Induction of immune tolerance to caseins and whey proteins by oral intubation in mouse allergy model.

This study was designed to evaluate the effect of oral tolerance of caseins (CSN) and whey proteins (WP) in alleviating the allergic response to cow's milk proteins in Swiss albino mice raised on a milk protein-free diet. Oral tolerance was induced by feeding mice with 20 mg of CSN or WP once in a day for 4 days consecutively before immunization with respective protein by intraperitoneal (i.p.) injections (20 µg 200 per µl of PBS) using 2% of alum Al(OH)3 as adjuvant. Three weeks later, oral tolerance induction was analysed in humoral and cellular compartments of CSN- and WP-fed versus saline-fed control mice groups by measuring seric and intestinal antibody responses, mRNA abundance in splenic tissue and cytokine secretion patterns. The specific serum immunoglobulin-E (IgE) levels were significantly suppressed (p < 0.05), while sIgA was enhanced in these groups when compared with their respective saline-fed mice. Moreover, the mRNA levels of interferon-γ (IFN-γ) and interleukin-4 (IL-4) in both CSN- and WP-tolerized mice were found to be significantly decreased, while the abundance of interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß) was increased significantly, as compared to respective control groups. Finally, cytokine profiles indicated a reciprocal decrease in IL-4 and IFN-γ versus an increase in IL-10 secretions in supernatants of cultured splenocytes of tolerized mice. Taken together, these results clearly showed that oral administration of cows' milk caseins and whey proteins can induce significant hyposensitization in mice, with the participation of suppressor cytokines.

Effect of thermal processing of cow and buffalo milk on the allergenic response to caseins and whey proteins in mice.

BACKGROUND: Heat treatment is the most common method for reducing pathogen load, but it remains controversial in reducing the incidence of hyperimmune reactions. The aim of this study was to compare the allergenicity of caseins (CSN) and whey proteins (WP) of thermally processed cow and buffalo milk in a mouse model. Swiss albino mice were sensitised by intraperitoneal injections (administered in three doses at weekly intervals) of CSN or WP from cow or buffalo milk for the evaluation of humoral response and splenocyte stimulation index. RESULTS: After 3 weeks of intraperitoneal stimulation of mice with milk proteins, the sterilised milk protein group displayed significantly lowered (P ≤ 0.05) serum IgG and IgE levels, while considerably increased cow milk protein-specific responses (IgE) were shown by proteins of pasteurised milk compared with those of raw milk. The stimulation index of splenocytes induced by CSN or WP of boiled and sterilised milk was also lower (P ≤ 0.05) than that of raw milk of both cow and buffalo. CONCLUSION: The experiment showed that boiling and sterilisation of cow and buffalo milk clearly affect the allergenicity by decreasing the humoral and cell-mediated responses in mice. All results indicated that CSN and WP of sterilised milk are less allergenic than those of raw milk in mice.

Probiotics: Potent Immunomodulatory Tool Against Allergy.

Probiotics are defined as live microbial food ingredients that produce several beneficial effects to human health. Probiotic bacteria have been mostly investigated in the prevention of and treatment for different gastrointestinal diseases and allergies. It is not fully clear how probiotics exert their beneficial effects on health, but one of the most probable mechanisms of action is the modulation of immune responses via the mucosal immune system of the gut. Commensal bacteria in the gastrointestinal tract play an integral role in both innate and humoral immunity. It is well established that this protective role can be maintained or modulated by the ingestion of probiotics. More recently, it has been shown that specific probiotic strains can influence the secretion of cytokines to help direct naïve T-helper cells toward either a Th1-dominant, cell-mediated immune response or toward a Th2-dominant, humoral immune response. This paper will review current knowledge of the Th1/Th2 model of humoral immunity as well as introduce how strain-specific probiotics can be used therapeutically to help balance this immune response and therefore help prevent allergy.

Synergistic effect of synthetic conjugated linoleic acid & non fat milk on fat deposition & lipid metabolism in mice.

