A novel pentasaccharide from immunostimulant oligosaccharide fraction of buffalo milk.

A processed oligosaccharide mixture of buffalo milk induced significant stimulation of antibody, delayed-type hypersensitivity response to sheep red blood cells in BALB/c mice. This also stimulated non-specific immune response of the animals measured in terms of macrophage migration index. A novel pentasaccharide has been isolated from the oligosaccharide containing fraction having immunostimulant activity of buffalo milk. This compound was isolated by a combination of gel filtration chromatography, silica gel column chromatography of derivatised oligosaccharides while the homogeneity was confirmed by high performance liquid chromatography. The results of structural analyses, i.e. proton nuclear magnetic resonance, fast atom bombardment mass spectrometry, chemical transformations and degradations are consistent with the following structure: GlcNAcbeta(1-->3)Galbeta(1-->4)GlcNAcbeta(1-->3)Gal beta(1-->4)Glc

Immunomodulatory activity of analog of muramyl dipeptide and their use as adjunct to chemotherapy of Leishmania donovani in hamster.

In search of a potent immunomodulator to be used as an immunoprophylactic agent and as adjunct to chemotherapy against Leishmania infection, two analogs of muramyl dipeptide, viz. N.Ac-norMur-MeVal-D-isoGln (86/448) and N.AcMur-Acc-D-isoGln (89/729) were evaluated for desired activity. Effect of these peptides on cell mediated and humoral immunity was studied by immunizing the peptide treated mouse with sheep red blood cells (SRBC) and determining HA-titer, plaque forming cells assay and delayed type of hypersensitivity (DTH) response after 4-5 days. Both the peptides stimulated cell mediated immunity (CMI), humoral response as well as macrophage function in terms of super oxide anion (O2-) and nitric oxide (NO) generation. Mitogen induced lymphocyte proliferation and production of IL-2 and INF-gamma increased while that of IL-4 and IL-10 decreased by both the peptides showing a typical Th1 type response. After establishing the immunostimulatory activity, these peptides were evaluated for immunoprophylactic efficacy as well as for use as adjunct to chemotherapy with stibanate (SSG) against Leishmania donovani infection in golden hamster. These peptides were found quite effective in both the modes. In adjunct use the treatment may require lower dose of SSG and thereby reduce the chances of drug toxicity.

Studies on ammonia-metabolizing enzymes during Plasmodium yoelii infection and pyrimethamine treatment in mice.

Blood ammonia content and enzymes involved in ammonia metabolism, namely glutamine synthetase (GS), glutamate dehydrogenase (GDH), monoamine oxidase (MAO), alanine amino-transferase (ALT) and aspartate aminotransferase (AST), were studied in Plasmodium yoelii-infected drug-treated mice tissues. The ammonia content in blood increased with the rise of parasitaemia. Hepatic GS, GDH and MAO showed a marked decrease in enzyme activity during parasitic infection. In contrast, cerebral GS and MAO showed a significant increase during infection. However, the parallel measurement of renal enzymes did not show any noticeable alterations except for ALT and AST. Oral pyrimethamine treatment (10 mg/kg for 4 days) in infected mice (5-10%) returned the altered levels of the above enzymes to almost normal 1 week after the cessation of drug treatment.

Effect of Plasmodium berghei infection and chloroquine on the hepatic drug metabolizing system of mice.

The hepatic microsomal mixed-function oxidase (MFO) system was markedly impaired during Plasmodium berghei infection in mice. Cytochrome P-450 and other mono-oxygenases, viz. aniline hydroxylase, aminopyrine-N-demethylase and benzo(a)pyrene hydroxylase, were significantly decreased while microsomal heme showed a four-fold increase at peak parasitemia (greater than 50%). Oral treatment with chloroquine (16 mg kg-1 body wt for 4 days) of P. berghei-infected mice cleared the parasitemia within 72 h and almost normalized the altered levels of MFO indices, a week after cessation of treatment. The findings were further supported by the isoenzymic profile and drug-binding properties of terminal mono-oxygenase, cytochrome P-450.

Amino acid transport in cerebral microvessels during Plasmodium yoelii infection in mice.

Plasmodium yoelii infected cerebral microvessels of mice had an enhanced time-dependent, temperature-sensitive, and saturable uptake of [14C]-amino acid. viz. leucine, valine and glycine. Metabolic inhibitors caused a noticeable inhibition of amino acid uptake in normal microvessels as compared to infected cerebral microvessels indicating that the uptake of [14C]-L-leucine, [14C]-L-valine and [14C]-glycine is an energy dependent process.

Permeability function related to cerebral microvessel enzymes during ageing in rats.

Cerebral microvessels from rats were prepared and characterized by their enrichment of specific markers, namely alkaline phosphatase (AP) and tau-glutamyl transpeptidase (tau-GT). Further, it was observed that AP and tau-GT registered marked increase in aged rats. On the contrary, lactate dehydrogenase (LDH) activity decreased with the increasing age. Monoamine oxidase A activity in the microvessels decreased with age whereas MAO-B moved in the reverse direction. No noticeable change was seen in acetyl-cholinesterase activity with increasing age of rats.

