+

Indian Journal of Experimenta l Biology Vol. 41. August 2003, pp. 805-8 13

Brain tumor inhibition in experimental model by restorative immunotherapy with a corpuscular antigen

Soven Kumar Gangopaddhyai . Susobhan Sarkar2. Zarina 8 egllm2. Swapna Challdhuri * & Samaresh Challdhuri .1

Ilmmunology Section. Departmelll of Pathology. Uni versity College of M ed ici ne, 244 B. A.J .e. Bose Road. Kolkata 700020. India

' Cellular & Molecular Immunology L ab, Department of Physiology, University College of M edicine. 244B. A.J.e. Bose Road. Kolkata 700020. India

' Section of Immunology. Department of Haematology , School of Tropica l M ed ici ne, e. R. Avenue, Kolkata 700073. India

Neceil'ed 17 December 2002; rel'ised April 2003

In view of Ihe advances in our understand ing ()f anti -tumor immune response, it is now tempting to contemplate the development of immunotherapies for malignant brain tumors, for which no cfTecti ve treatment ex ists. Immunotherapy. with agents known as biologica l response mod ifiers (BRMs) arc thus ga ining increasing interest as the fourth modality of treatmen t. A non -specific BRM. sheep ery throcy tes (S RBC) when administered ( ip. 7% PCVIV. 0.5 ml ) in a group of animals at the end of seventh month of ethy lnitrosourea administrati on. resul ted in significant increase in the mean su rvi val timc (>350 days). Studies conducted for growth kinetics pJttern w ith proliferati on index and fluorochrome (HO - 33342) uptake techniques at the ti ssue culture level ex hibited a regu lJtory inhibition of the cell s iso lated from tissue excised from the tumor susceptibl e area of brain of SRBC treated animals. M oreover, hi stologica l examination of brain from animals showed immunomodulatory ro le of SRBC in ex perimenta ll y induced brain tumor. Further probe into the mechanisms involving immunologica l investi gat ions at the ce llular leve l in these animals indicated an augmented and potentiated cell mediated immune response (CM I) as evidenced by enhanced spontaneous rosette forming capaci ty and cytotoxic act ivity of lymphocy tes and neutrophil (PMN) mediated phagocytos is respec ti vely. The observations suggest that SRBC down regulate malignant growth pattern of experimenta l brain tumors either by an imillunologicall y enhanced killing of tumor ce lls and/or by directl y inhi bi ting the tumor growth poss ibl y viJ a stimulated cy tok ine network . Thus, a corpuscular antigen, can potentiate CMI response in ex perimentally induced brain tumor animal model. in which response induced in the periphery are able to med iate anti-tumor effects in the brain .

Keywords: Anti-tumor immune response, Brain tumor. Corpuscu lar antigen. Immunotherapy. Restorat ive immunotherapy

Treatment of brain tumors presents unique challenge because of unique aspects of their biology and anatomic si te, and their overall poor response to curren t therapeutic approaches in vo lving surgery , radio therapy and chemotherapy which cause several adverse effects in addition to the lesion in the brain l

-4

.

The apparent lack of efficacy demonstrated by conventional therapies has presented a search for potentially beneficial alternative form of therapies. One of the most promising approaches for the Ireatme nt of malignant brain tumor is immunotherapy.

Patients with mali gnant brain tumors ex hibit a broad suppression of cell -mediated immune response (CMl)s. The impaired cell-mediated immunity observed in patients with malignant brain tumors appears to result from immuno-suppress ive fac tors(s)

*Correspondent author Ph: +91-33-2223-204112084, Fax : +91-33-22332084 e-mail : swapna_ chau@redifrlllail.colll

secreted by tumor6-9

, which have been implicated for the decrease in T-cell cytotoxici ty and proliferation potential <lnd systemic cytoki ne dysfunction. These findings, when taken together with the observation that ac ti vated lymphocytes are able to infiltrate effectively through the blood brain barrier (BBB )IO-12, suggest the feas ibility of app lying immunotherape utic approaches.

Numerous attempts have been made to treat malignant brain tumors using a variety of immunologically based strategies, including pass ive immunizati on, vacc ination with tumor cells, restorati ve immunotherapy with local and systemic delivery of biological response modifiers (BRMs), and adoptive cellular therap/ 3. 14. In experimental animal models, such attempts are directed towards man ipulating the immune systems to generate an overall effec tive anti -tumor immune response.

The unique observation that T-cells are capable to form spontaneous rosettes with sheep red blood cells 15-17

806 INDIAN J EXP SIOL. AUGUST 2003

and that sheep red blood cells can activate and enhance T-Iymphocytes proliferation 18 and can a lso

augment IFN -y production by lymphocytes 19 is possibly the crucia l point to consider sheep red blood ce ll (SRBC) as an immuno-provocating agent. SRBC has been shown to exert a strong immunostimulatory and anti-tumor property in diffe rent experimental animal models2o-22, which could be ex pl ained in terms of immuno-modulation .