BACKGROUND & OBJECTIVES: Conjugated linoleic acid (CLA) reduces fat deposition in the body, but the mechanism of action is not clear. The present study was conducted to investigate the effects of CLA on body fat metabolism. Since milk fat is the best natural source of dietary CLA, intervention of non-fat milk constituents on CLA treatment was also investigated. METHODS: Diets containing CLA (1%) with or without skim milk powder (SMP) was fed to male Swiss albino mice for 60 days. Adipose depots weight, faecal fat and the activities of selected enzymes of lipid metabolism were determined. RESULTS: The mice on CLA and CLA+SMP diets gained weight similar to those on control diet, despite higher feed intake in the former two groups. Total fat pad mass was significantly (P<0.05) less in CLA group than in control group, and inclusion of SMP in the diet enhanced the fat reducing effect of CLA. Adiposity index was also less on CLA and CLA+SMP diets than on control diet, and CLA+SMP was more efficacious in reducing adiposity index. The weight of liver and spleen was increased by CLA, and this effect was eliminated by inclusion of SMP in the diet. The fatty acid synthase (FAS) activity in liver and retroperitoneal adipose tissue decreased substantially on CLA and CLA+SMP diets compared to that on control diet. INTERPRETATION & CONCLUSIONS: Our preliminary data show that dietary CLA reduces body fat mass by decreasing fatty acid biosynthesis, and the effect is enhanced by inclusion of SMP in the diet.

Dark neurons in the ageing cerebellum: their mode of formation and effect of Maharishi Amrit Kalash.

Dark neurons are considered a manifestation of neuronal injury and although they cover various grades of damage their mode of formation is not yet clear. Age-dependent alterations in a dark purkinje neuronal population of guinea pigs (10 months and 32 months old) and rats (3 months, 6 months, 12 months, 15 months and 28 months) were studied. Light microscopical and electron microscopical observations revealed a significant increase (P < 0.05) in the number of dark purkinje neurons with age in both the guinea pigs and rats. Extraction of lipids from the cerebellum sections before processing for histochemical reaction resulted in a reduction of the dark neuronal population. In an other set of experiments, significant age-dependent increase in the cathepsin-D activity and lipid peroxidation was documented in the guinea pig cerebellum. Treatment of guinea pigs with Maharishi Amrit Kalash (MAK) (500 mg/kg body wt/day, for two months) significantly inhibited (P < 0.05) the activity of cathepsin-D and lipid peroxidation, and decreased the number of dark neurons. These findings suggest that the number of dark neurons increases with age and MAK prevents the conversion of light to dark purkinje neurons due to its inhibitory effects on cathepsin-D activity and antioxidant properties. We suggest that the conformational changes in the normal protein structure due to higher proteolytic activity and peroxidation of lipid in the aging cerebellum endangers a redundant capability for various staining agents and the Osimic acid molecules to react with proteins, lipids and other molecules, leading to an intensified cyto- and karyoplasms electron density.

Effect of Maharishi Amrit Kalash on age dependent variations in mitochondrial antioxidant enzymes, lipid peroxidation and mitochondrial population in different regions of the central nervous system of guinea-pigs.

Age related changes in the mitochondria of different regions of the CNS of two age groups of guinea-pigs (10 months and 32 months) were studied. The activities of glutathione peroxidase (GPx) and glutathione reductase (GRd) decreased significantly (p <0.05) with age in the mitochondrial fractions of cerebral cortex, hypothalamus, cerebellum and spinal cord. A significant (p <0.05) age related decrease in mitochondrial numerical density was observed in all regions studied. Electron microscopic observations revealed various degenerative changes in the mitochondria with age. Treatment of the animals with the Ayurvedic herbal mixture "Maharishi Amrit Kalash" (MAK), 500 mg/kg body wt. daily for 2 months, significantly induced the activity of antioxidant enzymes, and also reversed the pathological changes to a considerable extent. MAK increased the activity of GPx significantly only in the 32 month-old animals. This shows the specificity of the action of MAK.

