Early detection of tuberculous meningitis using one step competitive ELISA.

A one step competitive Enzyme-Linked Immunosorbent assay (ELISA) method was developed to detect mycobacterial antigen in cerebrospinal fluid (CSF) for the diagnosis of tuberculous meningitis and compared with a standard competitive ELISA method. Indigenously prepared soluble extract of Mycobacterium tuberculosis H37 Rv was used as antigen. The study was conducted using CSF of 230 clinically diagnosed cases of tuberculous meningit is and 208 control subjects. A cutoff value of 0.57 ng/ml by the one step ELISA and 0.5 ng/ml by the standard ELISA method were determined. The specificity of both methods were 100% and positivity was 68.26% and 70.43% respectively. A follow up study was conducted in 63 cases at various interval of time after starting anti-tubercular therapy i.e. at 3 weeks (63 cases), 6 weeks (27 cases) and > or = 4-12 months (13 cases). It was observed that antigen levels decreased gradually, but were much above the cutoff range. Indigenously prepared antigen was compared with antigen prepared in other laboratories and standard molecular weight markers using SDS PAGE (Sodium Do-decyl Sulphate Polycrylamide Gel Electrophoresis).

Interferon entry through the blood-brain barrier in glioma and its effect on lipoxygenase activity.

This study evaluates cerebral entry of mouse interferon alpha/beta (MuIFN alpha/beta) or mouse interferon gamma (MuIFN-gamma) following continuous (3 day), subcutaneous infusion of normal or glioma bearing mice. The intracerebral C57BL/6 mouse glioma-26 (G-26) model was used at days 10-14 post tumor implant, the advanced stage of glioma progression as defined by histology and the median survival time (27 +/- 3.8 days). The infusion of horseradish peroxidase (HRP) in vivo at day 10 or 11 post glioma implant showed strong staining in the tumor bed indicating compromised blood-brain barrier (BBB). In addition, histochemistry with Bandeiraea simplicifolia isolectin B4 demonstrated the accumulation and/or activation of macrophage/microglia. The 3 day infusion of mice (day 11-14 post tumor implant) via subcutaneous (sc) osmotic micro-pumps with MuIFN alpha/beta (8x10(5) - 1.7x10(6) international units [IU]/ml) or with recombinant mouse interferon gamma (rMuIFN-gamma) (1x10(6) IU/ml) resulted in a low but detectable (1-5 IU/ml) cerebral level of IFN. The IFN levels in the blood (20-40 IU/ml) and brain, measured by assay of inhibition of viral cytopathic effect (CPE) or ELISA assay for MuIFN-gamma, showed no difference between normal and glioma bearing mice. The lipoxygenase (LO) activity (dioxygenase) of glioma tissue and contralateral control was evaluated in non-treated and MuIFN alpha/beta continuously (3 day) treated mice. The LO activity in glioma tissue was significantly higher (p < 0.05) than the contralateral control in non-treated mice. However, following sc MuIFN alpha/beta infusion the LO activity of glioma decreased to control level.

Interferon effect on glycosaminoglycans in mouse glioma in vitro.

The effect of mouse interferon alpha/beta (MuIFN alpha/beta) on the production of glycosaminoglycans (GAGs) by mouse glioma G-26 in vitro was evaluated. Two GAG species secreted extracellularly by the mouse glioma G-26 were isolated using cellulose acetate electrophoresis. They were identified as hyaluronic acid (HA) and chondroitin sulfate (CS) following enzymatic digestion with enzymes: hyaluronidase and chondroitinase ABC. Further characterization of CS by enzymatic digestion with specific chondroitinases for chondroitin 4-sulfate (CSA) and chondroitin 6-sulfate (CSC), revealed that the isolated CS was neither CSA nor CSC. Therefore, it may be either chondroitin sulfate B (CSB) (dermatan sulfate) or one of the 'chondroitin sulfate isomers' (D-H). The three day incubation of glioma G-26 cells with 8 x 10-8 x 10(4) U/ml of MuIFN alpha/beta resulted in a dose dependent inhibition of cell proliferation measured by 3H-thymidine incorporation and the MTT assay. The significant decrease of the CS (p < 0.008) but not the HA level, (measured densitometrically), was observed following 72 hours (hrs) incubation of G-26 cells with 8 x 10(3) U/ml of MuIFN alpha/beta (IFN treated cells: 0.03 +/- 0.007 integrated optical density (IOD); control cells: 0.07 +/- 0.01 IOD). The decreased CS production may be the underlying cause of IFN mediated inhibition of glioma cell proliferation.

Inhibition of human glioma cell proliferation and glutathione S-transferase by ascorbyl esters and interferon.

The in vitro effect of ascorbyl esters (ascorbyl-stearate [As-S] and -palmitate [As-P]) and interferon (recombinant human interferon-a2b [rHuIFN-a2b]) on human glioma (U-373) cell proliferation, viability and glutathione-S-transferase (GST) activity was studied. The effect of As-S, As-P and rHuIFN-a2b on cell proliferation and viability was evaluated by [3H] Thymidine incorporation and colorimetric MTT assays, respectively. Incubation of glioma cells with As-S, As-P or rHuIFN-a2b for 24 h resulted in a dose dependent inhibition of cell proliferation (IC50 = 68.0 microM As-S, 86.0 microM As-P and 47.3 Units/ml rHuIFN-a2b), and moderate decrease of cell viability. It was found that As-S was a more efficient inhibitor of cell proliferation, viability and GST activity than As-P. GST from U-373 cells was purified. The activity of purified GST towards 1-chloro-2,4-dinitrobenzene (CDNB) was inhibited in a dose dependent manner by ascorbyl esters (I-50 = 27.5 microM As-S and 56.0 microM As-P) but not by rHuIFN-a2b. GST activity of cytosol isolated from U-373 cells which were previously treated with As-S (150 microM) or rHuIFN-a2b (150 units/ml) for 0, 2, 5, 10, 20 and 30 min was sharply decreased during 5 to 10 min of treatment and increased at longer durations of treatment.

