Potential allergens of green gram (Vigna radiata L. Millsp) identified as members of cupin superfamily and seed albumin.

BACKGROUND: No systematic study on allergenicity of green gram seed proteins have been performed so far, although incidences of IgE-mediated reaction to green gram seedlings have been reported. OBJECTIVE: We sought to investigate the allergenic potential of green gram, followed by identification and characterization of its relevant allergens using proteomic approaches. METHODS: BALB/c mice were sensitized intraperitoneally with green gram proteins, and levels of specific Igs, Th2 cytokines, histamine, anaphylactic symptoms and histopathological responses were studied. Twelve naso-bronchial allergic patients with a history of sensitization to green gram were selected on the basis of positive skin prick test and elevated specific IgE levels. Green gram allergens were identified and characterized by their ability to endure pepsin, by IgE immunoblot of two-dimensional (2D) gels in combination with mass spectrometry and by bioinformatics approaches. RESULTS: Increased specific IgE, IgG1, Th2 cytokine and histamine levels, high anaphylactic scores and histological changes in lungs and spleen of green gram crude protein extract-treated mice are indicative of its sensitization ability. Four proteins (molecular weights: 52, 50, 30 and 18 kDa) showed pepsin resistance and IgE-binding capability with sensitized human and mice sera. The four proteins tentatively named as Vig r2 (52 kDa, pI 5.7), Vig r3 (50 kDa, pI 5.8), Vig r4 (30 kDa, pI 6.6) and Vig r5 (18 kDa, pI 5.5) showed significant sequence similarity with known allergens of soybean, lentil, pea, lupin, etc. Mass spectrometric analysis identified Vig r2 as 8S globulin ß-isoform precursor, Vig r3 as 8S globulin α-isoform precursor and Vig r4 as seed albumin. CONCLUSION AND CLINICAL RELEVANCE: Green gram seeds contain at least four clinically relevant allergenic proteins, namely Vig r2, Vig r3, Vig r4 and Vig r5 that were capable of inducing strong IgE-mediated reactions. One of the most important steps towards diagnostic and therapeutic approaches to deal effectively with food allergy is continued identification of newer food allergens and their characterization. The significance of this study can be enormous as the data generated may work as basic biology data in developing a green gram species modified genetically that may have reduced allergenicity.

Culture-free detection and enumeration of STEC in water.

Shiga toxin-producing Escherichia coli (STEC) causes worldwide outbreaks of food and waterborne diseases. Rapid identification of causative agents is critical for early intervention in the case of widespread diarrheal epidemics to prevent mortality. In this study, a Molecular-Beacon targeting stx2 gene (highly associated with human illness) was designed to develop a culture-independent real-time PCR assay for detection and quantification of STEC in water samples. The assay could detect lowest 10 genomic equivalent (GE) of the reference strain (E. coli I.T.R.C.-18) per PCR or 100 GE/mL. The presence of 10(6)CFU/mL of non-pathogenic E. coli DH5α has no impact on sensitivity of the assay. The assay could successfully enumerate STEC in surface water (collected from a sewage impacted river) and potable water samples collected from Lucknow city without prior enrichment. The assay will be useful in pre-emptive monitoring of surface/potable waters to prevent waterborne outbreaks caused by STEC.

Chemoprevention of NMU-induced rat mammary carcinoma with the combination of melatonin and 9-cis-retinoic acid.

In experimental trials using the N-nitroso-N-methylurea (NMU)-induced rat mammary tumor model, a significant decrease in tumor incidence (to 5%) was observed in rats treated with melatonin and 9-cis-retinoic acid (9 cRA) compared to controls (55%). Although 9cRA alone decreased tumor incidence to 26%, this response did not reach statistical significance. Tumor incidence was significantly inhibited to 20% in the animals that received melatonin and 9cRA on alternating days. Latency to tumor onset was prolonged in animals receiving either of the combination treatments compared with controls, and tumor multiplicity was also significantly decreased.

Protein-A activates membrane bound multicomponent enzyme complex, NADPH oxidase in human neutrophils.

Protein-A, 42KD cell wall glycoprotein of S. aureus Cowan I enhance mononuclear and polymorphonuclear cell counts in vivo and possesses antitoxic, antitumor, properties. In order to explain the mechanism of its function, the respiratory burst phenomenon in cell and cell free system was studied using lucigenin-dependent chemiluminescence technique. A dose dependent increase in protein A-mediated generation of superoxide radical was observed in resting and PMA stimulated neutrophils. Superoxide dismutase (SOD) was used to confirm the production of superoxide radicals (O2-). To understand the mechanism of protein-A induced O2- generation; NADPH oxidase activity was measured in cell free system using NADPH as a substrate. A significant increase in NADPH oxidase activity was observed in the membrane and post-nuclear supernatant fraction of activated human neutrophils. Cytosolic fraction showed slight enzyme activation. Protein A (SpA)-induced NADPH oxidase activation in the membrane fraction was observed even in the absence of the substrate NADPH. These data indicate that protein A attenuate the NADPH oxidase system to produce O2- radicals.