Effect of Maharishi Amrit Kalash an ayurvedic herbal mixture on lipid peroxidation and neuronal lipofuscin accumulation in ageing guinea pig brain.

The effects of ayurvedic herbal mixture Maharishi Amrit Kalash(MAK) were studied on brain lipid peroxidation, oxygen consumption, and lipofuscin accumulation in 10 months and 32 months old guinea pigs. Brain regions studied were cerebral cortex, hypothalamus, cerebellum and spinal cord. Parameters assessed were lipid peroxidation, oxygen consumption, and lipofuscin accumulation. The endogenous lipid peroxide was found to be increased significantly (P < 0.05) in the 32-month-old animals. Neuronal lipofuscin accumulation in the neurons of cerebral motor cortex, cerebellum and cervical spinal cord was increased (P < 0.05) in the older animals. Oxygen consumption was found to be decreased significantly(P < 0.05) in the 32-month old guinea pigs. Treatment with MAK at a dose of 500 mg/kg body weight daily for two months reduced the lipid peroxidation and lipofuscin pigment accumulation significantly in brain regions and it also helped in restoring the normal oxygen consumption in the older animals. This indicates antioxidant properties of MAK.

Maharishi Amrit Kalash, an ayurvedic medicinal preparation, enhances cholinergic enzymes in aged guinea pig brain.

The effect of orally fed Maharishi Amrit Kalash was examined on the activities of cholinergic enzymes in the guinea pig brain. The activity of the cholinergic enzymes viz. choline-acetyltransferase and acetylcholinesterase enzymes was found to be reduced significantly (P<0.05) in the various regions of CNS of the aged guinea pigs. Oral administration of MAK(500 mg/kg body weight daily) for 2 months significantly increased (P<0.05) the activity of choline acetyltransferase and acetylcholinesterase in the older animals. The present study indicates that this food supplement can be helpful in alleviating the cholinergic deficits in the old age.

Heterogeneity of transport systems for L-glutamine in mouse mammary gland.

The characteristics of the transport systems of L-glutamine in lactating mouse mammary gland have been studied. L-glutamine uptake was mediated by three Na+-dependent and one Na+-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na+-dependent uptake exhibited the usual characteristics of system A. The other two Na+-dependent systems, which we have named BCI(-)-dependent and BCl(-)-independent, are the new systems identified. These are broad specificity systems and were discriminated on the basis of inhibition analysis, Cl- dependency and the effect of preloading mammary tissue with amino acids. While L-aspargine inhibited the uptake of L-glutamine via both these broad specificity systems, L-homoserine inhibited the uptake of L-glutamine via only BCl(-)-dependent system. The uptake of L-glutamine via the BCl(-)-independent system was upregulated by preloading mammary tissue with L-serine, while BCl(-)-dependent system was unaffected. The Na+-independent uptake of L-glutamine was inhibited by 2-aminobicyclo-(2,2,1)heptane carboxylic acid and other neutral amino acids, and identified as the system L.

Age-dependent variations in mitochondrial and cytosolic antioxidant enzymes and lipid peroxidation in different regions of central nervous system of guinea pigs.

The age-related changes in the activities of antioxidant enzymes of mitochondrial and cytosolic fractions were measured in different regions of the central nervous system (CNS) in 10 and 32 months old guinea pigs. In old animals, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were reduced (p < 0.05) in all the regions of CNS studied but catalase (CAT) declined significantly only in the cerebral cortex, hypothalamus and cerebellum. Glutathione reductase (GRd) activity declined in cerebral cortex and hypothalamus in the cytosolic fractions and only in cerebellum in the mitochondrial fraction. It is concluded that age-related decline in the activities of antioxidant enzymes is both region and enzyme specific. The endogenous lipid peroxide was found to be significantly higher (p < 0.05) in the 32 month old animals whereas, lipid peroxidation after incubating the tissue homogenate in air was found to be lower (p < 0.05). The in vitro mitochondrial lipid peroxidation decreased with age. The results indicate that accumulation of lipid peroxides takes place with ageing but the susceptibility of lipid peroxidation decreases in the older animals.

