T11 target structure induced modulations of the pro-inflammatory and anti-infammatorycytokine expressions in experimental animals for glioma abrogation.

Glioma angiogenesis is the result of the interaction between cancer cells with endothelial cells, and the surrounding inflammatory cells. This interaction plays a crucial role in directing the neo-formation of blood vessels. In the carcinogenic milieu, inflammatory cytokines secreted from inflammatory cells affect endothelial cell functions that are indispensable for tumor growth and metastatic propagation. TNF-α, referred to as the 'inflammatory switch', has shown its potential as an inflammatory agent by activation of IL-8 and IL-6 through NF-κB mediated pathway. Therefore, inhibitors of angiogenesis appear to be promising therapeutic agents for advanced gliomas. Previous studies from our lab showed that T11TS, a membrane glycoprotein, has antiangiogenic and antineoplastic activities in experimental animals and human samples. The present experimental study was designed to evaluate the effect of T11TS therapy on inflammatory cytokine expression of TNF-α, IL-8, IL-6 and their downstream associated molecule NF-κB in vivo. Our results revealed that T11TS therapy induced downregulation of TNF-α, IL-8, IL-6, and NF-κB confirmed by FACS assay and ELISA. In situ-immunofluorescence results hint that T11TS has the efficacy to stop the inflammation related to angiogenesis. Moreover, upregulation of IL-4 and IL-10 in microglia after T11TS therapy helps in complete abrogation of glioma inflammation and angiogenesis. These effects might contribute to the antineoplastic activity of T11TS.

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.

A novel monoclonal antibody inhibits the immune response of human cells against porcine cells: identification of a porcine antigen homologous to CD58.

BACKGROUND: Human CD58 is an adhesion molecule that interacts with CD2 on lymphocytes. We describe here an antibody that blocks responses of human peripheral blood mononuclear cells (PBMCs) to porcine cells and reacts with a porcine protein with homology to CD58. METHODS: Antibodies were isolated with a screen for inhibition of the human antiporcine response. One of these antibodies was used for immunoaffinity purification of a protein that was identified by molecular weight determination, endoglycosidase sensitivity, and microsequencing analysis as a porcine homologue of CD58. RESULTS: The antigen recognized by this antibody was a cell surface protein of relative molecular mass (Mr)=45,000 containing N-linked carbohydrate chains. Immunoaffinity purification of this protein and microsequencing revealed homology to sheep CD58 as well as sequences that were common to this protein and both sheep and human CD58. The protein was widely distributed on porcine cells, including lymphocytes, endothelial cells, muscle cells, and neuronal cells. This antibody efficiently inhibited lysis of porcine targets by human PBMCs in addition to preventing proliferation of the human PBMCs in response to the porcine cells. CONCLUSIONS: The CD2 interaction with porcine cells is important for the efficient recognition of porcine tissue, and inhibition of the human antiporcine immune response with the antibody is likely to be caused by the disruption of the human CD2 interaction with this porcine homologue of CD58. The antibody may prove to be useful for the blocking of this interaction without interfering with other functions of T cells.

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.

Characterization of a soluble form of CD58 in synovial fluid of patients with rheumatoid arthritis (RA).

Reduced levels of a soluble form of the adhesion receptor and CD2 ligand CD58 (sCD58) were previously described in RA patients. In order to understand the biological significance of this finding we biochemically characterized sCD58 in RA and asked how well sCD58 binds to CD2. sCD58 concentrations were measured in serum and synovial fluid (SF) samples of RA patients by two ELISAs, one detecting domain 1 of CD58 (CD58-D1), and the other one the complete molecule (CD58-D1 + D2). Small amounts of split sCD58-D1 were found in most RA sera, but not SF. In addition, split sCD58-D2 was detected in SF by affinity chromatography, SDS-PAGE, and Western blotting. Gel filtration gave similar peaks at 95-125 kD for RA sera, SF, and normal serum. Binding of SF-sCD58 to the CD2+ Jurkat variant JBB1 or recombinant CD2 was stronger than urinary sCD58 and reached binding of oligomeric recombinant CD58 at low concentrations. In conclusion, sCD58-split products were found in RA sera and SF. At concentrations as they occur in vivo, SF-sCD58 binds to CD2 much more strongly than urinary sCD58. It is conceivable that locally released sCD58 blocks the CD2/CD58 interaction under physiological conditions. Insufficient release of sCD58, e.g. in synovitis, might result in T cell accumulation and perpetuation of inflammation.