Effect of reserpine on immune reactions and tumour growth

Europ.~7.CancerVol. 2, pp. 253-261. PergamonPress 1966. Printed in Great Britain

Effect of Reserpine on Immune Reactions and Tumour Growth P. DUKOR, S. B. SALVIN, F. M. DIETRICH, J. GELZER, R. HESS and P. LOUSTALOT Research Laboratoriesof the PharrnaceuticalDepartraentsof CIBA Limited, Basle, Switzerlandand Summit, New ~ersfy, U.S.A.

IuuuNs responses may be suppressed by several types of chemical agent [1, 2]. The specific inhibition of autoimmune disease and of human homograft reactivity by immunosuppressive drugs is a major goal of experimental immunology. The clinical use of immunodepressants such as alkylating agents, antimetabolites, or tumour-inkibiting antibiotics is greatly restricted by their general toxicity, although their mode and site of inhibition within the host cell may be highly selective. Drugs such as corticoids, displaying mainly anti-inflammatory properties, suppress only some components of antibody mediated tissue injury, but fail to influence the specific immune mechanism itself [1]. Thus, any reports on immunosuppressive chemicals belonging to a new class of compounds are bound to arouse considerable interest. Recently, Dralkoci and Jankovid and their collaborators observed a marked inhibition of several immune reactions in laboratory animals following treatment with reserpine [3, 4]. The authors suggested that this drug might interfere with "the central or peripheral pharmacological control of immune response," or that the involution of lymphoid fiMues, produced regularly by sustained therapy with this compound, might provide a cellular b a.,i~ for the impaired immunological reactivity. In fact, reserpine has been reported to inhibit the development of routine lymphatic leukaemias [5, 6]. As reserpine is known to exhibit strong sedative effects [7, 8] and as a consequence does affect food intake [Sa, b], it

seemed necessary to re-evaluate its immunosuppressive and tumour-inhibiting properties. The effect of this drug and of some of its relatively less hypnotic analogues on body and organ weights and on the histology of the lymphoid tissues of rats and mice was examined. In addition, investigations were conducted on the influence of reserpine on haemagglutinin production to sheep red cells in mice, on delayed hypersensitivity to purified proteins and on autoimmune thyroiditis in guinea-pigs, as well as on the growth of a spontaneous mammary carcinoma in C3H mice. The data presented in this paper indicate that these actions of reserpine are probably an indirect result of the sedative effects of the drug and a more direct result of the subsequent starvation. M A ~

AND METHODS

Animals- (a) Colony-bred male Swim mice weighing 22-24 g and (b) inbred male and female C3H and C3HO mice [9] (obtained from the International Centre for the Provision and Study of Turnout-Bearing Animals, Netherlands) were maintained on mouse pellets (Nafag A.G., Switzerland). Colonybred albino rats weighing 180-240 g were given Nafag rat cubes. Guinea-pigs of the Hartley strain, weighing 300--400 g, were maintained on Purina guinea-pig chow. Drugs--The following compounds (CIBA) were used: 253

