Sheep erythrocyte demonstrated better effect than IL-2 and IFN-gamma as biological response modifier against glioma in experimental model.

The significant insights into the immunobiology of central nervous system (CNS) and brain tumor have opened up the feasibility of applying 'Immunotherapy' as an alternative to the poor prognosis of malignant brain tumor with conventional therapeutic approaches. Though cytokines like IL-2 and IFN-gamma used against glioma showed some favorable results by eliciting Th1 type immune response, a proper immunotherapeutic agent is still to be searched for. Sheep erythrocyte (SRBC), a corpuscular antigen showed a better therapeutic efficacy in terms of enhanced survival and augmentation of cell mediated immunity (CMI) in a glioma model developed by chemical carcinogen ethyl nitrosourea. Histological findings revealed most efficient glioma rejection in SRBC and combination biological response modifier (BRM) treated groups. Simultaneously E-rosetting, cytotoxicity of lymphocytes, phagocytosis and antigen presenting capacity of myeloid cells established the better therapeutic efficacy of SRBC alone than other BRMs viz. IL-2 and IFN-gamma. Even the effect of combination therapy of different BRMs showed marginal differences in facilitating glioma reduction than the single use of SRBC. These findings emphasized the application of SRBC as an exogenous BRM having the potential as a rational therapeutic adjunct against glioma.

CD2-SLFA3/T11TS interaction facilitates immune activation and glioma regression by apoptosis.

OBJECTIVE: Exogenous application of T11TS/SLFA3 in glioma model had shown the regression of tumor load through immunopotentiation. The mechanistic module of this interaction on immunological synapse formation and resulting effect in glioma regression is searched for delineating immunotherapeutic efficacy of T11TS. METHODS: After purification of T11TS/SLFA3 from sheep erythrocytes the glycoprotein was characterized by SDS-PAGE analysis and glycoprotein staining. The modulatory effect of T11TS application on glioma animals were studied by CD2 and MHC class II expression on peripheral lymphocytes, PMN, macrophages and intracranial microglia by flowcytometric analysis. Finally apoptotic killing of brain cells were studied through annevin-V expression and measuring fragmented cytoplasmic DNA by ELISA. RESULTS: Compared to healthy normal counter-parts the CD2 receptor downregulation by ENU treatment in lymphocytes and PMN were upregulated with three consecutive doses of T11TS. Splenic antigen presenting macrophages and intracranial mocroglia had shown CD2 and MHC class II modulation simultaneously in their different subtypes. These receptor studies revealed significant boosting of the immune competent cells most effectively in second dose of T11TS whereas the third dose had some regulatory effect. Sharp increase of apoptosis in brain cells was found by phosphatidylserine externalization and presence of fragmented DNA in cytoplasm with application of T11TS in consecutive doses as a result of immune potentiation. CONCLUSION: Receptor studies revealed modulation of CD2 and MHC class II, two important constituents of immunological synapse successfully help to form the TCR-p-MHC complex and provide required co-stimulation for activation. Potentiated immune effector machinery was then directed to abrogate glioma by apoptosis signifying T11TS as an immunotherapeutic probe.

Differential regulation of the protein tyrosine kinase activity following interleukin-2 (IL-2), interferron gamma (IFN-gamma) and SRBC administration in brain tumor-induced conditions: SRBC acting as a dual potentiator in regulating the cytokine profile.

Protein tyrosine kinases (PTKs) act as an important class of signal transducer in cytokine mediated signaling. Defects in phosphorylation of tyrosine residues of intracellular substrates of the immunocytes are a noted phenomenon in glioma induced immune suppression. Administration of BRMs like Interleukin2 (IL-2), Interferon gamma (IFN-gamma) and SRBC in glioma induced experimental models, improved their survival status by immune potentiation. It was shown that SRBC exerts the maximum anti-tumor immune boosting by augmenting the functional status of the two immunocytes-microglia and lymphocytes when compared with IL-2 and IFN-gamma. The present study focuses on the differential modulation of the protein tyrosine kinase activity in lymphocytes and microglia following the administration of the 3 BRMs. Our findings indicate that PTKs actively transduce signals on administration of exogenous IL-2. But exogenous IFN-gamma administration fails to elicit the enzyme activity. With SRBC administration, a differential PTK activity modulation was observed in the two immunocytes. SRBC not only shifted the cytokine profile to Th1 subset of lymphocytes but also simultaneously upregulated the expression of the activation marker IL-2Ralpha/CD25 thereby resulting in auto-activation of the hosts immunocytes.

Immunotherapeutic effects of T11TS/S-LFA3 against nitrosocompound mediated neural genotoxicity.

