Anti-Cancer Drngs1997, 8 (suppl DS Viscum album L. preparation /sorel modifies the immune response in normal and in tumour-bearing mice M. Jurin’, N. Zarkovic’, S. Borovic’ and D. Kissel? "Depariment of Experimental Biology and Medicine, Ruder Boskovie Insitute, Bjericks cesta 54, 10000 Zagreb, Croatia; ‘Vohanneshiaus, Oscheloronn, Germary. ‘The Viscum album (mistletoe) preparation fsorelis able to ck ‘troy tumour cells and to modify immune reactivity against a Particular antigen in normal and in tumour-bearing animals. CBAMZgr mice and methyicholanthrene-induced fibrosarco- ‘ma were used in these studies. A single dose of /sore/IM (140 ‘mg/kg or 1400 maikg body weight) significantly increased the ‘number of plaque forming cells if applied at the time of injoc- tion of sheep red blood calls or 1 day earlier The application of, ‘sore! 1 day after sheep red biood cells did not modi umber of plaque forming cell i ‘The higher the dose of Isorelthe stronger is the immu ro: ‘sponse to sheep red blood cells. Furthermore, one dose of, ‘sore! (140 mg/kg body weight) restored the suppressed im- "mune response of flbresarcoma-bearing mice toa significant ‘extent. Besides modification ofthe humoral immune respons the survival time of C57BUGoZgr malo skin grafts on syngeneic, female recipients was significantly shorter if/sorel was applied ata articular timeatter grafting, However, according to plaque forming cell numbers, a prolonged application of /sorel wae. significantly immunosuppressive in normal mice and particu- larly in tumour-bearing mice. It should be mentioned that the doses of /sore! usedin this experiment wore much higher than ‘gonerally used in cancer patients. In view of the immunomod= tlating effects of sore, the monitoring ofthe immune response of the patients treated with mistletoe preparations is to be recommended. ‘Keywords: immunomodulation, mistletoe, Viscum album, mouse. fibrosarcoma, skin grafting, plaque forming cel. Introduction Several Viscum albunr 1. (mistletoe) preparations are com= monly used for complementary cancer therapy [1-5]. Howev- cr, for centuries mistletoe plant extracts have been used 10 treat patients suffering from very different dis: hypertension, epilepsy, debility, or connective tissue disor ders [6.7] This wide area of application and the relatively successful results might indicate ts murval inflwence on the mechanisms regulating the complex function of the organ ism. Panicular attention is still focused on the immunomod- Ulatory action of mistletoe which, together with its pro- nounced cytotoxic action against tumour cells, leads to ‘Conespondence to. Jurin © 1997 Rapid Science Publishers potent therapeutic approach for treating cancer patients 8 10) Over the last few years we have studied the influence of the mistletoe preparation Jsore/on different experimental tumours in mice {1-14} In our defined experimental modes ‘we observed beneficial eects of Kore applied either alone Corin combination with loca rumour ination, chemoth apy, or surgery [11,13]. These findings support the u ‘Boren dlinical practice. Furheamnore, our previous results in experimental models indicate the use of sore! as.a potent mmunostimulator ifthe proper dose and time of application ¢ chosen [11,13], However, the application of fred could junosuppression {11,13,15) and this could be of particular interes for is clinical use in cancer patients To clavify this problem, we investigated the effects of cifer= een single and rozal doses of orefin tumour-bearing mice and varied the time of application in normal and tumour- bearing mice. The animals were injected with sheep red blood cells and the intensity of the humoral immune re- sponse was determined The reactivity of Korekreated mice to foreign skin graf was also determined. A sho eview of our results on the immunomodilating action of Isorel is presented in this ail, also result ini Materials and methods Animals CBA/H2ge and C57BI/GoZgr mice of both sexes were used inthe experiments. The animals were about 3 months old, weighing 20-22 g at the beginning of the experiment, The ‘mice were maintained in standard conditions with unsestict- edd access to food and water Isore! ‘The commercial preparation Jsore/M, strength 60, was used inthe experiments. According o the instructions, this mistle toc extract was produced by a special method in aqueous, solution avoiding any denaturating manipulation, The ex- tract from 60 mg of ‘plana tour (whole plane) ia I mul ster lized soxlium chloride was supplied in individual ampoules by Novipharm, Austria $27 AmieCancer Drags + Vol & Suppl 1 1907M. furinet a ‘Table 1. Plaque forming cell valuesin normal and ibrosarcome-bearing CBAHZg¢ nice immunized with sheep red blood cells and treated wit diferent single doses of sorefor saline applied al the indicated time ‘Animals jected with SRBC “Tealment Time oftreatment PEC (means + 8D x10) Nownal nice Tsore/(140 mgikg bw) 1 day priorto sheep red blood call injection 272432" (Onthe day of shaep red blood calinection staat 4 day ater sheep red blood cal injection 210428 Isorei(1200 gg) 1 day ptiorto sheep red blood cal injection Baan ‘On the day of sheop red bood cal injoction ai73 31" 1 day aftorsnaop redblood cellinjection 205242 Saline ‘On the day of sheep red bad celnjaction 2082 10 Mice beering forosarcoma Isoret(140mgikg bw) Onthe dayo! sheep red blood celnjection 21a280 {volume approximately 300mr®) Saline ‘Ontne day of sheep rod bead callnjocton ice bearing fbrosarcoma Isore!