Heterogeneity of cationic amino acid transport systems in mouse mammary gland and their regulation by lactogenic hormones.

The mechanism of cationic amino acid transport in lactating mouse mammary gland was investigated. Two Na(+)-independent systems of arginine transport were discriminated on the basis of their sensitivity to leucine. The leucine-sensitive uptake of arginine (Km 0.4 mM) was through a broad specificity system that interacted with both cationic and neutral amino acids, and was inhibited by preloading mammary tissue with neutral amino acids. The leucine-insensitive uptake was identified as the y+ system (Km 0.76 mM). Preloading mammary tissue with cationic amino acids increased the uptake of arginine by the y+ system. Decreasing the pH of the external medium to 6.0 suppressed the y+ system-mediated uptake by approximately 25%, whereas the broad specificity system remained unaffected. Lactogenic hormones upregulated the y+ system-mediated uptake of arginine in pregnant mouse mammary tissue cultured in vitro, although the broad specificity system remained unaffected. The y+ system-mediated uptake increased 2-fold with insulin alone and 4-fold with the combination of insulin, cortisol and prolactin.

Mechanism of glycine transport in mouse mammary tissue.

The mechanism of glycine transport in lactating mouse mammary gland was investigated. Three Na+-dependent systems of glycine transport, distinguished on the basis of their ionic requirement and sensitivity to 2-(methylamino)isobutyric acid (MeAIB), were A (Na+-dependent, MeAIB-sensitive); (Na++Cl-)-dependent, MeAIB-insensitive; and Na+-dependent, Cl--independent, MeAIB-insensitive. These systems were further distinguished on the basis of inhibition analysis and sensitivity to pH of the extracellular medium and preloading mammary tissue with amino acids. The uptake of glycine via the A system (Km 0.53 mM) was inhibited by preloading mammary tissue with alanine, while glycine uptake mediated by the (Na++Cl-)-dependent, MeAIB-insensitive system (Km 0.47 mM) was downregulated by preloading mammary tissue with all amino acids (alanine, sarcosine and histidine) tested. Treatment of mammary tissue with N-ethylmaleimide inhibited the uptake of glycine via both these systems. Decreasing the pH of the extracellular medium inhibited the uptake of glycine via the A system but not the (Na++Cl-)-dependent, MeAIB-insensitive system. On the basis of ionic requirement, system A appears to comprise two components, one dependent on Na+ plus Cl- and the other on Na+ alone. Insulin upregulated the A system-mediated uptake of glycine in pregnant mouse mammary tissue cultured in vitro, while the (Na++Cl-)-dependent, MeAIB-insensitive system remained unaffected.

Characterization of anionic amino acid transport systems in mouse mammary gland.

L-glutamate was transported into mammary tissue via Na(+)-dependent system XAG- that strongly interacted with both D- and L-isomers of aspartate but only with L-isomer of glutamate. Replacement of Cl- by gluconate from the extracellular medium did not affect the uptake of L-glutamate. Although neutral amino acids weakly inhibited the uptake of L-glutamate, there was no evidence for the heterogeneity of anionic amino acid transport system. The XAG- system was inhibited by sulfhydryl group blocking reagent N-ethylmalemide. Low pH (6) partially inhibited the uptake by L-glutamate by mammary tissue. Prior loading of mammary tissue with L-glutamate slightly down regulated its uptake. Culturing pregnant mouse mammary tissue explants in vitro in the presence of lactogenic hormones (insulin plus cortisol plus prolactin) did not affect appreciably the uptake of L-glutamate.

Characteristics of transport systems of L-alanine in mouse mammary gland and their regulation by lactogenic hormones: evidence for two broad spectrum systems.