254

P. Dukor, S. B. Salvin, F. M. Dietn'ch, aT. Gelzer, R. Hess and P. Loustalot

Ba-13,020 Reserpine [Serpasil~] Su-3118

Syrosingopine [SingoserpR] phosphate

Su-7192

Propyl 18-0- (2-tetrahydropyranoyl) reserpate

Ba-4311

Methylphenidate [Ritalin ~] hydrochloride

Reserpine and its analogues were dissolved in 0.65% ascorbic acid. Fresh solutions were made up daily. Methylphenidate was dissolved in water. Treatment schedules-- In experiments in which haemagglutination titres were determined, mice received subcutaneous injections of the drug on 5 consecutive days starting on the day of antigen administration (day 1). In order to gtudy suppression of delayed hypersensitivity to purified protein and of allergic thyroiditis, the drug was injected intraperitoneally into guinea-pigs daily for 7 days, starting on the day preceding antigen administration. Rats used for histology were injected intraperitoneally with the drug on 14 consecutive days. Mice bearing spontaneous mammary tumours were injected with the drug intrapefitoneally daily during the first week and every other day during the second and third week (except over the week-ends), eleven injections being given altogether. Starvation - - In some experiments, mice were kept individually in single cages on a wire net. Food was withdrawn as from the day of antigen administration. Water was given ad libitum. Force feeding--Groups of reserpine-treated mice were fed five times daily by stomach tube a mixture of commercial baby foods (approximately 200 mg Elonac R (Guigoz SA, Switzerland), and 25 mg Humana ~ (Schweizerische Milchgesellschaft, Hochdorf, Switzerland) per day. This diet was supplemented by a daily intraperitoneal injection of 0.5 ml Aminosol Vitrum ~ (Hausmann). Since mechanical force-feeding of the reserpine-anorectic mice was impractical over the 3-week period necessary for observation in the tumour experiments, the animals were stimulated for periods of about 30 rain by daily intraperitoneal injections of 5-10 mg/kg methylphenidate (by analogy with the procedure of Kurland [10]). The latter compound by itself had no effect on tumour growth, but enabled the animals to obtain some food and water during the limited periods of alertness due to methylphenidate.

Body and organ weights - - In most experiments, total body-weights were recorded daily. After the animals had been sacrificed, weights of thymus and spleen, and in some groups, of heart, kidney, liver, and testes were recorded. Histology--Animals were killed for histological studies at various intervals after the onset of drug treatment. Tissues were fixed in Carnoy's or Bouin's fluid, cut at 5 la and stained either with haematoxylin and eosin, or with Sudan II. Haematocrit values were determined bystandard haematological procedures. Haemagglutinin production - - Haemagglutinins to sheep red blood ceils (SRBC) were determined with the aid of a previously described technique [11]. Briefly, mice were immunized with a single intraperitoneal injection of SRBC and bled 4 and 9 days later by orbital puncture Sera were titrated using a Takatsy microtitre kit with titres expressed as reciprocals of the end point dilution (powers of the base 2). Controls matched for body weight and receiving appropriate injection of solvent were included in all experiments. Delayed hypersensitivity - - For the induction of delayed hypersensitivity, guinea-pigs were injected in the footpads with 5lag hen egg albumin in incomplete Freund's adjuvant. The animals were tested intracutaneously with 5 lag hen egg albumin in saline containing 1% guinea-pig serum on days 6 and/or 7 to determine the extent of delayed hypersensitivity and on days 13 and 14 to measure Arthur-type hypersensitivity. Reactions were observed at 4, 18 and 24 hr [12]. Autoimmune thyroid#is--Guinea-pig thyroglobulin was prepared by the modified [13] method of Derrien et al. [14]. On the second day of drug administration, the guinea-pigs were injected in the footpads with 2"5 mg thyroglobulin in incomplete Freund's adjuvant. Four to six weeks later, the animals were challenged in the footpads with 100 lag guineapig thyroglobulin in Freund's complete adjuvant containing 2 mg tubercle bacilli. At irregular intervals thereafter, the guinea-pigs were skin-tested for delayed or Arthus-type hypersensitivity, and bled for determination of antithyroid antibody by passive cutaneous anaphylaxis [15], and thyroid glands were extirpated for histological examination. Spontaneous mammary tumours - - A modification of the test system described by Le Page et al. [16] was used. Briefly, groups of fifteen

255

Effect of Reserpine on Immune Reottions and Tumour Growth animals with single primary tumours of comparable size (5-15 mm dia.) were treated and observed during a 3-week period. The average weight of the animals was recorded weekly and the growth of the tumours was measured simultaneously in two diameters by calipers. Both values were compared with those obtained in control animals injected with solvent. Statistical analysis - - Results were analysed by Student's "i"-test.

organ weights was dose-dependent (Fig. 1). During the early days of treatment, as seen on day 4, thymus weights were more depressed than spleen weights. Later, as seen on day 9, spleen and thymus showed a comparable weight loss. Starvation, in the absence of drug administration, produced a decrease in weight of the same order of magnitude as that seen following daily .administration of the highest dose (1 mg/kg per day) of reserpine tested (Fig. 1).

