each when infection followed hexoestrol ad- group. 41
9C.
.5-98
KITTAS AND L.
DISC USSION
Britain, toxoplasmic lymphadenopathy is seen more frequently in m.ales under 15 years, in females over 25 years and equally in the two sexes between these ages (Beverley et al., 1976). In the present experiment it is shown that the morphology of the lymphoid organs and their response to toxoplasma infection is changed by altering the sex-hormonal milieu. Thus the thymus has a higher relative weight in female than in male controls and the converse holds in gonadectomized mice. Gonadectomy increases the relative thymic weight in both sexes but significantly more so in males. Gonadectomy also increases the relative splenic weight and the area of the lymph nodes occupied by paracortex and produces enlargement of the thymic cortex, the lymph node paracortex and the area of the splenic white pulp round the central arteriole (Dougherty, 1952; Castro, 1974a). These findings suggest a depressive effect of sex hormones on the thymus itself and on the thymus-dependent areas of both lymph node and spleen, areas generally accepted as being involved in cell-mediated immunity (Parrott and de Sousa, 1971). Similar findings have been previously reported (Reilly et al., 1967; Sobhon and Jirasattham, 1974; Kalland, Fossberg and Forsberg, 1978). It has also been suggested (Money, Fager and Rawson, 1952) and supported by the present findings that androgens have a more powerful suppressive effect on the thymus than In
oestrogens.
Infection with Toxoplasma gondii induces an increased paracortical but a less marked medullary reaction in gonadectomized than in control animals of both sexes. In addition, there is a slight, but constant decrease in numbers of brain cysts in gonadectomized mice as compared to those of the controls. The response to toxoplasma infection may well be mediated by a better cellular immunity in gonadectomized than in control mice. This is supported by the changes induced in the thymus-dependent areas of the
IIENRY
lymphoid organs by gonadectomy. Moreover, it has been previously shown that gonadectomy induces a more rapid rejection of skin grafts (Graff et al., 1969) and tumours (Castro, 1974b). In the present experiment, oestrogens were given by implantation of a pellet of 12 mg of hexoestrol, the oestrogenic activity of which has been previously compared to that of stilboestrol (Harmer and Broom, 1948). Implantation ensures a constant effect for a long period of an estimated dose of 130 itg daily. This dose is higher than that necessary to induce oestrus (Nicol et al., 1964), but lower than that capable of producing severe liver damage (Reilly et al., 1967). Complete atrophy of the thymic cortex occurs after hexoestrol in both sexes with preservation of only medullary elements. Involutionary changes of the lymph nodes and reduced splenic white pulp are also seen. Despite these changes there were no deaths or other signs of severe illness if infection did not supervene. There were no histological changes suggesting adrenal involvement, in line with previous work stating that oestrogens have a direct effect on the thymus with no mediation through the adrenals (Pereira-Luz et al., 1969; Kalland et al., 1978). When hexoestrol-treated male and female mice are infected with Toxoplasmra gondii a mortality of 5000 is seen despite the presence of high levels of antibodies. In the former group there are signs of overwhelming toxoplasmosis such as numerous toxoplasma cysts in the brain and other organs including peripheral nerves. Severe lung and liver lesions are also found. It is proposed that these changes are due to severely depressed cellular immunity in the hexoestroltreated animals resulting in almost complete elimination of resistance to toxoplasmic infection. The complete atrophy of the thymus, and the other histological changes seen in the thymus-dependent areas of the spleen and lymph nodes after hexoestrol administration, support this hypothesis. It is suggested that cellular
SEX HORMONES AND TOXOPLASMA GONDII