Syntheses and immunomodulatory activity of 3-O-[2'-hydroxy-3'-N,N-disubstituted aminopropan-1'-yl]-alpha-D-glucofuranoses.

A number of 3-O-[2'-hydroxy-3'-N,N-aminopropan-1'-yl]-alpha-D-glucofuranoses were synthesised by regioselective oxirane ring opening in compound 2 with different secondary amines followed by selective deacetalisation. All the compounds were tested for their immunomodulatory potential in vitro; seven of them expressed significant immunostimulant activity.

Suppression of release of microfilariae in vitro from Setaria cervi (Nematoda: Filariodeae) by chlorpromazine.

Chlorpromazine (CPZ) caused suppression in release of microfilariae in vitro from Setaria cervi at 10(-5) M concentration. The inhibitory effect of CPZ was further stimulated by Ca2+ (2 mM), whereas addition of EGTA (2 mM) resulted in partial reversal. Addition of dopamine (1 mM) along with CPZ (10(-5) M) caused similar inhibition as was observed in the presence of CPZ (10(-5) M) alone. CPZ did not effect in vitro oxygen uptake by worm homogenates up to 0.5 mM concentration. The results indicate the Ca2+/calmodulin-dependent anthelmintic action of CPZ on S. cervi.

Neuropathological studies on Plasmodium yoelii nigeriensis-induced malaria in mice.

Malaria infection in mice was produced by intraperitoneal inoculation of 10(6) erythrocytes parasitized with Plasmodium yoelii nigeriensis, a virulent strain of murine malaria. About one week after infection parasitaemia ranged between 60 and 80%, and 100% mortality was observed. Infected animals were killed 6 days after infection to allow the examination of brain tissue. Electron microscopical observations revealed marked damage to cerebral vascular vessel walls with separation of muscular layers, media and adventitia. The endothelial cell layer was discontinuous in places. Activated fibroblast cells producing collagen fibres were seen around the necrotic region of cerebral vasculature. Some parasitized erythrocytes were also seen attached to the endothelial cell lining. Cerebral oedema was prominent around the blood vessels.

Status of urea and related enzymes during Plasmodium yoelii infection and pyrimethamine treatment in mice.

Plasmodium yoelii infection alters the hepatic levels of key enzymes of urea cycle, viz.carbamoyl phosphates synthetase (EC 6.3.4.16) and ornithine transcarbamoylase (EC 2.1.3.3) and urea levels in mice. The urea level was found elevated in liver, brain and plasma during P. yoelii infection. However, carbamoyl phosphate synthetase and ornithine transcarbamoylase were noticeably decreased during P. yoelii infection. Pyrimethamine treatment (10 mg/kg body weight for 4 days) brought back the altered parameters to normal a week after cessation of drug treatment.

Status of ammonia, glutamate, lactate and pyruvate during Plasmodium yoelii infection and pyrimethamine treatment in mice.

Ammonia, lactate, glutamate and pyruvate levels in blood, liver, brain, spleen and kidney were determined during Plasmodium yoelii infection and pyrimethamine treatment in mice. Ammonia and lactate levels showed significant increase with rise in parasitaemia except in spleen where decrease in the lactate levels was observed. The glutamate level displayed a marked decrease in blood, liver and splenic tissues, whereas, significant increase in glutamate level in kidney was observed, although its level in cerebral tissue remained unaltered. The pyruvate level in blood and liver showed a noticeable decrease but brain, spleen and kidney registered an elevation of the same due to the parasitic infection. Pyrimethamine (oral) treatment (10 mg/kg body weight) to infected mice (5-10%) for four days brought back the altered levels of the above cellular constituents in different tissues to normal, a week after cessation of drug treatment.

Implication of intracellular glutathione and its related enzymes on resistance of malaria parasites to the antimalarial drug arteether.

The control of malaria has been complicated by the increasing resistance of malarial parasites to multiple drugs. However, artemisinin-based drugs offer hope in the fight against drug-resistant parasites. The mode of action of these drugs remains unclear, but evidence suggests a role for free radicals in their mechanism of action. In this study, we examined the relationship between the intracellular levels of glutathione (GSH) and antioxidant enzymes and resistance to the artemisinin-based drug arteether in experimentally selected arteether-resistant Plasmodium vinckei. GSH plays a critical role in the detoxification and protection of cells against oxidative stress. Our comparative studies showed a significant (2.9-fold) increase in the GSH level in arteether-resistant parasites as compared to arteether-sensitive parasites. Simultaneously, significantly increased activities of glutathione reductase, glutathione-S transferase and glucose-6-phosphate dehydrogenase and decreased activity of superoxide dismutase were recorded in resistant parasites; the activity of glutathione peroxidase was comparable in arteether-sensitive and -resistant parasites. Artemisinin derivatives act by generating free radicals and our results indicate that glutathione's antioxidant effects may counteract that drug effect and thereby contribute to the parasites' resistance to arteether and other artemisinin-based antimalarials.