Glutathione reduces the toxicity associated with antitumor therapy of ascites fluid adsorbed over Staphylococcus aureus Cowan I in tumor bearing mice.

It has been well documented in the literature that the removal of circulatory immune complexes (CICs) from the host circulation leads to the immunopotentiation as well as generation of antitumor responses in a variety of tumors in rats, cats, dogs and human patients. CICs are the major immunosuppressive factors in tumor bearing host. Protein A (PA) has been extensively used for the removal of these CICs from the sera/plasma of tumor bearers, because PA has the ability to bind with the Fc portion of mammalian immunoglobulins. Previously, we reported for the first time a potent antitumor response by the inoculation of cell free Ehrlich's ascites fluid adsorbed in vitro over PA containing Staphylococcus aureus Cowan I (SAC) in Ehrlich's ascites tumor model. However, there was toxicity associated with this form of therapy in terms of early death of treated animals and the depletion of hepatic glutathione pool as well as phase I biotransformation enzyme and increase in glutathione-S-transferase (GST) activities. In the present investigation, tumor bearing animals were treated intraperitoneally (i.p.) on alternate days for 15 days with adsorbed ascites fluid (ad-ASF) (0.1 ml) and glutathione (GSH) (250 mg/kg body weight) separately. We found that GSH supplementation increases mean survival time of GSH and ad-ASF treated mice up to 37.2 days in comparison with 19.9 days for only ad-ASF treated animals, while percent increase in body weight was found to be not affected by the GSH substitution, which remains significantly lower (P < 0.01) in comparison to the tumor control animals. GSH supplementation causes a significant decrease (P < 0.05) of glutathione-S-transferase and restoration of aniline hydroxylase activity (P < 0.05) and aminopyrine-N-demethylase activity. We have also observed that GSH supplementation does not alter the tumor cell viability and tumor cell counts in ad-ASF treated animals in comparison to only ad-ASF treated animals, which indicates that GSH supplementation does not alter the antitumor effect of the therapy. Treatment of Ehrlich's ascites tumor bearing mice with ad-ASF and glutathione increased their survival, but did not reduce the mortality of animals because of tumor.

Ehrlich's ascites fluid adsorbed over protein A containing Staphylococcus aureus Cowan I produces inhibition of tumor growth.

Our earlier studies have shown that removal of various blocking factors from the sera of tumor-bearing animals and humans by adsorption over heat-attenuated and formalin-fixed-Staphylococcus aureus Cowan I (SAC) containing Protein A (PA) causes antitumor immune response. It was also shown that this procedure caused regression of a wide variety of established animal and human tumors. In the present investigation, the therapeutic potential of inoculation of ascites fluid adsorbed in vitro over non-viable SAC containing PA has been demonstrated in Ehrlich' s ascites tumor (EAT) in mouse. The antitumor effect was evident by a significant decrease in body weight (p<0.001) as well as significant reduction in viability of ascites tumor cells (p<0.001) in peritoneal cavity. However, some of the responding animals died earlier than controls, this may be due to the toxicity associated with therapy. The toxic effects were evident in decreased contents of glutathione, and increased activity of glutathione-S-transferase, decreased activity of microsomal enzymes and also in an early death of some of tumor regressed animals. The probable causes of toxicity of the therapy and prospects of reversing these toxic effects are discussed.

Role of cytochrome P-450 in quinalphos toxicity: effect on hepatic and brain antioxidant enzymes in rats.