In the present study, restorative immunotherapy has been attempted with sheep red blood cells (S RBC) in bra in tumors, ex perimenta ll y induced with ethy lnitrosourea (ENU ) in animals. In order to explore the immunostimulatory and anti-tumor property o f sheep red blood cells in bra in tumor, attempts have been made to evaluate its role in the growth kinetics of experime ntal bra in tumors. A conseq uent survival study and immunolog ical parameters have been conducted to hint at the mechani sms invo lved. Moreover, hi sto log ical study has been carri ed out in orders to ascertain its anti­tumor property conclusively.

Materials and Methods

Animals - Healthy new born Druckrey rats ( 120) of both sexes, aged 7 ± 3 days, o ri g ina lly supplied by Central Drug Laboratory, Calcutta, Indi a and main­tained subseq uently in the Laboratory were used . These an imals formed following five ex perimental groups of 24 each: (1) normal untreated contro ls (N), (II) A-ENU: with inocu lation of an acute dose of 50 + 30 mg/kg body weight of ENU; (III) F-ENU: with inocul ation of fractionated doses of 15 mg/kg body weight! week with a tota l of 120 mg/kg body weight in 8 consecuti ve weeks ; (IV) SRBC-A-ENU: animals prepared as per gr. (II ) and received 7% SRBC (0.5 ml , ip.) at the end of 7 th mo nth of ENU adm ini strati on and ; (V) SRBC-F-ENU: animals pre­pared as per g r. (111 ) and received SRBC (7%), 0.5 ml , ip., at the end of 7 th month ENU treatment. The ani ­mals in each group were weaned at 30 days of age and housed separatel y in isolated cages. All animals were fed autoclaved Hind-Lever pe llet and water ad libitum. Rats were examined daily and weighed weekly throughout the experi mental peri od. Prog ress ive neu­rologic signs and alterati ons of body weight were considered as criteria for animal selection. M ainte­nance and animal experiment procedure s tri ctly fol­lowed "Princ iples of laboratory animal care (NIH)" and also local "ethical regul ations" .

Induction of brain tumors: ENU administration­ENU was freshly prepared by di ssolving 10 mg/ml in sterile saline and adjusting the pH to 4.5 with crystalline ascorbic acid. ENU was injected (ip) to 48 animals with an acute dose of 50 mg/kg body weight in the first week after birth and 30 mg/kg body weight in the second subsequent week respect ive ly. Animals (48) of another batch were injected, (ip) on the same day with equal fractionated doses of 15 mg/kg body weight per week with tota l 120 mg/kg in 8 consecutive weeks. From the above two groups, 24 animals each were kept for SRBC treatment to constitute group IV and group V animals, whereas , the rest 24 animals each in acute and fractionated ENU groups served as group I and group II animals respecti vel/3

-27

.

Adrninistratiol1 of SRBC-The meth ods have been described elsewhere2o

.21

. Briefl y, 7% SRBC suspensio n was

prepared through PCV/(N) sa line volume d ilution , 0 .5 ml of it was inoculated (ip) to 24 animals each belonging to A­

ENU and F-ENU res pecti vely to serve as group IV (S RB C­A-ENU) and group V (S RBC-F-ENU) animals.

Survival study - Of all the animals prepared as above, observations were made to account for the total number of days survived by indi vidual animals and the mean survival time in each group were determined. Further, progressive neurol ogic signs and weight loss were taken into account in se lecting the animals for the tumor development study.

Histological studies - Porti o ns of brain ti ssues from respective g roup of animals were prepared for rou tine hi stolog ica l studi es : tissues were fixed in 10% formal­buffer overni ght and finally dehydrated and embedded in

paraffin through histokinet processing. Sections were cut at

5 j..UTI thickness and finally stained with routine

haematox iIi ne/eosine.

Growth kinetics of tumors - Small portions of tumor susceptible areas of cerebral cortex and subcortical white matte r from all g roups of animal s were tqken as the sample of ti ssue culture; primary explant technique was employed28 . After the steady state is achieved, the growth kinetics of the ce lls as maintained from all the above groups of animals were studied in terms of Proliferation Index (PI)28 , which was calculated from the ratio of counts at 24 hr to that at "0" hr. ; and also by a fluorescent dye, Hoechst-33342 uptake28

, where counts were taken in a spectrofluorimeter (Beckman , USA) using a scanning wavelength in between 400-500 nm (365 nm excitation/435 nm em iss ion28 .

Studies on immune function - Spleen ce lls isolated from animals of different groups treated as above

~' . ,

•

+

were subjected for cellular immune response studies. For each study, cells from normal group of animals served as normal control.

E-Rosette formation - Lymphocytes were separated from spleen cell suspension on a percoll density gradient elut method29

. Lymphocytes (0.25ml of 3-4 x 106 /ml. ) were mixed with 0.25 ml of 1 % (PCV/saline vo l. ) sheep erythrocytes (SRBC) and incubated at 37°C for 15 min. Following brief centrifugation, the preparation was kept overnight at 4°C. Number of rosettes formed were counted per 200 lymphocytes and expressed as rosette %.