The characteristics of the transport systems of L-alanine in lactating mouse mammary gland and their regulation by lactogenic hormones have been studied. L-alanine uptake was mediated by three Na(+)-dependent and one Na(+)-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na(+)-dependent uptake exhibited the usual characteristics of system A. Cl- dependency has been established for system A. The other two Na(+)-dependent systems, which we have named BCl(-)-dependent and BCl(-)-independent, are described for the first time. These are systems with broad specificity and were distinguished on the basis of inhibition analysis, Cl- dependency and the effect of preloading mammary tissue with amino acids. The Na(+)-independent route was identified as system L, which operates independent of Cl-. The A, L and BCl(-)-independent transport systems were upregulated in pregnant mouse mammary tissue cultured in vitro in the presence of lactogenic hormones (insulin plus cortisol plus prolactin). Insulin alone also upregulated systems A and L to some extent in pregnant mouse mammary tissue. BCl(-)-dependent activity was not detected in pregnant mouse mammary tissue and was not induced by lactogenic hormones in vitro.

Maharishi Amrit Kalash rejuvenates ageing central nervous system's antioxidant defence system: an in vivo study.

The oxygen-free radical involvement in various deteriorative processes and in ageing is unquestionably established. In the present study age-related changes in antioxidant enzyme activity in the different regions of CNS of 10-month and 32-month-old guinea pigs were studied. Maharishi Amrit Kalash has shown promise in inhibiting the in vitro and in vivo lipid peroxidation. Therefore in the present study the effect of MAK on the activity of antioxidant enzymes was checked. Our results indicate that the activity of superoxide dismutase and glutathione peroxidase, was found to be reduced (P < 0.05) in all the regions of CNS studied, The activities of catalase declined significantly only in the cerebral cortex, hypothalamus and the cerebellum. Whereas glutathione reductase activity declined in the cerebral cortex and hypothalamus. It is concluded that the age-related decline in the activities of antioxidant enzymes is region-specific as well as enzyme-specific. The endogenous lipid peroxide was found to be increased significantly (P < 0.05) in the 32-month-old animals. Whereas the lipid peroxidation after incubating the tissue homogenate in the air was found to be decreased (P < 0.05) in the older animals. The results indicate that the accumulation of lipid peroxides take place with age but the susceptibility of lipid peroxidation decreases in the older animals. The treatment of MAK 500 mg kg-1 body wt. for 2 months could augment the activities of antioxidant enzymes (P < 0.05). The effect of MAK was more pronounced in older than younger animals. It is concluded that the MAK can be used in compensating the decline in the activities of antioxidant enzymes in CNS and thereby it reduces the risks of lipid peroxidation.

Discrimination of transport systems of L-tyrosine in mouse mammary gland: characterization of system T.

On the basis of inhibition analysis, tyrosine uptake in mouse mammary gland was found to be mediated by Na(+)-dependent and Na(+)-independent systems. Na(+)-dependent system was insensitive to 2-(methylamino) isobutyric acid (MeAIB), with an apparent Km of 1.67 mM and maximal velocity 74.5 nmol.g-1 cell. min-1. Competition experiments showed the presence of two distinct Na(+)-independent components of tyrosine uptake. One component was sensitive to 2-aminobicyclo (2,2,1) heptane-2-carboxylic acid (BCH) and was similar to the system L, with an apparent Km of 0.23 mM and maximum velocity of 31 nmol.g-1 cell.min-1. Second component was BCH-insensitive but tryptophan-sensitive, with an apparent Km of 15.75 mM and Vmax of 157.5 nmol.g-1cell.min-1. BCH-insensitive, tryptophan-sensitive system was a low affinity system. It approached steady state slowly and was more sensitive, relative to the system L, to n-ethylmaleimide inhibition. Tyrosine uptake through this system did not respond to trans-stimulation, whereas system L mediated uptake responded considerably. BCH-insensitive, tryptophan-sensitive component of Na(+)-independent tyrosine uptake is attributed to the system T, previously described only in human red blood cells and rat liver cells.

Lack of functional correlation between gamma-glutamyl transpeptidase and amino acid transport in the lactating mouse mammary gland.

