T11TS immunotherapy augments microglial and lymphocyte protective immune responses against Cryptococcus neoformans in the brain.

Cryptococcus neoformans, the encapsulated yeast acquired through inhalation, remains localized in lungs, but harbours the CNS in immunocompromised individuals. Several treatment regimes have failed combating this disease totally, but long-term usage of drugs leads to organ damage. As T11-target structure (T11TS) has documented profound immune potentiation, we aimed to investigate the role of microglia, pivotal immune cells of brain in ameliorating cryptococcosis, with T11TS immunotherapy. Murine model with C neoformans infection was prepared by intraperitoneal injection and the brains of rats examined 7 days post-infections for histopathology by PAS and Alcian blue staining corroborated with organ fungal burden evidencing restorative T11TS action on Cryptococcal meningitis. Immunotherapy with three doses of T11TS, a CD2 ligand, in C neoformans infected rats, upregulates toll-like receptors 2, -4 and -9 of microglia, indicating increased phagocytosis of the fungus. Flowcytometric analysis revealed increased numbers of T11TS treated brain infiltrating CD4+ and CD8+ T-lymphocytes along with increased MHC I and MHC II on microglia, activating the infiltrating lymphocytes aiding the killing mechanism. Present study also indicated that T11TS increased production of Th1 inflammatory cytokines conducive to fungal elimination while the inhibitory Th2 cytokines were dampened. This preclinical study is first of its kind to show that T11TS effected profound immune stimulation of microglial activity of C neoformans infected rats eradicating residual fungal burden from the brain and can be a useful therapeutic strategy in fighting against this deadly disease.

T11TS inhibits glioma angiogenesis by modulation of MMPs, TIMPs, with related integrin αv and TGF-ß1 expressions.

During glioma development, angiogenesis plays a crucial role in growth and vascularization of primary brain tumors. T11 target structure (T11TS), a bioactive molecule, has been documented as an anti-neoplastic agent in glioma-induced rats and also in human glioma in vitro. This novel molecule induces apoptosis of tumor cells by way of immune potentiation and impairs the glioma cell cycle, but its role in glioma angiogenesis has not been worked out in detail. Matrix metalloproteinases (MMPs) are enzymes promoting tumor angiogenesis by enzymatically remodeling the extracellular matrix and altering surface protein expression such as integrin αv and the matrix-bound proteins like TGF-ß1. The present study was formulated to assess the efficacy of T11TS in the modulations of MMP-2 and -9 and their endogenous inhibitors (TIMP-1 and TIMP-2) as well as modulations of integrin αv and TGF-ß1 in glioma-induced rats and also on the phenotypic markers of endothelial cells (CD31 and CD34). The parameters used were zymography, western blot, and flow cytometric analyses. It was observed that T11TS administration significantly downregulates the expression of matrix metalloproteinase-2 and -9 along with its ligand integrin αv and upregulates TIMP-1 and TIMP-2. In situ immunofluorescence and FACS results revealed that T11TS administration decreased the expression of the phenotypic markers (CD31/PECAM1, CD34), inhibiting the cell grip and also downregulating TGF-ß1 expression (ELISA) from microglia cells in the glioma microenvironment. These results suggest that T11TS suppresses the expression of positive angiogenic growth factors and potentiates the expression of negative regulators in glioma-associated endothelial cells (ECs), resulting in an anti-angiogenic effect on glioma-induced angiogenesis.

The novel immunotherapeutic molecule T11TS modulates glioma-induced changes of key components of the immunological synapse in favor of T cell activation and glioma abrogation.

T-cell-mediated immune responses are typically low in conditions of malignant glioma which has been known to cause marked immunesuppression and dysregulate major T-cell signaling molecules. Thus, T-cell-based immunotherapies are currently in vogue in the treatment of malignant glioma. The novel glycopeptide, T11TS/S-LFA-3/S-CD58 has previously been shown by our group to be highly efficacious in glioma abrogation in in vivo and in vitro conditions. This glycopeptide ligands to the costimulatory CD2 molecule on T-cells, causing profound immune stimulation leading to glioma abrogation, suggesting probable involvement of T11TS in modulation of the T-cell signaling pathway. The present study offers a multi-targeted approach towards repair of some of the key components of the immunological synapse at the T-cell-APC interface and is therefore the first of its kind to offer a holistic model of restoration of immunological synapse components so as to trigger T-cells towards activation against glioma. The study thus indicates that the totally dysregulated molecular events at the immunological synapse in glioma are restored back to normal levels with the administration of T11TS, which finally culminates in glioma abrogation. The present study thus delineates an important T-cell signaling approach whereby T11TS acts as an anti-neoplastic agent, thus helping to chart out newer avenues in the fight against gliomas.

T11TS impedes glioma angiogenesis by inhibiting VEGF signaling and pro-survival PI3K/Akt/eNOS pathway with concomitant upregulation of PTEN in brain endothelial cells.