immunity is of greater importance in this infection than the presence of circulating antibody (Lindberg and Frenkel, 1977). Depression of cellular immunity by oestrogens has been previously shown (Stimson and Huinter, 1976). The present findings suggest that sex hormones have a suppressive effect on thymus and thymus-dependent areas of peripheral lymphoid organs with androgens having a greater effect, leading to a female superiority over male in cellmediated immunity. This may be responsible for the higher incidence of toxoplasmic lymphadenopathy in women of 25 years and over than in men of the same age, in view of the immunological rather than the inflammatory character of the lymph-node response to toxoplasmic infection (Henry et al., 1973). Androgens have been reported to be an inhibitor of the humoral immune response (Kotani, Nawa and Fujii, 1974). By contrast oestrogens enhance phagocytic (Dobson and Kelly, 1973; Nicol et al., 1964), and B-lymphocyte activity (Stimson and Hunter, 1976) inducing an increased antibody response to many antigens (Eidinger and Garrett, 1942; Thanavala, Rao and Thakur, 1973; Rangnekar, Rao and Joshi, 1974). Oestrogens do not always increase antibody levels (Vasilakis et al., 1974) as was seen in the present experiments, but in general adult females have a better response than males in both cell-mediated and humoral immunity. No evidence was obtained for the interpretation of the higher incidence of toxoplasmic lymphadenopathy seen in males under the age of 15 years. A sex-hormonal effect on the immune system cannot be entirely excluded since differences in sexhormone secretion have been reported in infants. Thus it has been shown that the plasma levels of testosterone are low in both male and female infants under the age of 12 years (Winter and Faiman, 1972, 1973) while a greater oestrogenic effect has been found in girls than boys before puberty (Preeyasombat and Kenny, 1966) even in those prematurely born (Silver and
599
Kirchner, 1966). These oestrogens could have a greater suppressive effect on cellmediated immunity in females than males under the age of 12 years, leading to a male superiority in this respect. Thus, in contrast to the mature subject, male infants may well have a better response than females in cell-mediated immunity, leading to a higher incidence of toxoplasmic lymphadenopathy. However, a genetic control independent of the sex-hormonal system must also be considered. Dr Kittas was in receipt of a fellowship from the State Scholarship Foundation of Greece. The work was financed by a grant from the Medical Research Fund of Sheffield University. Thanks are due to Mr C. Day for technical, Mrs M. Hogg for secretarial and Mr M. J. Eaton for photographic assistance. The hexoestrol was obtained with the help of Mr D. A. Atkin, Boots Company Limited, Nottingham. The toxoplasma dye tests were performed by Dr D. G. Fleck. REFERENCES BEVERLEY, J. K. A., FLECK, D. G., KWANTES, W. & LUDLAM, G. (1976) Age-Sex Distribution of Toxoplasmic Lymphadenopathy. J. Hygiene (Cambridge), 76, 215. CASTRO, J. E. (1974a) Orchidectomy and the Immune Response. I. Effect of Orchidectomy on Lymplhoid Tissues. Proc. Roy. Soc. B, 185, 425. CASTRO, J. E. (1974b) Orchidectomy and the Immune Response. The Effect of Orchidectomy on Tumour Induction and Transplantation. Proc. Roy. Soc. B, 186, 387. DOBSON, E. L. & KELLY, L. S. (1973) The Combined Stimulation of the Reticuloendothelial Svstem by Estradiol and Endotoxin. J. Reticuloendoth. Soc., 13, 61. DOUGHERTY, T. F. (1952) Effect of Hormones on Lymphatic Tissue. Physiol. Rev., 32, 379. EIDI-NGER, D. & GARRETT, T. J. (1972) Studies in the Regulatory Effects of the Sex Hormones on Antibody Formation and Stem-cell Differentiation. J. exp. Med., 136, 1098. GRAFF, R. J., LAPPIE, M. A. & SNELL, G. D. (1969) The Influence of the Gonads and Adrenal Glands on the Immune Response to Skin Grafts. Transplantation, 7, 105. HARMER, G. L. Al. & BRooM, W. A. (1948) Activity of Some Synthetic Oestrogens Determined by Experiments on Rats. Lancet, 2, 766. HENNIG, A. & MEYER-ARENDT, J. R. (1963) Microscopic Volume Determination andl Probability. Lab. Invest., 12, 460.