Cytotoxic effi cacy of spLenic Lymphocytes by HO-33342 reLease assay- A newer approach to this method using a fluorochrome dye Hoechst 33342 (H033342, Sigma, USA) which binds to DNA of cells irreversibly without leakage until lysed was adopted30

. Tumor cell s (target; a steady glial tumor line, syngeneic in nature) were labeled with HO-33342 fluorochrome dye (6 ~106/ml ) for 15 min at 37°C and excess was washed off (total incorporation). Cytotox icity assay was performed by maintaining an effector (splenic-Iymphocytes)-target ratio at 10: I through an incubation (37°C, 4% CO2 - air environ­ment) period of 18 hr. Fluorochrome released as per target lys is (experimental lysis) measured in a spectrofluorimeter (Hitachi , Tokyo) provided an index of cytotoxic efficacy of effectors31

. A labeled target group maintai ned alone serves for spontaneous lysis.

C . ft·· Experimental lysis - Spontaneous lysis 100

ytotox IC e Icacy = x Total incorporation

PMN-mediated phagocytosis - Neutrophils (PMNs) were isolated from the splenic ti ssues from different experimental groups by percoll densi ty grad ient (density 1.089) and were allowed to phagocytose the target brain tumor cells (from a steady glial tumor line, syngenic in nature) in presence of nitroblue tetrazolium chloride at the effector: target ratio of 100: I. The preparation was incubated for 18 hr at 37°C in 4% CO2 -humidified atmosphere. Finally, the reaction was stopped by adding O.lN chilled HCI and pellet was extracted with boiling pyridine for the reduced blue formazan.Reduction of yellow NBT to blue formazan indicated the extent of phagocytic brust at 530 nm by effectors concerned . Thus, results were evaluated from the extent of oxidative brust as revealed for blue formazan formed (relative 0 .0 .)2 1.31.

Statistical analysis-Statistical analyses of the data were based on standard dev iations of means and level of significance were followed through Student 's t test. All results were evaluated stati stically by applying the SPSS-PC package (Version 9.0,SPSS, Chicago, Illinois, U.S.A.). A probability of <0.0 1 was considered stati st ically significant.

Results and Discussion The present study has provided several

experimental evidences to conclude that administration of sheep red blood cells (ip) in experimentally induced brain tumor, can potenti ate the cell mediated immune response (CMI) resulting in an effective tumor regress ion through effector mechani sms. Such therapeutic efficacy has been ev idenced by the improvement of survival in animals (Fig. 1) as well as by hi stological findings (Fig. 2) .

Administration of 7% SRBC (0.5 ml) in ENU treated animals revealed some interesting results: total survival period significantly increased (Fig. 1), tumor induction was delayed or absent over the period of one year after ENU administration as observed macroscopically/microscopically. Thus, signi ficant increase in survival period in ENU-SRBC group supports the immunotherapeutic effect of SRBC which has been shown to exert strong anti-tumor property in ENU-treated tumor bearing animals as evidenced through histological findings (Fig. 2), where the reversion of neoplastic g lial features to normal glial features has been shown to occur in

800 ± 35 ±40

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>-III 500 c -0 400 j

300 E ± 30

± 30 :::J -

Z 200 -100

0 N A-8IJU F-ENU A-8IJ~ F-EN~

SRBC SRBC

Fig. I - Survival study - survival data of ENU induced rats (A­ENU and F-ENU) compared to normal animals (N). Mean sur­vival period was significantly decreased in tumor bearing anima ls (A-ENU , F-ENU). SRBC administration demonstrated significant increase in mean survival period in tumor bearing animals. for both A-ENU and F-ENU group, although the best protective ef­fect was observed in F-ENU-SRBC group.

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INDIAN J EXP BIOL, AUGUST 2003

a

Fig. 2 - Histolog ical cvidences showing normal brain tissue with the presence of few astrocytes, oligodendrocytes and few neurons (a); The effect of A-ENU (acute dose) showed grade I V ol igodendrogl ioma with typical honeycomb li ke appearance and presence of mitoti c figures, giu nt cell s and absence of intercellu ­lar spacing (b), and hyper proliferution of the o li godendroglia cells wi th the presence of mitotic figures were observed with the frac tionated doses of ENU (F-ENU) (c).s ignificant decrease in the number of oligodendrogl ial cell s was observed with the SRBC ad mini stration in A-ENU grouped), however, the best effect was observed in the F-ENU group, where reversion of neoplastic glial features to normul glia l features was observed

following SRBC administ ra tion(e) x 400.

J.

GANGOPADDHYA Y el al. : BRAIN TUMOR IMMUNOTH ERAPY WITH A NOV EL CO RPUSCULAR ANTIGEN 809

SRBC-treated ENU-induced animals. Thi s finding is well corroborated with the immunological findings as well as growth kinetic study .