The crucial role of angiogenesis in malignant glioma progression makes it a potential target of therapeutic intervention in glioma. Previous studies from our lab showed that sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) has potent anti-neoplastic and immune stimulatory effects in rodent glioma model. In the present study we investigated the anti-angiogenic potential of T11TS and deciphered the underlying molecular mechanism of its anti-angiogenic action in malignant glioma. Vascular endothelial growth factor (VEGF) signaling is crucial for initiating tumor angiogenic responses. The present preclinical study was designed to evaluate the effect of T11TS therapy on VEGF and VEGFR-2 expression in glioma associated brain endothelial cells and to determine the effects of in vivo T11TS administration on expression of PTEN and downstream pro-survival PI3K/Akt/eNOS pathway proteins in glioma associated brain endothelial cells. T11TS therapy in rodent glioma model significantly downregulated expression of VEGF along with its receptor VEGFR-2 and inhibited the expression of pro-survival PI3K/Akt/eNOS proteins in glioma associated brain endothelial cells. Furthermore, T11TS therapy in glioma induced rats significantly upregulated brain endothelial cell PTEN expression, inhibited eNOS phosphorylation and production of nitric oxide in glioma associated brain endothelial cells. Taken together our findings suggest that T11TS can be introduced as an effective angiogenesis inhibitor in human glioma as T11TS targets multiple levels of angiogenic signaling cascade impeding glioma neovascularisation.

Regulation of key molecules of immunological synapse by T11TS immunotherapy abrogates Cryptococcus neoformans infection in rats.

Cryptococcus neoformans infects and disseminates in hosts with diminished T cell responses. The immunomodulator T11TS (T11 target structure) had profound potential in glioma as well as C. neoformans infected model for disease amelioration. It is been established by our group that T11TS potentiates Calcineurin-NFAT pathway in T cells of C. neoformans infected rats. We investigated the upstream Immunological Synapse (IS) molecules that are vital for the foundation of initial signals for downstream signaling, differentiation and proliferation in T cells. Improved RANTES level in the T11TS treated groups suggests potential recruitment of T cells. Down-regulation of TCRαß, CD3ζ, CD2, CD45 and CD28 molecules by cryptococcus were boosted after T11TS therapy. Heightened expression of inhibitory molecule CTLA-4 in cryptococcosis was dampened by T11TS. The decline of MHC I, MHC II and CD80 expression on macrophages by C. neoformans were enhanced by T11TS. The dampening of positive regulators and upsurge of negative regulators of the IS during cryptococcosis was reversed with T11TS therapy resulting in enhanced clearance of fungus from the lungs as envisaged by our histological studies. This preclinical study with T11TS opens a new prospect for potential immunotherapeutic intervention against the devastating C. neoformans infection with positive aspect for the long-term solution and a safer immunotherapeutic regimen.

Development of cholesterol biosensor based on immobilized cholesterol esterase and cholesterol oxidase on oxygen electrode for the determination of total cholesterol in food samples.

The development of a cholesterol biosensor by co-immobilization of cholesterol esterase (ChEt) and cholesterol oxidase (ChOX) on oxygen electrode is described. The electrode consists of gold cathode and Ag/AgCl anode. The enzymes were immobilized by cross-linking with glutaraldehyde and Bovine Serum Albumin (BSA). The immobilized enzymatic membrane was attached to the tip of the electrode by a push cap system. The optimum pH and temperature of the sensor was determined, these are 6 and 25 degrees C respectively. The developed sensor was calibrated from 1-75 mg/dl of cholesterol palmiate and found linear in the range of 2-50 mg/dL. The calibration curve was drawn with V(i) (ppm/min)(initial velocity) vs different concentrations of cholesterol palmiate (mg/dL). The application of the sensor to determine the total cholesterol in different real food samples such as egg, meat was investigated. The immobilized enzymatic layer can be reused over 30 times and the stability of the enzymatic layer was studied up to 9 weeks.

A biosensor based on co-immobilized L-glutamate oxidase and L-glutamate dehydrogenase for analysis of monosodium glutamate in food.

A monosodium glutamate (MSG) biosensor made by co-immobilized L-glutamate oxidase (L-GLOD) and L-glutamate dehydrogenase (L-GLDH) as the bio-component based on substrate recycling for highly sensitive MSG or L-glutamate determination, has been developed. Regeneration of MSG by substrate recycling provided an amplification of the sensor response. Higher signal amplification was found in the presence of ammonium ion. The sensor was standardized to determine MSG in the range of 0.02-3.0 mg/L. Linearity was obtained from 0.02 to 1.2 mg/L in presence of ammonium ion (10 mM) and NADPH (reduced nicotinamide adenine dinucleotide phosphate) (2 mM), but in absence of L-GLDH, the detection limit of MSG is confined to 0.1 mg/L. The apparent Km for MSG with L-GLOD-L-GLDH coupled reaction was 0.4451 mM but 1.9222 mM when only L-GLOD was immobilized. Cross linking with glutaraldehyde in the presence of bovine serum albumin (BSA) as a spacer molecule has been used for the method of immobilization. The response time of the sensor was 2 min. The optimum pH and temperature of the biosensor has been determined as 7+/-2 and 25+/-2 degrees C, respectively. The enzyme immobilized on the membrane was used for over 50 measurements. The standard error of the sample measurement was 4-5%. The activity of the enzyme-immobilized membrane was tested over a period of 60 days.