RESULTS

Histologically, typical alterations could be observed on day 4. Administration of 0.3 and 0.4 mg/kg reserpine subcutaneously per day induced marked narrowing of the thymic cortex with a partial depletion of lymphocytes which accumulated in peripheral parts of the medulla. Spleens and lymph nodes retained their normal structure. Daily injection of 0.5 mg reserpine or more per kg subcutaneously (causing a reduction of

Body and organ weights- Prolonged administration of high doses of reserpine and some of its analogues in mice and rats produced diarrhoea and a soporous state, which was associated with a reduction in food intake, a rapid deterioration in the animal's general condition, and a doserelated increase in mortality. Reserpine-induced reduction of body and

DAILY DOSE OF RESERPINE IN mg/kg s.c.

0.1

0.2

0.3

0.4

0.5

0.7

1.0

I starvation

~4

10/10

10/10

10/10

10/10

10/10

10/10

10/10

12/12

100%

day 9

10/10

10/10

3/10

3/10

2/10

2/10

+

+

0/10

o/12

II111111~body weight t ~ thymus weight I~R'spleen weight in % of controls ~ a n t l - s h e e p red cell haemagglutlnln tlter~ n

numberof surviving animals F/g. 1. Effect of reserpine or starvation on weights and haemagglutinin formation in mite.

256

P. Dukor, S. B. Salvin, F. M. Dietrich, 07. Gelzer, R. Hess and P. Loustalot

3/4-4/5 in thymic weight) was associated with complete lymphoid depletion of the thymic cortex, which became wholly epithelial and contained scattered pyknotic cells, fragmented nuclei, and cellular debris. On the other hand, the medulla was packed with lymphocytes, thus displaying a reversal of the normal thymic architecture (Figs. 2A and 2B). The spleens showed only moderate depletion of the white pulp, with dissociation of lymphoid follicles, as well as hyperplasia of red pulp and reticular elements (Figs. 3A and 3B). In the lymph nodes, a moderately decreased cellularity was apparent (Figs. 4A and 4B). Starvation alone produced qualitatively and quantitatively the same histological changes as the highest doses of reserpine employed (Figs. 2C, 3C and 4C). On day 9, histological alterations of the lymphoid tissues were even more pronounced. As little as 5 × 0.3 or 0.4 mg/kg reserpine subcutaneously caused complete thymic involution, extensive reduction of the lymphoid follicles of the spleen, and marked hypoplasia of the lymph nodes (Figs. 2D, 3D and 4D). Since the degree of weight loss seemed to be linked to the drug-induced sedation and thus to the reduction in food intake, force feeding of reserpine-treated mice was attempted. Body, thymus, and spleen weights of the force-fed groups were somewhat higher than those of animals treated with reserpine alone (Fig. 5). However, the differences were not statistically significant.

Essentially identical histological alterations and weight changes were obtained when mice were treated with sublethal doses of the reserpine analogues Su-3118 and Su-7192. Again, rats given daily intraperitoneal injections of 1 mg/kg reserpine over a 2-week period showed a similar involution of their lymphoid tissues, as well as of heart, liver and kidneys. Haemagglutinin production ~ The effect of various dosages of reserpine on haemagglutinin production in mice is shown in Fig. 1. In view of the limitations of the method, only a reduction in titre of more than 25% was considered to be significant [11]. Thus, reserpine doses of 0" 5 mg/kg per day or more were necessary to suppress antibody formation on day 4 (titre 2.1 -4- 1.3 vs. 5 . 0 + 1.0 in control mice). However, starvation alone (Fig. 1) produced a similar reduction (titre 1.8 4- 1.6). On day 9, the titres of all surviving mice were again within the range of that of the controls (7"2 + 0.7). A similar pattern was observed with the reserpine analogues Su-3118 and Su-7192 which are both substantially less soporific than reserpine itself. Only near-lethal doses, causing marked sedation and diarrhoea, produced a temporary reduction in haemagglutinln titres on day 4. On day 9, all the survivors from the experimental groups (1/5 in the case ofSu-3118 and 5/5 in that of Su-7192) showed normal antibody titres.

DAILY DOSE OF RESERPINE IN mg/kg s.c.

.

lo/lo

I I I n

lo/1o

body weight 1 thymusweight i i n % of spleenweight anti-sheepred cell haemagglutinintiter numberof surviving animals

lo/lo

8/lo

controls

Fig. 5. Effect of reserpine on u~ights and haemaJ~glutininformation in force-rid mice.

Fig. 2.