Histological studies show normal glial cell populations in white matter of cerebral cortex with few astrocytes oligodendrocytes and a few neurons (Fig. 2a). The effect of A-ENU (acute dose) showed grade IV oligodendroglioma with typica l honeycomb like appearance and presence of mitotic figures, giant cells and absence of interce llul ar spac ing (Fig.2b), and hyper proliferation of the oligodendroglia cells with the presence of mitotic figures were observed with the fractionated doses of ENU (F-ENU) (Fig. 2c). Significant decrease in the number of oligodendroglial cells W3<; observed with the SRBC admini stration in A-ENU group, however, the best effect was noted in the F-ENU group, where revers ion of neoplasti c g li al features to normal glial features was observed following SRBC admjnistration (Fig. 2d & e).

Malignant cell s of the brain have been shown to be regulated by SRBC. As found with other studies32

.34

the proliferation kinetics o f such tumor cells in vitro culture almost conclusively demonstrated the mali gnant nature of the tumors. The growth kinetics study as conducted in ti ssue culture system showed a significant increase in proliferation capacity of the tumor ce ll s both in terms of their proliferation index

(PI) (Fig. 3a) and per cent fluorescence uptake (HO-33342) (Fig. 3b & c) of cells at 24 hr culture. The degree of malignancy as determined by the above methods also corresponded well with the higher morbidity rate in the group of animals concerned. These results were comparatively more pronounced in the animals receiv ing acute doses of ENU rather than those receiving fractionated doses. It seems likely from the present data that SRBC has the capability to inhibit or block tumor proliferation not only by potentiation of the CMI response but also, presumably by interfering with the mitotic rate of the malignant cells. This may be possible by 1) inducing a cytostatic effect, 2) differentiation induction, 3) blocking D A sy nthesis and growth factor interact ions, and 4) interacting with cellular oncogene ex press ion . Several in vitro studies have revealed that all these manifestations can be achieved by interferon type I (alpha and beta) and type 2 (gamma) depending on h f . I ' . I d35-37 t e type 0 ti ssues or tumor me mvo ve .

However, there are ample supports in favour of the facts that interferons generated in vivo can induce gene activation causing Il1Itl atlon of latent endoribonuclease and thus interfering with tumor replicating processes37

; spontaneous action of interferon in vivo can induce re-ex press ion of characteristic differentiation phenotype marker; moreover, in vivo

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n n n N A· EM) F·1N..J A·ENJ. F· ENJ.

SRBC SRBC

Fig. 3 - (a) : Cell proliferation Index (PI) of Glial cells before and after induction of tumors and that following application of Sheep Erythrocyte. The figure shows high counts in tumor induced animals (b,c) and near normal counts in SRBC treated tumor groups.( b): Fluorescent spectra of HO-33342 uptake of Glial ce ll s under variable conditions (vide text). The spectra shows a s ignifi cantly higher dye uptake in tu mor induced gli al cells and near normal values fo llowi ng SRBC treatment in tumor groups.(c): Cytokinetic Index of Glia l cells reveal ed through HO-33342 uptake in different groups of animals (see text) . The quantitative data confirms the tumor reducing e f­fect of SRBC in tu mor bearing animals.

8 10 INDIAN J EXP BIO L, AUG UST 2003

res istance to growth facto r receptor ex pression by tumor ce ll s is also prono unced by interferon38

. It seems reasonable to argue that SRBC by stimulating i ll vivo e laboratio n of IFN-y I9 can down regulate the growth character of the brain tumo r cells through modula tion of gene express ion at diffe rent levels39

.

The poss ibi lity whether the S RBC mediated stimulation to astrocytes in brain might have interplay in tumor inhibitory process is a subject of further research.

Mechanism of anti -tumo r ac ti vity of sheep erythrocytes stud ied earli er2 1 showed some immunomodul atory fu nctio ns as being responsib le fo r tumor in hibiti on in the host concerned . In the present context , s tudies conducted with the splenic ly mphocy tes (S L) and PMNs in diffe rent groups of experi mental animals revea led inte resting correlations as well : spontaneous E- Roselting, the cy totox ic efficacy of SL and phagocy tic capac ity of PMNs were significantly decreased in a ll the corresponding ENU groups o f animals (Figs 4-6) indicating inh ibitory effects of developing intracrani al tumor in systemic immuni ty. Although it raises questi ons about the res idua l e ffec ts of ENU o n the immunocytes concerned , such poss ibiliti es can be ru led out as the in vestigations were conducted more than 6 months after ENU admini stration when the immunocytes were represented by fresh progeny afte r several regeneratio ns. Admini stration of S RBC o n the anima ls receiving both acute and fractionated doses of E U was found to be s ig ni ficantly protecti ve; the S L and PMNs show s ig nificantly hi gher cy totoxic (Fig. 5) and phagocy tic efficacy (Fig . 6) respecti vely, the sti mul atio n being more in animals receiv ing frac ti onated doses of EN U; may be due to a greater

25 ±2.84

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a::: 10 ~ 0

± 1.26

5 ± 1.03 n 0 n N A-ENU F-ENU A-ENlJ.. F-ENlJ..

