Iranian Biomedical Journal
Year:2005 | Volume:9 | Issue:1|Papers Count:8

Toxoplasmosis is a worldwide infection which is commonly asymptomatic but can cause serious medical problems in immunocompromised individuals and fetus. The infection also causes considerable economic loss because of abortion in livestock, mostly in sheep and goats. DNA vaccination may be a powerful approach against intracellular parasites such as Toxoplasma gondii. The goal of this study was to construct and evaluate the functionality of an eukaryotic expression plasmid pRC/CMV-GRA2, harboring dense granule antigen-2 (GRA2) gene of T. gondii and to perform preliminary studies on its immunogenicity in a mouse model. The GRA2 complete cDNA was inserted in PCR2.1 plasmid, sequenced, then cut and inserted in pRC/CMV plasmid, to produce the recombinant plasmid pRC/CMV-GRA2 (pGRA2). To verify that the plasmid construct pGRA2 was capable of expressing GRA2 in mammalian cells, it was transfected into 293-T cells, an embryonic kidney cell line. Western-blot analysis of the transfected cells using a monoclonal antibody specific for GRA2 indicated specific expression of GRA2 protein. CBA/J mice were subcutaneously immunized three times with 100 µg of pGRA2 plasmid. The obtained sera recognized GRA2 that is shown by Western-blotting. These findings indicate that pGRA2 plasmid directs high-level expression of antigenic GRA2 protein in mammalian cells and is immunogenic in CBA/J mice.

New Page 1 Transforming growth factor beta (TGF-b) is a mediator released by nearly all cell types. It has suppression activity on the immune system, but exactly how this effect is carried out is not clear. Previous experiments showed that IgG interacts with or carriers active TGF-b, that could suppresses cytotoxic T-cell responses to an immunogenic tumor in mice. Since T cell receptor (TCR) has structural similarities with IgG, we asked the question whether a specific TCR could interfere with and enhance the suppressive effect of TGF-b on T-cell proliferation. T-cell lines were established by limiting dilution and specific TCR were extracted and purified. Mixed lymphocyte reaction (MLR) was carried out using DA (RT1a) vs. LEW (RT11) lymph node cells and DA vs. PVG (RT1u) lymph node cells. DA cells were used as responder cells and PVG/LEW as stimulator cells. Proliferation of DA cells was examined with different concentration of TGF-b by adding 1mci 3H-thymidine 24 hours prior to harvesting the cells.  The results showed that the presence of a specific TCR does not have any effect on the percentage of suppression when already fully suppressed by TGF-b.  However, it does have an effect on TGF-b stimulated suppression under certain conditions. When TCR was added at the same concentration as TGF-b (1-2 ng/ml), inhibited TGF-b stimulated suppression of proliferation, but when added at higher concentration than TGF-b, this effect disappeared, and the proliferation was suppressed in the same way, as TCR was absent. Thus, TCR interaction with TGF-b could play an important role in the homeostasis of immunity by augmenting the proliferation of activated dominant lymphocyte clones. This would promote suppression of activation/proliferation of new specific antigen-reactive clones that may arise during ongoing immunity, and also suppressing some autoimmune diseases

Change in transmitting time of impulses along axons is traditionally attributed to two parameters: the myelin formation and the diameter of neurite, both rising during the postnatal development. In the previous study, we showed that conduction velocity of the fibers projecting from the thalamus to the layer IV of the somatosensory (barrel) cortex increases as a function of age. However, the conduction velocity change is not parallel outside and inside of the barrel cortex. Here, we tried to find a probable relationship between disparity of the conduction velocities and the extent of myelination of the thalamocortical pathway. Our results indicate that myelin is evident on the extra-cortical but not intra-cortical fibers at ages >10 days. At the older age, however, myelin wholly covers the fibers both outside and inside of the cortex, more considerably in the former. Our results demonstrate that difference in the conduction velocities of the extra-cortical and intra-cortical axons, at least to more extent, can be attributed to myelination dissimilarity along the thalamocortical fibers

It is well known that glycoconjugate components of the cell surface and extracellular matrix, play an essential role(s) in many developmental phenomena such as cell differentiation, migration, and cellular interactions. The purpose of this study was to investigate distribution of this macromolecules during differentiation of the notochord and paraxial mesoderm. Formalin fixed paraffin sections of 9 to 16 days of BALB/c mouse embryos were processed for histochemical studies using four different horseradish peroxidase conjugated lectins including wheat germ agglutinin (WGA), Griffonia simplicifolia (GSA1-B4), Arachis hypogaea (PNA) and Lotus tetragonolobus (LTA), specific for sialic acid, galactose, N-acetylgalactosamine (GalNAc) and fucose terminal sugar, respectively. Our results showed that in primordial of the developing vertebrae PNA sensitive glycoconjugate appeared on gestational day 13 and increased to gestational day 15 significantly (P<0.05) and disappeared later. GSA1-B4 revealed no reaction and LTA and WGA reactions were observed only in notochord. Among the lectins that were used in this study, only PNA showed continuous and strong reaction just during gestational day 13 to 15. This finding suggests that PNA temporally regulated changes occurred during early vertebral development in mouse embryos and probably their specific terminal sugar plays a key role(s) during prechondrogenic vertebrae