Mouse thymus (magnification 26 ×, H.E.)

A : Control:

Typical correlation between predominantly lymphoidal cortex and mainly reticulo-epithelial medulla B: Reserpine 1 mg/kg per day (4 x ) s.c. day 4: "]Cortex depleted of lymphocytes, relative increase C: Starvation : day 4: k.~in lymphoid cells in medulla. D : Reserpine: 0" 4 mg/kg per day (5 x ) s.c. day 9:

(to face p. 256)

Fig. 3.

Mouse spleen imagnification 2 6 x , H.E.)

A : Control : Red and white pulp in typical proportions B : Reserpine : 1 rng/kg per day (4 × ) s.c. day 4 : ) Diminution of lymphoid follicles, increase m C: Starvation day 4: f haemopoietic activi~, reticular hyperplasia D : Reserpine 0-4 mg/kg per day (5 ~ ) s.c.day 9: Complete loss of follicular structure, marked lymphoid depletion.

Fig. 4.

Mouse inguinal lymph-node (magnification × 60, H.E.)

A" Control Cortical area with denselypacked lymphocytes B : Reserpine 1 mg/kg per day (4 × ) s.c. day 4: "~Cortical area showing moderate reduction in C: Starvation day 4: f smaU lymphocytes D : Reserpine 0 . 4 mg/kg per day (5 × ) s.c. day 9 : Substantial lymphoid depletion of whole lymphnode.

257

Effect of Reser/Kne on Immune Reactions and Tumour Growth

Skin hypersensitivity to hen egg albumin. Autoimmune thyroiditis

Force feeding effectively offset the temporary immunosuppression produced by reserpine (Fig. 5). The differences in titres between reserpinetreated mice with and without force feeding were statistically significant (p < 0.05 for mice given 0.5 mg/kg per day, p < 0.01 for animals treated with 1.0 mg/kg per day). All haematocrit values on day 4 of reserpinetreated, starved and force-fed animals were within the range of control animals.

The results are summarized in Table 1. Guinea-pigs treated with the maximal tolerated dose of reserpine (0.3 mg/kg per day) developed delayed and Arthus responses to hen egg albumin that were virtually identical to those in untreated controls. Similarly, this drug had no effect on the

Table 1. Effect of reserpine on hypersensitivityin guinea-pigs Skin reactions to hen

Autoimmtme thyroiditis

egg albumin Treatment

Delayed*

Arthus

Delayed*/ Arthus

PCA

Cellular infiltration

Rmerpine 0.3 mg/kg per day

8/12t

7/7

5/7

5/7

5/7

Controls

9/12

8/8

8110

8110

81to

! I

,

t

* S k i n reaction, l 0 m m o r m o r e i n diameter.

tNumerator: number of ~

with Ix~ifive responses.

D e n o m i n a t o r : m u s h e r o f azJhz~s tested.

DOSE OF RESERPINE IN m g / k g

|.p.

--

.

~- 14/15

15/15

14/15

100%

15/15 100%

day 21

n .....

-

I

12/15

+ 0/15

11/15

11/15

body wll|ght ~t in % of controls tumor growth J

i n

numberof survlvlng an|reals

Fig. 6. Effect of reserpine on growth of spontamous mammary carcinomas in C3H-raice.

258

P. Dukor, S. B. Salvin, F. M. Dietrich, 07. Gelzer, R. Hess and P. Loustalot

development of skin hypersensitivity, circulating antibody, and cellular infiltration in autoimmune thyroiditis.