SRBC SRBC

Fig. 4 - E-Rosette Formation -Rosell ing fo rming capac ity of the ly mphocytes was drastica lly reduced in_E NU groups (A-ENU and F-ENU), SRBC adm inistra ti on was fo und to be effec tivc in bri ng· ing back the va lue 10 norma l leve l.

residual suppressive effect of tumo r/ENU in acute dose recipient. Correspo nding greater surviva l period in these animals furt her supported the immuno­therapeutic effects of SRBC in ani mals with ENU induced brain tumor. Precisely , the therapeuti c results can be considered to be effecti ve th rough enhanced immune effec tor mechani sm under the influence of SRBC which generate a sig ni ficant number of activated lymphocytes capable of cross ing the blood­brain-barrier I2A0

.

In the present course o f in vest igati on, the sig nificant enhancement o f E-rosseLe fo rmati on in ENU -treated animals foll owing ad mi nistrati on of sheep red blood cells (Fi g . 4) sugges ts lymphocy te activation and prolife ratio n lR

• Presumably, the up regulatio n of C 0 2 (T I l) molecules occuring in individual lymphocytes indi cated by super rosette formation and quantitative lymphocyte pro life rati on activity was indicated by s igni ficant increase in rosette forming ce lls (C0 2 + lymphocy tes). Th is study also indicates possible up regulati on of C 0 25 (lL-2R) express io n and IL-2 liberatio n fro m lymphocytes (unpub li shed data). The acti ve compo nent o f sheep red blood ce ll which is presumed to be responsible for such acti vity has been fo und to be a ce ll surface g lycoprotein molecule, know n as T II Target Structure (T II TS) or sheep lymphocytes form of LFA-3 or mo re currentl y C D58 that binds with the E­receptor or C0 2 (T I I) molecule of the lymphocytes4 1

.44 has been shown to send mitogen ic

stimu lus to acti vate them, supporting the findin gs of the previo us workers who demo nstrated ac tiva ti on of T cell s through an a lte rnati ve path way th rough SO KD T I I sheep eryth rocy te receptor prote i n -15.5 1.

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,-± O.OO4 r-

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Fig. 5 - Cytotox ic ity assay (CTL-Assay) of spleni c lymphocy tes (SL) as delCrmined th rough HO-31342 re lease in diffe rent g roups o f ra ts. S ignifican t decrease in cy totox ic efficacy o f SL is no ted in bo th the ENU treated g roups. SRBC significant iy sti mulated CTL ac ti vity in both the tumor groups.

GANGOPADDHYA Y el al. : BRAIN TUMOR IMM UNOTHERAPY WITH A NOV EL CORPUSCULAR ANTIG EN Sil

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0 n N A-ENU F-ENU A-ENU- F-ENU-

SRBC SRBC Fig. 6- Phagocytic activity of PMN determined by NBT reduc­tion assay in different groups o f animals: Significant suppress ion of activity is noted in acute - ENU treated rats: SRBC restored the ac ti vi ty more in fractionated ENU group than the acute grou p.

Administrat ion of sheep red blood cells (SRBC) in tumor bearing animals augmented the cytotox ic efficacy of lymphocytes (Fig.S). The non spec ific lytic activity carried out by lymphocytes possibly in volves CDS+, CD4+ T-Iymphocytes and natural killer ce ll s (N K cell s), wh ich were presumed to be acti vated foll owing SRBC admini strati on, since they were all CD2+ cells- showing activation of CTL and NK funct ion through sheep ely th rocyte receptor (C02, Til )52.

Finally, the enhancement of phagocytic activity of PMN (Fig. 6) seemed to occur by SRBC ac ti vated CD4+lymphocytes indirectly through cytokine network (neutrophil activating factor) . Chaudhuri et of?t have shown that tumoricidal phagocytic activity is increased in SRBC treated animal s2t

. Moreover, direct activation of PMN phagocytic activity wi th SRBC is also poss ible, through C02 receptor reported

b . f 5\ to e present on Its sur ace" . Although immunomodulatory activities are h'b' d b 13 54-S6 h d' d ex lite y m<rny agents . - , t ose me tate

through SRBC were found to be unique in that they have direct stimulatory effects on lymphocytes. Thus, interaction of TIlTS (LFA-3) of sheep red blood cell s with C02 (TIl) receptor is supposed to act ivate a large number of il11munocytes not only of lymphoid but also of myeloid lineage. Moreover, acti vat ion of CD4+ lymphocytes with SRBC also implies activation of immunologica l network indirectly through cytokine network , recruiting various immune cells. Further, it is now known that acti vated-T cells can enter the brain across the blood- brai n-barri er, thus act ivation of CD2+ lymphocytes (CD4+, CDS+, NK) with sheep red blood cell s at the peripheral

extent in an immunosuppressive state is an important finding, that facilitates infiltration of act ivated lymphocytes across blood brain barrier, favoring antigen presentation by the APC cells of the brain , particularly microglia and generating an effective an ti-tumor immune response (unpublished data).