(140mqikg bx) nthe day of hoop rod blood cel injection (volume approximatoly2000mm") Sane (Onihe cay sheep red blood cellinjaction ‘SHGG,eheap ed t000 ols; PFCY, plague orming cell vaussbw, body Wg "Pe 0.02, “P0001 Tumour ‘The tumour used in these experiments was a fibrosarcoma, induced in the CBA’HZgr female mouse by injecting methyl cholanthrene into the subcutaneous tissue of dhe flank. ragments from this original tumour had been transplanted to syngeneic hosts and the first-generation specimens were then kept in liquid nitrogen [11], By injecting these speck. ‘mens into the recipient, source mice, second-generation transplant tissue was obtained, From these specimens, grow ng tumour cell suspension was made as described previous Jy 11], Each of the experimental mice received 10° viable tumour cells subcutaneously into the sight thigh. By measur ing three tumour diameters, the volume was calculate described elsewhere [11 Skin grafting Pullthickness grafts of tail skin were transplanted onto the dorsolateral side on the recipient unk as described previ- ously 1111 Plaque forming cell assay Sheep red blood cells were injected intraperitoneal and the, number of haemolytic antibodies producing cells in the spleen determined 4 days later [11] Statistics Mann-Whitney and tests were used Results The influence of single doses of Isore! on the intensity of the immune reaction to sheep erythrocytes in normal and tumour-bearing mice CBA/EIZgr mice (8-12 per group) were injected intraperito- peally with sheep red blood cells and treated with ore! 828 Auti-cancer Drugs * Vol 8, Suppl t © £997 (C140 mg/kg or 1400 mp/kg body weight) 1 day prior to sheep red blood cell injection, on the day ofthe injection, ot 1 day after the injection, Furthermore, CBA/HZer mice b ing CMC-1 fibrosarcoma transplanted inthe sight thigh were injected with sheep sed blood cells and treated with {sore [As shown in Table 1, the mice injected with 140 mg/kg of Jsorel day before or on the day of sheep red blood cell, injection produced a significantly higher (P< 0,02) number (of plaque forming cells than the other animals (untreated, controls or these treated 1 day after sheep red blood cell injection). Moreover, the higher dose of oref(1400 mg/kg) was more effective and the numbers of plaque forming cells obtained were significantly higher than in the control group (P<0,001) and the group treated with 140 mg/kg of the dng (P-<0,02) bore! was further able to restore the immune reaction of tumour-bearing mice. As presented in Table 1 the immune reaction to sheep red bloc cells i significantly (P<0.001) suppressed, but only one injection of 140 mg/kg. kovel completely cestored the reactivity of timour-bearing mice, regardless of tumour size The influence of multiple doses of fsarefon the intensity of the immune reaction to sheep erythrocytes in normal and tumour-bearing mice CBA/HZgr mice (8-12 per group) were treated with re peated doses of Jsore/and injected with sheep red blood, cells. As shown in Table 2, the mice receiving 14 mg/kg, ‘Kovelcaly, over 14 consecutive days, reacted significantly (P ‘© 0.01) stronger to sheep red blood cells than control ni mals, After the addition of 140 mg/kg, no iniluence was ob- served on plaque forming cell numbers, ie. on the intensity ‘of immune response 10 sheep red blood cells. However, the prolonged application of sere! was significantly suppres- sive, As shown in Table 2, either 14 mg/kg or 140 mg/kg, Borel was significantly (P < 0,001) suppressive in mice treated with « caily injection over 5 weeks (except at the weekends, i¢, fot of 25 doses). However, in tumourbearInmemnomoduiating activities oftsorel Table 2. Plaque forming cel values in normal and fibrosarcoma-bearing CBA/HZgr mice treated with diferent doses of Isorafor with saline aver incicated time intervals immunized with sheep red blood cals onthe day af the lest sore/or saline injection ‘Aniralsinjected with SABC Teatmentiniewvals| Daily dose FOV {means = 80 x10") Normal mice “consecutive days (otal 14 injections) ‘sorel(i4marg bs) 120.82 232° ‘gorei(140 maka bw) 7664 198 Saline 83.15 942 Daily overS weeks (except weekends, Isorei(14imakg bw) 725 27" {otal of28 injections) ‘sorei(140 mgikg bw) 507+ 198" ‘Sane 108.22 200 Mice bearing fibrosarcoma ‘4.consacutive days (total 14 injections) Isorei(14 mag bu) 414s 269 (olume approximately 800 mm) 'sorei(140 mgikg bw) 1762 78 Isorei(1400 morkg ts} eas 54° ‘Ssaine 502296 ‘SRBC, sheep red boos cal PFCV, plaque arming calvaluesow, body woight P01, “Ppopaation in patients ‘with acre leukersi. ek Res 1994.38 BIS-822 Franz Request fran imparial discussion ofthe sovalled mistle- tne therapy. 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