Spontaneous mammary turnouts- The effect of reserpine alone and in combination with methylphenidate hydrochloride on body weight and on the growth of primary breast tumours in C3H mice is summarized in Fig. ,6. Administration of 0.3 mg/kg reserpine alone (left side of Fig.6) caused almost a 50 per cent inhibition of tumour growth and 0.7 mg/kg a more than 80 per cent inhibition after 1 week of treatment. With the lower dose, this inhibitory effect remained constant throughout the 3-week period of observation. Mice treated with 0" 7 mg/kg died before the end of the second week of the experiments. At both dose levels of reserpine, the animals became increasingly more somnolent. The tumour-inhibiting effect of reserpine was virtually eliminated in mice injected simultaneously throughout the experiment with 5-10 mg/kg per day methylphenidate hydrochloride (see right side of Fig. 6). Under these conditions, 0.3 mg/kg reserpine had no effect after 7 days of treatment and caused a tumour inhibition of less than 25% after the third week. This degree of inhibition is considered not to be significant. Mice injected with 0.7 mg/kg reserpine and stimulated simultaneously showed larger tumours than their controls after the first week. These differences, however, are within the limits of experimental variations. Moreover, such animals survived the 3 week period of observation, at the end of which time the final inhibitory effect of reserpine on tumour growth and body weight was virtually identical. I]~[SG~ION (1) Prolonged treatment of rats and mice with reserpine was associated with a dosedependent involution of lymphoid structures. Since the lymphatic organs play a central role in immune reactions, the belief may be held that lymphoid depletion per se may pro-tide a cellular basis for immunological impairment [3, 4]. However, the data presented in this paper clearly show that mice with only moderately reduced peripheral lymphatic tissue, as is found early on during a course of reserpine administration, showed a highly significant impairment of their immune response. On the other hand, at a later stage, animals presenting extreme involution of their lymphoid tissues did

not show significant depression of their capacity to form antibodies. As thymectomy in adult life is not associated with any immediate impairment of immune reactivity [17-19], mere thymic involution cannot be expected to interfere directly with an animal's immune capacity. Furthermore, it has been demonstrated that correlation is poor between the cellularity of peripheral lymphoid tissues and the immunological potential of neonatally thymectomized mice bearing thymus tissue in diffusion chambers [20]. Thus, the findings in this paper further support the concept that immunological reactivity depends rather upon the qualitative than the quantitative state of the lymphatic system [21]. (2) The immunosuppressive effect of reserpine and of the analogues studied in this report appears quite marginal. Only very high dosages within the sublethal range were shown to lower haemagglutinin production against heterologous red cells in mice. On the other hand, both skin hypersensitivity towards hen egg albumin and development of autoimmune thyroiditis in guinea-pigs, which are known to he suppressed by specific [22] and non-specific [23, 24] methods, were not influenced at all. Furthermore, the immune response was only temporarily suppressed by reserpine during the early log phase [11] of antibody formation (day 4) and did not even last until day 9, when normal antibody titres developed. In most of the experimental groups receiving high doses of the drug, a proportion of the animals died between the 2 days of antibody determination. The question therefore arises as to whether the apparent normalization of the immune response was simply due to a selection of resistant mice. However, in experments using the reserpine analogue Su-7192, the same individual animals showed reduced antibody titres when bled on day 4, but did no longer differ from their controls on day 9. Hence, this temporary suppression of the immune response may reflect either a mere delay in, or else a rapid recovery of, antibody production. (3) The relevance of reserpine's action on lymphoid depletion and immunosuppression becomes even more doubtful when due account is taken of the pharmacological effects [7-8b] of this group of drugs. Thus, significant impairment of the immune response was regularly associated with marked sedation and the development of diarrhoea, both of which necessarily interfered with normal food and water balance. It was therefore important to

Effect of Reserpine on Immune Reactions and Tumour Growth investigate at the same time not only the effect of starvation alone, but also the countereffect of force feeding during the treatment with reserpine. Our data show that both reduction of body weight and involution of lymphoid tissue with immune suppression were produced by mere starvation of the animals. After mechanical force feeding, inhibition of antibody production by reserpine could no longer be detected.

259

(4) Similarly, the tumour-inhibiting effect of reserpine appears to be directly linked to its sedative action [25-28] and the resultant anorectic state, for this effect could be successfully neutralized by simultaneous administration of methylphenidate hydrochloride, which acts as a central stimulant.