Acknowledgement The work was supported by grants obtained from

the Department of Science & Technology , Mini stry of Science & Technology, Government of Indi a. and R.D. Birla Smarak Kosh , Bombay. India.

References I Bartel A L. Heilbroon Y D & ShifTer J. Ex tensive resection

of primary mali gnant tumor of the left cerebral hemi sphere. Sur!:: Nellrol . I ( 1973) 337.

2 Diengdoh J V & Booth A E. Post-irradia tion nccrosis of the tempora l lobc prescnting as a glioma. J Neurusllr!::, 44 (6) ( 1976) 732.

3 Glantz M J. Kim L, Choy H & Akerley W. Concurrelll chemotherapy and rad iotherapy in patients wi lh brain tumors. Ol/colugy (H/IIuil/gT) 10 ( 1999) 78.

4 Nakngaki M. Brunharl G, Kempen & Caveness W F, Monkey brain damage from rad iation in the therapeutic range, J Nell roslI rg. 44 ( 1976) 3.

5 Di x A R. Brooks W H, Roszman T L & Morford L A. Immune defects observed in patients with primary mali gnant brain tumors. J Nellro illlll/ul/ ol , 100 ( 1999) 2 16.

6 Hi shii M. Nitta T, & Ishid a H. Human glioma derived interl eukin- IO inhibits antitumor immune responses ill ,·ilru. J Nellmsurg. 37 ( 1995) 11 60.

7 Ku ppncr M C, Hamou M F & Sawamura Y. Inh ibition of lymphocyte function by glioblastoma-derived transforming growth factor beta 2, J Nellrusllrg, 71 ( 1989) 2 11.

8 Maxwell M, Galanopou ios T & evi l Ie-Go lden. Effect of the ex pression of transforming growth factor beta2 in primary human gli ob lastomas on immunosuppress ion and loss of immunosurvei ll ance. J Nellro.wr!::. 76 ( 1992) 199.

9 Roszman T L & Brooks W H. Immunob iology of primary intracrani al tumors. Ill. Demonstrati on of a qualitative lymphocy te abnormality in pat ients with prilllary bra in tumors. Ciill Ex!, 1111111111101. 39 ( 1980) 395.

10 Bradle M. Immu ne cont ro l of the brain. in 11I /lIIl1l/ollellrologv edited by M CholTl on & L Steinman (Springer- Verlag A Illsterda m. New York) , 1966, 153.

II Selmaj K, Pathophysi ology of the blood-brai n barrier. In IIIIIIIIII/ol/ellrology edited by M Chofnon & L Stein man (S pringer-Verlag, Amsterdam, New York), 1966, 175.

12 Weller R 0, Engelhardt B & Phillips M J, Lymphocytes targetling of the central nervous system:a review of afrarelH and efrcrcnt CNS -immune pathways. Braill Palhol, 6 (1996) 275.

13 Gillespie G Y & Mahalay M S. Biological response mod ifier therapies ror patients with malignant gli omas: In Nellro ­

Ol/ cology, edited by G.T.Thomas, (Churchill Livingstone. New York, London) 1995 , 242.

14 Pollack I F. Okada H & Chambers W II , Exploitation of immune mecha ni sms in the treatment of celllral nervous sys tem cancer, Sell/ ill Pedimr Nellrol. 7(2) (2000) 131.

812 INDIAN J EX P BIOL, AUGUST 2003

15 Bernard A L , Tran H C & Boumse ll L , Three di fferent erythrocyte surface molecules are required for spolllaneous T cell roselle formation, J 1111111111101, 139( I ) ( 1987) 18.

16 Ogasawara H, Kusuy K & Takasaki S. Role of terminal galactose residues in N-linked sugar chains of sheep erythrocy te membrane glycoproteins in roselle formation with T lymphocytes. 1111111111101 Lell. 48 ( 1995) 35.

17 Pang G T. Wiggins P M & Wil son J D. atu re of the union between sheep red blood cells and T lymphocy tes. 1111 Arch AI/ergy Appl 1111111111101. 5 1 (6) ( 1976) 75 1.

18 Eberl E C. Sheep red blood cells enhance T- Iymphocytes proliferation, Clill "11111111101 111111111/ 101)(111101.37(2) ( 1985) 203.

19 Wilkinson M & M orris A G. Role of the E-receptor in i ntcrferon- gamma exprcssion: Shccp crythrocytes augmcnt intcrfcron-gamma production by human lymphocytes. Cell 1111111111101.86( 1) ( 1984) 109.