Lithium is widely used in medicine as an anti-depressive drug. In spite of abundant literature, questions on the side effects of lithium ions are far from being answered. In this study, the effects of lithium on biochemical parameters related to lipid metabolism were investigated. Male Wistar rats were treated with different doses of lithium for a period of up to 60 days. Blood samples were collected and livers were removed for analysis. Lipid related parameters in plasma and livers were measured by standard methods. Epididymal fat pads were used to investigate the mechanism of lithium action on lipolysis. It is shown that the major effect of lithium is reduction of HDL-C concentration and the increase of LDL-C only in high doses. Lithium treatment led to a decrease in liver content of triglycerides but phospholipids increased significantly. Lithium also showed to inhibit lipoprotein lipase activity. This inhibitory effect is potentiated in the presence of citrate. Fat cell lipolysis is also inhibited by lithium, which is not reversed by alpha, and beta-receptor blockers indicating that lithium may exert its effect beyond these receptors. Lithium changes the metabolism of lipoproteins. The finding that lithium decreases HDL and increases LDL concentrations should be considered seriously, especially in patients using this drug for a long period.

Angiotensin-converting enzyme (ACE) inhibitors including quinapril could exert a protective effect on cardiovascular system through endothelial system in normoglycemic and diabetic rats. The present experimental work was designed to study the vascular reactivity of aortic ring segments isolated from streptozotocin (STZ)-diabetic rats treated for 4 weeks with nitro-L-arginine-methyl ester (L-NAME; 50 mg/100 ml) or L-NAME plus quinapril (10 mg/100 ml) in drinking water. The results showed that quinapril treatment significantly attenuated the augmented contractile response to phenylephrine and KCl in diabetic rats. In addition, quinapril treatment partially restored the reduced contractile response in diabetic animals treated chronically with L-NAME. It can be concluded that quinapril could partly counteract the effect of long-term L-NAME administration on vascular reactivity in STZ-diabetic rats.

New Page 1 This study was conducted to determine the location of DNA segment with homology to the rat conserved genomic DNA in human chromosomes. The labeled rat genomic DNA was hybridized with normal human (male) metaphases. The study of 74 metaphases after fluorescence in situ hybridization showed 371 twin-spot signals on human chromosomes. Statistical analysis indicated that the specific accumulation of signals on 1q22-qter, 2p2, 3p21-p23, 4q3, 6q2, 8p12-pter, 11p12-pter, 11q12-qter, 12q2, 13p, 15p, 16q2, 21q12-qter, Yq1-qter, and Xq2 was not random. Results of stepwise multiple linear regressions indicated that number of mapped oncogenes (Beta = 1.092; t = 7.552; P<0.001) and density of mapped oncogenes on chromosomes (Beta = -0.832; t = -5.751; P<0.001) have significant effects on number of double-spots on human chromosomes. These data reflects the evolutionary conservation between rat DNA and human DNA at the above-mentioned bands.

Recently it has been reported, that immunoglobulin Y (IgY) can be used instead of polyclonal antibodies extracted from mammals (IgG) for the purpose of diagnosis and therapy. These antibodies are found to have better properties in terms of specificity and ease of large-scale production. In addition, IgY binds neither to mammalian complement or Fc-receptors nor does it interfere with rheumatoid factors (RF), which has proven to be advantageous in many immunological tests. Proteinase 3 (PR3), a constituent of azurophil granules of neutrophils, is the target antigen for most anti-neutrophil cytoplasmic antibodies (c-ANCA) in Wegener granulomatosis (WG). Capture ELISA was found to be the method of choice in case of c-ANCA determination for the diagnosis and management of WG. However, in this method, the reaction of RF with the Fc portion of IgG in capture ELISA leads to false positive in the assay of c-ANCA and is found to be the most important short-comings of available diagnostic immunochemical tests using mammalian antibodies. To avoid such unwanted interactions, laying hens were inoculated with PR3, and IgY was purified from egg yolk by acidic extraction with chloroform. The aqueous phase was treated with sodium sulphate and the precipitate collected, was dissolved in buffer and was purified using a T-gel chromatography method. The prepared IgY-anti-PR3 was used to set up a capture ELISA. Our results showed that the prepared IgY-anti-PR3 had good titer (1µg/ml in a coating system) and specificity. Hence, IgY based immunoassay would be a useful alternative to mammalian IgG antibody used in PR3 immunoassays.