Z~ m t c a n i ~ d' action de l' uctivit~ immuno-suppressive et cytostatique de la rgserpine a gtg gtudiL Chez des rats et des souris, l'administration prolongge de trks hautes doses s'est accompagn& de l'apparition d'un dtat stuporeux, avec chute considgrable du poids corporel et du poids des organes, ainsi que de profondes modifications dans la structure des tissus lymphofdes. Cependant, la privation de nourriture, ~ elle seule, a produit une involution comparable des tissus de la rate et des ganglions lymphatiques, En dlpit d' une extrgrae dgplgtion du tissu lymphofde, les souris trait&s ~ la rgserpine ou g~ l'un de ses d&ivgs, ontfinalement dg,veloppg des quantitgs d'hhnagglutinine aux Mmaties de mouton, proches de la normale. Au cours de la phase de production des anticorps, on a observg un effet immuno-suppressif, d~pendant de la dose, de manikre significative, et qui a pu ~tre reproduit aishnent par simple privation de nourriture. D'autre part, un gavage prdalable a emp~cM l' action suppressive de la rgserpine. Chez les cobayes, la rgserpine n' a eu aucune influence sur le dg'veloppement, soit d'une hypersensibilitg cutange g~lYgard de l' albumine des poulets, soit d' une thyroktite d' auto-immunisation. Les doses maximales de rgserpine tolg'rges, administrges ~ des souris porteuses de cancers mammaires sponta~s, avaient un lgger effet cytostatique qui pourrait ~tre en relation avec l' importancee de la perte de poids et qui est contrarig efficacement par un stimulus central. Il appara~t que les faibles proprigtgs immuno-suppressives et cyto-inhibititrices de la rgserpine ne semblent ~tre dgclencMes que par un ragcanisme indirect. SUMMARY The immunosuppressive and tumour-inhibiting actions of reserpine were investigated. In rats and mice, prolonged administration of very high doses was associated with the development of a soporous state, with a considerable drop in body and organ weights, and with profound histological changes in lymphoid structures. However, starvation alone produced a comparable involution of thymus, spleen, and lymph nodes. In spite of extreme lymphoid depletion, mice treated with reserpine or some of its derivatives eventually produced near normal amounts of haemagglutinins to sheep red cells. During the log-phase of antibody production, a significant dose-dependent immunosuppressire effect was observed, which was easily reproduced by mere starvation. Moreover,force feeding prevented the suppressive action of reserpine. In guinea-pigs, reserpine did not have any influence on the development either of skin hypersensitivity to hen egg albumin or of autoimmune thyroiditis. Maximal tolerated doses of reserpine given to mice bearing spontaneous mammary carcinomas had a slight tumour-inhibiting effect, which could be correlated with the extent of body weight loss and antagonized effectively by a central stimulant. Hence, the minor immunosuppressive and tumour-inhibiting properties of reserpine seem to be initiated only by an indirect mechanism.

260

P. Dukor, S. B. Salvin, F. M. Dietrich, J. Gelzer, R. Hess and P. Loustalot ZU~.t/181JI~

Die immunosuppressiven und tumorhemmenden Eigensehaflen yon Reserpin wurden auf ihren Mechanismus kin untersucht. Bei Ratte und Maus f'~rte ltingerdauernde Verabreichung hoher Dosen bald zu einem soporiisen Zustand mit betrachaichem Abfall des Kiirpergewichts und einer weitgehenden Involution der lymphatischen Organe. Blosses Hungern hatte iihnliche Ver/inderungen yon Thymus, Milz und Lymphknoten zur Folge. Trotz der hochgradigen lymphatischen Atrophie bildeten Reserpin-behandelte M~use schliesslich fast normale Haemagglutinintiter gegen Schaferythrocyten. ?Cur wiihrend der initialen log-Phase der Antikiirperbildung wurde unter Reserpin voriibergehend dne signifikante dosisabh~ngige Titerverminderung beobacktet. Ein vergleichbarer Titerabfall land sich freilieh auch bei Hunger-Kontrollen. Ferner verhinderte gleichzeitige Zwangsfiitterung die ohnehin nut bescheideneimmunosuppressive Wirkung'von Reserpin. Beim Meerschwdnchen verm~hte Reserpin weder die allergischen Hautreaktionen gegeniiber Eialbumin noch die Entwicklung einer Autoimmunthyreoiditis zu unterdratken. Schliesslich zeigte Reserpin einen gewissen HemmtJekt auf das spontane Mammakarzinora der CsH-Maus, der fieilich wi~,.aum dem K~pergeuridaroerlust entsprach und iiberdies dutch Go.be eines zentralen Stimulus aufgehoben werden konnte. Die Befunde sprechenfiir einen indir~ra Widumgsraechanismus des Reserpin in den untersuc,hten Moddlen.

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261