Quinalphos (QP), an organophosphate pesticide, is used in controlling the pests of a variety of crops. To understand the mechanism of the metabolic basis of the toxicity of QP it was thought pertinent to study the role of cytochrome P-450 (P450) and antioxidant enzyme systems. Albino rats treated orally with QP (0.52 and 1.04 mg/kg body weight) for 60 days showed a significant decrease in body, brain and liver weights. Hepatic P450 content and its dependent monooxygenases, namely aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin-O-deethylase (ERD), were induced to 1.8-2.5-fold, while neuronal AHH was induced to 1.8-fold following QP treatment (1.04 mg/kg) to animals. The hepatic antioxidant defence system, comprising catalase, glutathione (GSH) reductase, superoxide dismutase (SOD) and GSH peroxidase, was also significantly increased in QP-treated animals, while in the brain only catalase was increased and GSH reductase decreased. There was no significant change in hepatic GSH content and lipid peroxide levels in QP treated animals at any dose group in comparison with the control group. Pretreatment of rats with phenobarbitone (PB) or 3-methylcholanthrene (MC) (P450 inducers) prevented mortality caused by the LD50 dose of QP, whereas pretreatment with cobalt chloride (a P450 inhibitor) enhanced the mortality rate to 100% within 3 days. From the above study it can be inferred that the toxicity of QP may be due to the parent compound or its metabolite(s) produced prior to P450 oxidation and that the induction of P450 system by QP may be a defence mechanism.

Dibutyltin dilaurate induced thymic atrophy and modulation of phosphoinositide pathway of cell signalling in thymocytes of rats.

A marked dose dependent reduction in thymus weight and its nucleated cell counts with histological alterations was observed in rats exposed to oral dibutyltin dilaurate (DBTL) for 2 weeks at 2, 4, 8 or 16 mg/kg body weight. The incorporation of [3H]-inositol into all the three major phosphoinositides was drastically reduced in thymocytes in a dose dependent manner. Furthermore, the basal and the mitogen (Con A) stimulated [3H]-inositol phosphates generation was diminished significantly in 8 mg DBTL group. However, in vitro incubation of DBTL with thymocytes failed to evoke any change in phosphoinositide hydrolysis. Similarly, a time and dose dependent inhibition in phosphoinositide synthesis with as high as 80% by 10 microM DBTL was exhibited under in vitro conditions. A 130% and 600% enhancement of protein kinase C (PKC) activity in thymocytes was seen in 4 mg and 8 mg DBTL group, respectively. Addition of DBTL to the cell free assay system of thymocytes resulted in a concentration dependent activation of the enzyme activity. A dose dependent increase in intracellular calcium was also evident when DBTL was added to thymocytes under in vitro conditions. These results are of significance and may bear close relationship to the observed thymic atrophy by DBTL.

Mechanism of enhanced phagocytic response in protein a treated rat macrophages.

Protein A of S. aureus Cowan I has been shown to stimulate macrophage mediated phagocytosis. The present study was undertaken to understand the mechanism involved in the enhancement of phagocytosis of peritoneal macrophages by protein A. The lucigenin and luminol-dependent chemiluminescence (CL) of rat peritoneal macrophages, after incubation with various concentrations of protein A, flow-cytometric studies using DCFH-DA as a fluorescent compound and phagocytosis of sheep red blood cells (SRBCs) by rat peritoneal macrophages were studied. A significant increase in lucigenin dependent CL due to formation of superoxide anions (O2-.) and in luminol dependent CL due to formation of hydrogen peroxide (H2O2) was observed in protein A treated macrophages. A significant increase in intracellular hydrogen peroxide (H2O2) was also observed along with an increase in phagocytosis of SRBCs by protein A treated macrophages. The present findings indicate that protein A helps to increase phagocytosis and triggers respiratory burst of macrophages. Thus, both increased phagocytic response and respiratory burst of macrophages in protein A treated animals may be contributing to the antitumor property of protein A reported earlier.

Induction of immune rejection of tumors by protein A in mice bearing transplantable solid tissue Dalton's lymphoma tumors.

In a transplantable solid tissue Dalton's lymphoma tumor model in mice we have studied the mode of antitumor action of protein A, a well known biological response modifier. Protein A (15 ug) was administered intravenously in normal and solid tissue Dalton's lymphoma tumor bearing mice on day 3 and 7 after tumor inoculation. Incidence of mortality was more in untreated tumor bearing group than that in PA treated tumor bearers. There was a significant decrease (p less than 0.001) in tumor diameter in PA treated group compared to untreated group. Protein A treatment significantly enhanced the delayed type hypersensitivity (p less than 0.01), T-cell number in spleens (p less than 0.001) and lymph nodes (p less than 0.05) as well as phagocytosis (p less than 0.001) of opsonized SRBC by peritoneal macrophages of tumor bearing animals. Apart from the nonspecific immunopotentiation, Protein A also activates natural Killer (NK) cell activity and also splenic lymphocytes mediated killing of autologous tumor targets in a significant (p less than 0.001) manner. These results suggest that PA treatment activates cellular arc of the immune system in general, and macrophage, T cells and NK cells specifically. In the present communication, we have attempted to provide the information that these immune activations appear to be related to antitumor response induced by Protein A.