20 Chaudhu ri Swapna, DUlla S K. Roy N, Gangopadhayay S K & Chaudhuri S. Immunomodulation and la variab ilit y in dendrit ic lymph ce ll s: radiobinding immunoassay versus immunc rcsponse study. Biochelll Mol Bioi 1111. 30 (3) ( 1993) -171.

2 1 Chaudhuri Swapna. Sinha A. Sengupta A & Chaudhuri S, Sheep cry th rocytcs providc mctabolic triggers for tumor phagocytos is in polymorphonuclea r neutrophils: A possible mcchanism of tumor inhibition in mice. Biochelll 1111, 23 (2) ( 1991 ) 23 1.

22 Roy R U, SarkaI' S, DUllachaudhuri M , Dutta S K . Chaudhuri Swapna & Chaudhuri S, Antigen prescnting capaci ty of mononuc lcar phagocytes in expcrimelllal Icukemia and in pati ents with hematological malignancics, in India. J Helllalol Blood Trall .I!, 15 ( I ) ( 1997) 33.

23 , Barth R F. Rat brain tumor models in cxpcrimcntal neuro­oncology: thc 9L. C6. T9, F89. RG2 (074), RT-2 and CNS- I gliomas. J Neul"Oollcol , 36( I ) ( 1998) 9 1.

24 Druckrey H, Ivancovic S & Preussman R. Teratogen ic and carcinogenic effects in the offspring aftcr single injection of cthy ln itrosourea to pregnant rats, Nalllre, 2 10 ( 1966) 1378.

25 Kocstner A, Swenbcrg J A & Wechsler W . Transplacental production with ethylnit rosourca of ncoplasms of thc ncrvous system in Sprague-Dawley rats, ;\111 J ParllOl, 63 ( 1971) 37.

26 Lantos P L. VandcnBerg S R & Paul Kleihues. Tumors of the nervous system, in Gree/([ie lr/ 's Neuropalhology, cd ited by David I Graham & Pctcr Lantos, Ed iti on 61h

, Volumc 2. (Arnold , London, Sydney, Auckland), 1997.583.

27 Lantos P L. Chemica l induction of tumors in ncrvous systcm. in Nellrooll cology, cd ited by G. T. Thomas, (Churchill Livingstone New York . London). 1993.85.

28 Chaudhuri Swapna, Ganguly S. Ghosh S . DUlla S, Sarkar S. Begum Z, Roy R U. Bhallacharya M K, DUlla S K & Chaudhuri S. Immunological insurgence during intracranial tumor developmcnt: Cellular immunity in astrocytoma patients. J Physiol Appl Sci, 54(3) (2000) 11 8.

29 Raha S K, Dey S K , Roy S K , Chaudhuri S & Chakrabort y S L. A ntitumor acti vity of L-Asparaginasc from Cylil1drocarpol1 obslurisporum MB 10 and its effects on the immune systcm. Biochem In l. 2 1(6) ( 1990) 987.

30 Law Sujata, M aiti 0 , Palit Aparna, M ajumder 0 , Basu K. Chaudhuri Swapna & Chaudhuri S, Facili tation of func tional compartmentalization of bone marrow cell s in Icukemia mice by biological response modifiers: An immunotherapeutic approach, 1111111111101 Lell. 76 (200 1) 145.

3 1 Hudson L & Hay F C. Ccll dynamics ill vivo, in Practi cal Immunolo~y (Blackwell Scientific Publications, Oxford. London, Edinburg, Boston, Melbourne), 1989, 182.

32 Hoshino T . Commentry on the biology and growth kinetics of low gradc and high-grade gliomas. J Nellrosllrg, 61 ( 1984) 895.

33 Hoshino T , Barker M. Wilson C B. Boldrey E 13 & Fewer D. Cell kineti cs of human gliomas, J Nellrosurg, 49 ( 1972) 13.

34 Stce l G G, Growth kinetics of brain tumors. in Braill I!/IlIon;: Scielllific basis, clillical ill vesligalions alld currelll Iherapy. (Buttcrworths London). 1980. 10.

35 l3alkwill F R, Cytokincs cancer thcrapy (Ox ford Uni vcrsity Press , Oxford) 1989,8.

36 Clcmcns M J. Cytok ines (B iose iemifi c Publi cations, Oxford) 199 1. 103 .

37 Samucl C, in Mechanism of intcrferon actions. ed ited by LM Pfeffer, (C RC press Inc. Florida). 1987. III.

38 Lc vy 0 E, The rcgulation of gcnc exprcssion by intcrferons. in l lIfelferoll: To-do." alld TO-IIW/TOII '. cdi ted by Stark. G.R (Medi script. London), 1988.4.

39 Palma L, Di Lovenzo N & Guidctti B. Lymphocytic infi ltrates in primary gl ioblastomas and residual gliomas. Incidence, fate, and rcvelenec to prognosis in 288 opcratcd cases . .I Nelll"OslIrg, 49 ( 1978) 854.