600
C. KITTAS AND L. HENRY
HENRY, L., BEVERLEY, J. K. A., SHORTLAND, J. R. & Coup, A. J. (1973) Experimental Toxoplasmic Lymphadenopathy in Rabbits. Br. J. exp. Path., 54, 312. HENRY, L. & BEVERLEY, J. K. A. (1976) Age and Sex Differences in the Response of Lymph Node Post-capillary Venules in Mice Infected with Toxoplasma gondii. Br. J. exp. Path., 57, 274. KALLAND, T., FOSSBERG, T. M. & FORSBERG, J. G. (1978) Effects of Estrogen and Corticosterone on the Lymphoid System in Neonatal Mice. Exp. mol. Path., 28, 76. KENNY, F. G. & GRAY, A. J. (1971) Sex Differences in Immunological Response: Studies of Antibody Production by Individual Spleen Cells after Stimulus with Escherichia coli Antigen. Pediat. Res., 5, 246. KITTAS, C. & HENRY, L. (1979a) Effect of Gonadectomy and Oestrogen Administration on the Response of Lymph-node Post-capillary Venules to Infection with Toxoplasma gondii. J. Path., 127, 129. KITTAS, C. & HENRY, L. (1979b) An Electron Microscope Study of the Changes induced by Oestrogens on the Lymph Node Post-capillary Venules. J. Path., 129, 21. KOTANI, M., NAWA, Y. & Fujii, H. (1974) Inhibition by Testosterone of Immune Reactivity and of Lymphoid Regeneration in Irradiated and Marrow Reconstituted Mice. Experientia, 30, 1343. LINDBERG, R. E. & FRENKEL, J. K. (1977) Toxoplasmosis in Nude Mice. J. Parasitol., 63, 219. MONEY, W. C., FAGER, J. & RAWSON, R. W. (1952) The Comparative Effects of Various Steroids on Lymphoid Tissue of the Rat. Cancer Res., 12, 206. NICOL, R., BILBEY, D. L. J., CHARLES, L. M., CORDINGLY, J. L. & VERNON-ROBERTS, B. (1964) Estrogen: The Natural Stimulant of Body Defence. J. Endocrinol., 30, 277. PARROTT, D. M. V. & DE SOUSA, M. A. B. (1971) Thymus Dependent and Thymus Independent Populations: Origin, Migratory Patterns and Lifespan. Clin. exp. Immunol., 8, 663. PEREIRA-Luz, N., MARQUES, M., AYUB, A. C. & RIET-CORREA, P. (1969) Effects of Estradiol upon the Thymus and Lymphoid Organs of Immature Female Rats. Am. J. Obst. Gynec., 105, 525. PREEYASOMBAT, C. & KENNY, F. M. (1966) Urocytograms in Normal Children and Various Abnormal Conditions. Pediatrics, 38, 436.
RANGNEKAR, K. N., RAO, S. S. & JOSHI, V. M. (1974) Influence of Oral Contraceptives on Circulating Immune Response. II. Effect of Progestogenic and Estrogenic Components. Contraception, 10, 527. REILLY, W. R., THOMPSON, S. J., BIELSKI, K. R. & SEVERSON, C. D. (1967) Estradiol-induced Wasting Syndrome in Neonatal Mice. J. Immunol., 98, 321. SABIN, A. B. & FELDMAN, H. A. (1948) Dyes as Microchemical Indicators of a New Immunity Phenomenon Affecting a Protozoon Parasite (Toxoplasma). Science (N. Y.), 108, 660. SILVER, H. K. & KIRCHNER, R. L. (1966) Cytologic Study of Urinary Sediment in the Evaluation of Sex Hormone Production in Childhood. Pediatrics, 38, 886. SNELL, G. D. (1958) Histocompatibility Genes of the Mouse. I. Demonstration of Weak Histocompatibility Differences by Immunization and Controlled Tumour Dosage. J. Natl Cancer Inst., 20, 787. SOBHON, P. & JIRASATTHAM, C. (1974) Effect of Sex Hormones on the Thymus and Lymphoid Tissues of Ovariectomized Rats. Acta anat., 89, 211. STEINBERG, A. D., PINCUS, T. & TALAL, N. (1971) The Pathogenesis of Autoimmunity in New Zealand Mice: III. Factors Influencing the Formation of Antinucleic Acid Antibodies. Immunology, 20, 523. STERN, K. & DAVIDSOHN, I. (1955) Effect of Oestrogen and Cortisone on Immune Haemoantibodies in Mice of Inbred Strains. J. Immunol., 74, 479. STIMSON, W. H. & HUNTER, I. C. (1976) An Investigation into the Immunosuppressive Properties of Oestrogen. J. Endocrinol., 69, 42. THANAVALA, M. Y., RAO, S. S. & THAKUR, A. N. (1973) The Effect of an Oestrogenic Steroid on the Secondary Immune Response under Different Hormonal Environments. Acta endocr., 72, 582. VASILAKIS, J. G., KuNz, W. H. & GILL, T. J., III (1974) The Effect of Gonadectomy on Antibody Production by Inbred Rats. Arch. Allergy, 47, 730. WINTER, J. S. D. & FAIMAN, C. (1972) PituitaryGonadal Relations in Male Children and Adolescents. Pediat. Res., 6, 126. WINTER, J. S. D. & FAIMAN, C. (1973) PituitaryGonadal Relations in Female Children and Adolescents. Pediat. Res., 7, 948.