Development of biosensor based on immobilized L-glutamate oxidase for determination of monosodium glutamate in food.

A monosodium glutamate (MSG) biosensor with immobilized L-glutamate oxidase (L-GLOD) has been developed and studied for analysis of MSG in sauces, soup etc. The immobilized enzymatic membrane was attached with oxygen electrode with a push cap system. The detection limit of the sensor was 1 mg/dl and the standard curve was found to be linear upto 20 mg/dl. Response time of the sensor was 2 min. Cross-linking with glutaraldehyde in presence of Bovine Serum Albumin (BSA) as a spacer molecule has been used for immobilization. Optimization of the sensor was done with an increase in L-GLOD concentration (6.3-31.5 IU) and also with increase in loading volume of enzyme solution (5-20 microl). Optimization of pH and temperature was also studied. The permeability of O2 through different membrane was studied with and without immobilized L-GLOD. The enzymatic membrane was used for over 20 measurements and stability of the membrane was observed.

Development of an amperometric hypoxanthine biosensor for determination of hypoxanthine in fish and meat tissue.

A hypoxanthine (Hx) biosensor based on immobilized xanthine oxidase (XO) as the bio-component was developed and studied for the rapid analysis of fish (sweet water and marine) and goat meat samples. The biosensor was standardized for the determination of Hx in the range of 0.05 to 2 mM. Crosslinking with glutaraldehyde in presence of BSA as a spacer molecule was used for the method of immobilization. One layer of gelatin (10%) was applied over the immobilized enzyme layer to reduce the leaching out of enzyme from the membrane (cellulose acetate) matrix. The optimum pH of the immobilized system was determined to be 8.5 at 25 degrees C instead 7.0-7.2 for free enzyme system. Km and Vmax values were determined for the immobilized system. The developed sensor was applied to determine the amount of Hx present in fish and meat over a period of time. The stability of the enzyme immobilized membrane was also tested over a period of 30 days.

Isolation and characterization of Pseudomonas sp. STM 997 from soil sample having potentiality to degrade 3,6-dimethyl-1-keto-1,2,3,4-tetrahydrocarbazole: a novel approach.

A pure colony of a bacterium from contaminated soil was isolated by exploiting 3,6-dimethyl-1-keto-1,2,3,4-tetrahydrocarbazole, a novel carbazole derivative, having indole moiety as well as 3-methyl functionality both in aromatic and hydro-aromatic moiety, as a sole source of carbon and energy. Taxonomical studies, biochemical analysis, and 16S rDNA sequence analysis indicated that the isolated strain has close similarity with Pseudomonas sp. Thin-layer chromatography followed by HPLC and mass spectroscopic study indicates that the isolated Pseudomonas sp. STM 997 degrades 3,6-dimethyl-1-keto-1,2,3,4-tetrahydrocarbazole, and this strain may be useful in the bioremediation of environments contaminated by the compounds containing carbazole moiety with methyl substituents at various reactive sites. This study also provides an evidence in favor of the suggested biodegradation of 3-methylcarbazole to carbazole in plants.

Therapeutic profile of T11TS vs. T11TS+MiADMSA: a hunt for a more effective therapeutic regimen for arsenic exposure.

Arsenic exposure is a serious health hazard worldwide. We have previously established that it may result in immune suppression by upregulating Th2 cytokines while downregulating Th1 cytokines and causing lymphocytic death. Treatment modalities for arsenic poisoning have mainly been restricted to the use of chelating agents in the past. Only recently have combination therapies using a chelating agent in conjunction with other compounds such as anti-oxidants, micronutrients and various plant products, been introduced. In the present study, we used T11TS, a novel immune potentiating glycopeptide alone and in combination with the sulfhydryl-containing chelator, mono-iso-amyl-dimarcaptosuccinic acid (MiADMSA) as a therapeutic regimen to combat arsenic toxicity in a mouse model. Results indicated that Th1 cytokines such as TNF-α, IFNγ, IL12 and the Th2 cytokines such as IL4, IL6, IL10 which were respectively downregulated and upregulated following arsenic induction were more efficiently restored to their near normal levels by T11TS alone in comparison with the combined regimen. Similar results were obtained with the apoptotic proteins studied, FasL, BAX, BCL2 and the caspases 3, 8 and 9, where again T11TS proved more potent than in combination with MiADMSA in preventing lymphocyte death. The results thus indicate that T11TS alone is more efficient in immune re-establishment after arsenic exposureas compared to combination therapy with T11TS+MiADMSA.