Inhibition of cell proliferation and glutathione S-transferase by ascorbyl esters and interferon in mouse glioma.

Mouse glioma-26 (G-26) cell line established in this laboratory was used in the study. The in vitro effect of ascorbyl esters, viz., ascorbyl-palmitate (As-P), -stearate (As-S) and mouse interferon-alpha/beta (MulFN-alpha/beta) on the glioma cell viability, proliferation and glutathione S-transferase (GST) activity was investigated. Cell viability and proliferation were examined by colorimetric MTT assay and [3H]-thymidine incorporation, respectively. Incubation (24h) of G-26 cells with As-S, As-P or MulFN-alpha/beta, resulted in a dose dependent decrease in cell viability (IC50 = 125 microM As-S; 175 microM As-P and 3.6 x 10(4) U/ml MulFN-alpha/beta) and proliferation (IC50 = 157 microM As-S; 185 microM As-P and 3.6 x 10(4) U/ml MulFN-alpha/beta). A combined exposure to 175 microM As-S and 800 U/ml of MulFN-alpha/beta resulted in a greater than an additive effect on cell viability and proliferation. The inhibition of cell proliferation/viability by interferon was species specific and was observed only with homologous MulFN-alpha/beta, but not with human interferon-alpha lymphoblastoid or human interferon-beta. Ascorbyl esters inhibited cytosolic GST activity (1-50 = 15.0 microM As-S and 28.5 microM As-P) towards 1-chloro-2,4-dinitrobenzene in a dose dependent manner. The apparent Ki values for affinity purified GST, deduced from Dixon plots were 0.95 microM and 2.0 microM for As-S and As-P, respectively. Significant inhibition of GST was also observed in the cytosol isolated from G-26 cells exposed to 300 microM As-S or 800 U/ml MulFN-alpha/beta.

Dioxygenase and hydroperoxidase activities of rat brain cytosolic lipoxygenase.

Dioxygenase and hydroperoxidase activities of dialyzed rat brain cytosolic lipoxygenase were studied using linoleic acid as the substrate. The formation and utilization of linoleic acid hydroperoxide was monitored spectrophotometrically at 234 nm. Dioxygenase activity was dependent upon the concentration of linoleic acid and enzyme. The optimum assay conditions necessary for maximal dioxygenase activity included the 0.5 mM linoleic acid, cytosol (100 micrograms of protein) as the enzyme source and pH 7.4. All the classical inhibitors of lipoxygenase, nordihydroguaiaretic acid, phenidone and 5,8,11-eicosatriynoic acid significantly inhibited dioxygenase activity in a dose dependent manner. A significant hydroperoxidase activity was observed towards xenobiotic substrates viz. benzidine, guaiacol, tetramethylphenylenediamine, thiobenzamide in the presence of linoleic acid. NDGA at 1 microM concentration inhibited benzidine oxidation (2.45 nmol product formed/min/mg protein) by 75%, while Phenidone at 0.125 microM and ETI at 20 microM inhibited benzidine oxidation by 30% and 25%, respectively.

Aldrin epoxidation. Catalytic potential of lipoxygenase coupled with linoleic acid oxidation.

Epoxidation of aldrin was studied using highly purified soybean lipoxygenase in the presence of linoleic acid. Dieldrin, the primary stable reaction product, was quantified by electron-capture gas chromatography. The oxidation of aldrin to dieldrin was dependent on the concentration of linoleic acid, aldrin, and enzyme. The epoxidation was linear with time and exhibited a pH optimum of 7.4. The optimal conditions to observe maximum enzyme velocity included the presence of 0.25 mM linoleic acid, 200 microM aldrin, and 20 nM enzyme. Lipoxygenase inhibitors nordihydroguaiaretic acid, phenidone, 5,8,11-eicosatriynoic acid, and 5,8,11,14-eicosatetraynoic acid significantly inhibited epoxidation in a dose-dependent manner. Catalytic potential of lipoxygenase as expressed in terms of its turnover numbers was approximately 4.0 nmol/min/nmol of enzyme, and it appears that lipoxygenase is up to 20 times a better catalyst of aldrin epoxidation than cytochrome P-450. These results suggest that lipoxygenase, which is widely distributed in plants and animals, may represent yet another important pathway for epoxidation of aldrin.

Role of lipoxygenase in xenobiotic metabolism: sulfoxidation of thiobenzamide by purified soybean lipoxygenase.

Earlier this laboratory recognized lipoxygenase catalyzed reactions as a novel pathway for xenobiotic metabolism. To further explore the spectrum of reactions catalyzed by lipoxygenase, sulfoxidation of thiobenzamide was studied. Purified soybean lipoxygenase was found to oxidize thiobenzamide to thiobenzamide sulfoxide in the presence of linoleic acid at a rate of 241 nmoles/min/nmole enzyme. The reaction was dependent upon enzyme, pH, thiobenzamide and linoleic acid concentration. Other polyunsaturated fatty acids namely arachidonic acid, cis, 11, 14-eicosadienoic acid and linolenic acid also supported the sulfoxidation reaction. Nordihydroguaiaretic acid and phenidone, the classical inhibitors of lipoxygenase, significantly blocked the sulfoxidation of thiobenzamide.