Nitrosocompounds formed by the interaction of nitrites and secondary amines are neurotoxic in human and different rodent species. Human exposure of nitrosocompounds are widespread affected by different modes like nitrite/nitrate preserved foods, beverages like beer, formed in the stomach following uptake of the precursors nitrates, nitrites and secondary amines. The productions of alkylating metabolites during the breakdown of nitrosocompounds are the causative agents for the neurotoxic changes of the neural cells. An attempt has been made in our lab to study the effect of nitrosocompound mediated toxicity and the gradual toxic effects of these neurotoxic agents to transform the normal glial cells to a neoplastic one. The present study indicated that a transmembrane glycopeptide of sheep red blood cell (SRBC), known as S-LFA3 or T11 target structure (T11TS) applied to nitrosocompound induced animals manifesting a full grown intracranial malignancy can revert back tumor-bearing condition to the normal physiological state. Young Druckray rat of both sexes aging 3-5 days were injected with N'-N'-ethyl nitrosourea (ENU) intraperitoneally (i.p.) at a single dose of 80 mg/kg body weight to simulate nitrosocompound mediated neurotoxicity. 2-,4-, 6-, 8-, and 10-month-old neonatal ENU induced animals were sacrificed for growth kinetics, functional immunological parameters and receptor studies to hint at the changes during tumor development. In order to determine the immunomodulatory role of T11TS, 7-month-old ENU induced animals were injected with T11TS at a dose of 0.41 mg/kg body weight, in three consecutive doses at an interval of 6 days maintaining normal control as untreated control and ENU induced animals of age-matched rats as tumor-bearing control. All the immunological parameters, growth kinetic study, receptor-based study by FACS directly established the immunomodulatory, anti-toxic and anti-tumor property of T11TS/S-LFA3. Finally, formation of DNA ladder along with the FACS-based apoptosis study clearly indicated that T11TS is a potent apoptotic inducer in neoplastic neural cells.

Evaluation of anti-tumor property of specific and non-specific BRMs in experimental gioma by assessing the microglial cell functional and phenotypic modulations.

BACKGROUND: Microglial cells are considered to be the chief immunomodulatory cells of the brain. These cells play a crucial role against various neurodegenerative diseases. When modulated microglia have been shown to exert a potential anti-tumor immune response against brain neoplasms. Although several specific BRMs like IL-2, IFNgamma have been shown to modulate the microglia to get an effective anti-tumor immune response, associated toxicities and detrimental side effects have posed severe limitation in there use particularly for therapeutic purposes. OBJECTIVES: In the present study, attempts have been made to elicit the modulations of microglia cell function and phenotypic expression following specific (IL-2, IFNgamma and a novel nonspecific BRM (corpuscular antigen) in order to determine their anti-tumor property in experimental glioma model. MATERIALS AND METHODS: Brain was experimentally induced in young Druckray rats of both sexes with N-N-ethyl nitroso urea (ENU). These ENU treated animals were administered with both specific and nonspecific BRMs like IL-2, IFNgamma and SRBC either singly or in combination 5 months after ENU administration and after ascertaining its degree of malignancy. RESULTS: Microglial cells separated from different experimental groups were found to be positive for CD11b, MHC II, CD4 and negative for GFAP.FACS analysis demonstrated that different subtypes of microglial cell populations (CD25+, MHC II+ and CD25+MHC II+) in the brain tissue based on phenotypic expression, which were downregulated in tumor bearing animals, and subsequently restored with increased expression, particularly with SRBC administration. The Scanning Electron Microscopic (SEM) study also depicted modulation of cellular morphology of microglial cell predominantly with SRBC administration. Studies conducted ton evaluate immunological functions at the cellular level showed increased antigen presenting caopacity of microglial cell with SRBC administration, which was significantly greater then IL-2, IFNgamma and combined doses (E2_S). However phagocytic functions of microglial cells were found not to be significantly modulated with BRM treatment. CONCLUSION: The results suggest that the nonspecific BRM SRBC can exert greater modulatory effect on microglila cell function and phenotypic expression than the other BRMs used, and in doing so culminate in a potent anti tumor immune response in experimental glioma with compatible tolerance profile.

Preclinical Changes in Immunoreactivity and Cellular Architecture during the Progressive Development of Intracranial Neoplasms and an Immunotherapeutic Schedule with a Novel Biological Response Modifier, the T11TS / S-LFA3.

Among neoplasms, brain tumors are particularly " difficult to treat" because of the partial immune privileged status of the brain and the presence of the blood brain barrier (Selmaj, 1996). Many details of progressive development of brain tumors remain unexplored and elucidation of consequent changes of the immune system with correlated cellular architecture and cell kinetics were the major objectives of the present course of investigations. Our studies have indicated that the primary resistance by the immune system to a growing tumor declines after a certain point, resulting in an immune suppressed state in a tumor bearing individual. The poor prognosis of malignant brain tumors with classical treatments like surgery, radiotherapy and chemotherapy has led to interest in immunotherapeutic protocols. In the present study, an attempt was made to determine the immunomodulatory and antitumor properties of a transmembrane glycopeptide of sheep red blood cells (SRBCs), known as S-LFA3 or T11TS. Young Druckray rat of both sexes aged 3-5 days were injected with N'-N'-ethyl nitrosourea (ENU) (i.p) to induce brain tumors and at 2,4,6,8 and 10 months of age were sacrificed for study of survival, tumor growth kinetics and immunological parameters like lymphocyte rosette formation, denoting CD2 - CD58 interactions and phagocytosis by peripheral macrophages and PMN hint at the changes during tumore development. In order to determine the immunomodulatory role of T11TS, 7 month old ENU induced animals and controls were injected with the compound (1 ml., i.p). The data obtained indicate that administration of T11TS results in increased survival of rats along with a decrease in growth kinetics of tumor cells to the normal level when compared to ENU induced animals of the same age. Pointers to mechanisms involving immunological investigations at the cellular level in these animals indicated improved lymphocyte function in terms of E-rosetting, augmented cytotoxicity and enhanced PMN and macrophage function in terms of phagocytosis. Finally histological examination showed complete reversal from the hyperplastic state to normal cellular homeostasis, indicating antitumor efficacy of T11TS, correlating very well with the data from survival and cell kinetic studies.