Gamma-glutamyl transpeptidase (EC 2.3.2.2) in lactating mouse mammary gland was inhibited by affinity labelling of the tissue with 6-diazo-5-oxo-L-norleucine. Amino acid (L-alanine, L-methionine) uptake by the affinity labelled mammary gland tissue and the control tissue was measured in vitro. Uptake of amino acids by the affinity-labelled tissue was comparable to that of control tissue. These findings suggest that gamma-glutamyl cycle is not involved in amino acid uptake by the mammary gland.

Characterisation and starvation induced regulation of methionine uptake sites in mouse mammary gland.

The sites of methionine uptake by 10 day lactating mouse mammary gland were determined in vitro. Four modes of methionine entry characterised were: (i) A sodium-dependent, N-(methylamino) isobutyric acid (MeAIB)--sensitive system with a Vmax of 18.8 nmol/g cells/min (this mode of entry was similar to the A site in other tissues); (ii) A sodium-dependent, MeAIB--insensitive uptake system with a Vmax of 12.4 nmol/g cells/min); this mode of entry was inhibited by substrates preferred by ASC system); (iii) A sodium-independent, 2-amino-bicyclo heptane 2-carboxylic acid (BCH)-sensitive system L with a Vmax of 30 nmol/g cells/min; and (iv) A sodium-independent entry which was not inhibited by high concentrations of MeAIB or BCH. The Km value of each of the former three carrier mediated transport systems was 0.46 mM. Starvation of animals brought about important increase in the Vmax of the A system by 97% and that of ASC system by 1003% which was accompanied by similar increases in the Km values of these systems. These results show an adaptive regulation of these two sodium-dependent sites as a result of starvation.

Characterisation of the routes of methionine transport in mouse mammary glands.

The sites of methionine uptake by mammary glands from late pregnant and lactating mice were studied in vitro. Using the specific A system inhibitor, N-(methylamino) isobutyric acid (MeAIB) and the specific L system inhibitor, 2-amino-bicyclo (2.2.1) heptane 2-carboxylic acid (BCH), we have defined four modes of methionine entry into these tissues. (i) A sodium-dependent A system with a Vmax of 13.4 and 18.8 n mol/g cells/min in pregnant and lactating mice, respectively. This mode of entry was completely inhibited by MeAIB and its Km value was similar (0.45 mM) in both groups. (ii) A sodium-dependent mode with a Vmax of 6.7 and 12.4 n mol/g cells/min and a Km of 0.24 and 0.46 mM in pregnant and lactating mice, respectively. This mode of entry was insensitive to inhibition by MeAIB, and was similar to the ASC (alanine, serine, cysteine) system in other tissues. (iii) A sodium-independent L system with a Vmax of 13.8 and 30.0 n mol/g cells/min and a Km of 0.27 and 0.46 mM in pregnant and lactating mice, respectively. This mode of entry was completely inhibited by BCH. (iv) A sodium-independent non-specific entry amounting to 25 per cent of the total entry at 0.1 mM external methionine which was not inhibited by high concentration of BCH. The results of our studies show an increase in the number of active carriers of the A, ASC and L systems of methionine uptake in mammary glands of mouse during lactation.

Effect of skim milk on progression of atherosclerosis in cholesterol-fed rabbits.

Effect of skim milk on progression of atherosclerosis was studied in cholesterol-fed rabbits. Rabbits were given a high cholesterol food (0.5%) with skim milk powder (16%) or no milk (control group). At 12 wk, the plasma cholesterol level was significantly higher in the control group (1605 mg/d1) than in the milk-fed group (1146 mg/d1). The contents of esterified cholesterol and elastin in the aorta were higher in the control group than in the milk-fed group by 28 an 94 per cent, respectively. The differences between the two groups in the contents of aortic triacylglycerols, mucopolysaccharides, collagen and unesterified cholesterol were not significant. The difference in sudanophilic area in the aorta between the control (35%) and the milk-fed groups (31%) was not significant. However, intimal proliferation and medial involvement in the aortic lesions were more severe in the control group. These findings suggest that skim milk can slow down the process of cholesterol induced atherogenesis.