40 Hunig T, Thc ce ll surface molecule recogn izcd by thc eryth rocyte reccptor of T lymphocyte. J Ex!, Ma l. 162 ( 1985) 890

4 1 Sclvaraj P, Plunkctt M L, Dustin M, Sandcrs ME, Shaw S & Springer T A, The lymphocyte glycoprotein C02 binds the cell surface li gand LFA-3, Nalure, 326 ( 1987) 400.

42 Sun Z J. Dotsch V, Kim M Li J. Reinherz E L & Wagner G. Functi onal glycan-free adhesion domain of human cell surface receptor CD58 : Dcsign. production and NMR studics, EM BO. 18 ( 1999) 2941.

43 Wallener B P. Frcy A Z. Tizard R. Mallalians R J. Hession C. Sanders M E. Dustin M L & Springer T A. Primary structure of lymphocy te associated antigen -3 (LFA-3), The ligands of thc T lymphocytes C02 glycoprotein, J Exp M ed, 166 ( 1987) 923.

44 Dcnning S M. Dustin M L, Spri nger T A. Singer K H & I-laynes B F, Purified lymphocyte funct ion assoc iatcd anti gen - 3 (L FA-3) acti vates human thymocytcs via the C02 pathway. J 1111111111101.14 1(9) ( 1988) 2980.

45 Hunig T , Tiefenthaler G. Mitracht R. Kholer C, Lottspeich F & Meuer S, The erythrocyte rcceptor of T -Iymphocytcs and T- II target structure (Til TS): Complcmentary ccll interacti on molecules invol vcd in T -cell activat ion. Behring 111 .1'1 Mill 8 1 (1987) 3 1 . .

46 Hunig T , Mitracht R, Tiefenthaler G. Kohlcr C & Miyasaka M , The cell surface' molecu le binding to the 'ery throcyte receptor of T -Iymphocytes, Ce llular distribution. purifica tion to homogeneity and biochemical propcrti es. Eur J Immul1oi, 16( 12) ( 1986) 16 15.

47 Meucr S C, Roux M V & Schraven B, The alternative pathway of T cell acti va ti on : Biology, pathophys iology and perspect ive for immunopharmacology, Clill 1111111111111101 IlIIlIIul1opalhol , 50 ( 1989) 133.

48 Meuer S C. Hussey R E, Fabbi M. Fox D. Acuto O. Fitzgerald K A, Hodgdon J C, Protentis J P, Schlossman S F & Reinherz E L , An alternat ive path way ofT-ce il activation: A functional role for the 50 KD Til sheep erythrocyte receptor protein , Cell , 36 ( 1984) 897.

GANGOPADDHY A Y el al. : BRAIN TUMOR IMMUNOTH ERAPY WITH A OV EL CORPUSCU LAR ANTIG EN 8 13

49 Semnani R T, Nutman T B, Hochman P. Shaw S. van Scventer G A, Co-stimulati on by purifi ed intercellular adhes ion molecule I and lymphocy te function-assoc iated anti gen 3 induces di stinct proliferation, cytokine and cell surface anti gen profil es in human "na'ive" and "memory" CD4+ T-cells, J Exp M eel , 180 (1994) 2 125.

50 Wingren A G. Parra E, Varga M. Kalland T. Sjogren H O. Hedlum G & Dohl sten M. T-ce ll acti va tion path ways: B7. LFA-3, and ICAM- I shape uni quc T cell pro fil es. Cri l Re\' //1/11/1111 0 1, 15 ( 1995) 109.

51 Siliciano R F. Prall J C. Schmi dt R E, Ritz J & Reinherz E L, Acti vation of cy tolyti c T lymphocyte and natural killer ce ll function through the Til sheep crythrocyte binding protein . Nalll re. 3 17 (1985) 428.

52 Niiazova 0 P, Osipova S O. Badalova I S & Dekhkan-Khodzhaeva A. Earl y and late E-rosclle forming ncutrophil s in persistent lamblias is. Med Parazi lOl (Mosk), I ( 1995) 43 .

53 Berquist B J. Mahalcy M S. Jr. , Steinbook P & Dudka I. Treatment of brain tumor with BCG ce ll wall preparati on. Surg Nell ro l. 18 ( 1980) 197.

54 Endo Y, Matsushima K & Oppenhcin J. Mechani sm of ill vilro antitumor cffects of interl eukin I (IL- I). illlIl ll ll lObiol. 172 ( 1986) 3 16.

55 Ishi zawa A, Immunotherapy fo r mali gnant glioma. Nell ro log ica M edico - Chirugica (Tokyo) ( 198 1) 179.

56 Nakagawa Y, Kirakawa K. Ueada S, Suzuki K, Fukullla S, Ki shida T. Imanishi J & Aillagai T. Loca l ad mini stration of interf"cron for malignant brain tumors. COllcer Treat Rep. 67 ( 1985) 833 .