Protection against 7,12-dimethylbenzanthracene-induced tumour initiation by protein A in mouse skin.

Protein A is an immunostimulating glycoprotein obtained from Staphylococcus aureus Cowan I. Its antitumour activity is proven in various tumour models. Its ability to provide protection against tumour initiation by the chemical carcinogen 7,12-dimethylbenzanthracene (DMBA) has been investigated in the present study using a mouse skin model of two-stage carcinogenesis. Protein A was administered intraperitoneally (1 microgram/animal 20 g body wt.) twice a week for 2 weeks, prior to initiation by DMBA. The promotion was performed by twice weekly applications of 12-O-tetradecanoyl phorbol-13-acetate (TPA) (3 or 5 micrograms/animal in 100 microliters acetone). Protein A provided significant protection to animals from DMBA-induced tumour initiation as was observed by the decrease in cumulative number of tumours, percent of animals developing tumours, number of tumours per animal and rate of tumour growth. Our data indicate that protein A has anticarcinogenic properties.

Protection against carbon-tetrachloride-induced lymphoid organotoxicity in rats by protein A.

In a previous study we described protection of rats from carbon tetrachloride (CCl4)-induced hepatotoxicity by protein A (PA). In the present report we described protection against CCl4-induced lymphoid organotoxicity in rats by PA. Our data indicate that CCl4 administration produced a significant increase in the number of total leukocytes and polymorphs in blood, a significant decrease in leukocyte count in bone marrow, and a significant loss in weight of spleen and adrenals. In the animals receiving PA prior to and after CCl4, these values were found to be near those of controls. Histological examination revealed thymocyte depletion in thymic lobe and degenerative changes of tissue in spleen of CCl4-exposed animals. PA treatment to CCl4-exposed animals exhibited improvement and recovery from the damage caused by CCl4. These observations throw a new light on the field of CCl4 toxicity to lymphoid organs and their protection by PA.

Protein A protects mice from depletion of biotransformation enzymes and mortality induced by Salmonella typhimurium endotoxin.

Changes in hepatic microsomal mixed-function oxidase enzyme levels (aniline hydroxylase, aminopyrine demethylase, glutathione S-transferase), glutathione content, total sulphydryl content, and plasma enzyme levels of aspartate transaminase, alanine transaminase and alkaline phosphatase were studied in male Swiss albino mice exposed to Salmonella typhimurium endotoxin (50-150 micrograms per mouse, LC50 141.82 micrograms). Animals exposed to the same dose of endotoxin but pretreated with protein A of Staphylococcus aureus (5 micrograms/per mouse) protected the animals from both mortality and depletion of biotransformation enzymes.

Protection against carbon tetrachloride-induced hepatotoxicity by protein A.

Protection from carbon tetrachloride (CCL4)-induced hepatotoxicity by protein A was assessed histologically in rats. Carbon tetrachloride exposure produced swollen, vacuolated and necrotic cells in the centrilobular region of the hepatocyte in rats. Animals given protein A prior to and during CCL1 treatment showed a complete absence of hepatic lesions. Our study showed that protein A, a potent immunomodulator, has the potential to reduce liver injury caused by carbon tetrachloride, a known hepatotoxin in the rat.

Effect of protein A on mast cell numbers and macrophage phagocytic activity.

Swiss albino male mice were given Protein A (PA) treatment ip (0, 1.0, 6.0 or 12.0 micrograms per mouse) twice weekly for two weeks. Alterations in white blood cell counts, peritoneal cell number, peritoneal macrophage and mast cell number were found. Macrophage induced phagocytosis of sheep red blood cells was also found enhanced in PA group. The maximum effect was found in 1.0 microgram of PA dose group of mice. From our studies it can be derived that PA induced tumor regression may be due to potentiation of macrophage induced antitumor activity.

Effect of leprosy sera and foetal calf serum (FCS) on the T cell number of peripheral blood of leprosy patients.

Study was conducted in 24 cases of various types of leprosy and 10 healthy controls to find out the effect of various sera on the T cell count of peripheral blood lymphocytes by sheep erythrocyte rosetting method. The percentage of T lymphocytes in lepromatous and tuberculoid cases were significantly lower compared to that in normal healthy controls. All sera except FCS had a stimulatory effect on the number of T cells. The cells incubated for 24 hours in FCS did not show any stimulatory effect on the number of T cells, however, these FCS incubated cells showed a significant elevation in the number of T cells when further incubated in sera either from leprosy cases or from healthy subjects.