Background & Objectives: Brucellosis is one of the most important zoonotic disease caused by genus of Brucella. Lipopolysaccharides (LPSs), outer membrane proteins and several ribosomal or cytosolic proteins of Brucella have been considered for diagnostic and subunit vaccine purposes. The objective of this experiment was to identify and compare immunogens of Brucella abortus (S19) in infected human and goat and immunized rabbit.Materials and Methods: Brucella abortus (S19) was obtained from Pasture Institute of Iran. The bacterial proteins were extracted by lysozyme, urea and CHAPS, then analysed by two-dimensional gel electrophoresis. The resolved proteins were transferred to nitrocellulose membrane by tank blotting and their reactions with the sera preformed by immunoblotting. Results: The proteins of the (S19) strain were often acidic pI 4.5-5.7) in range of 10-100 kDa. The most aboundant proteins which were immunogenic in goat and rabbit but not in human was a group of 5-6 spots observed in acidic side (pI 4.5-5.7) and molecular mass of 32-34 kDa. In human a group of 4-5 proteins with mass of 44 kDa and pI ranging from 4.5 to 5.5 and several low molecular weight proteins were immunogenic. Furthermore several proteins of Rubella had common reactions with human, goat and rabbit sera.Discussion: Lipopolysaccharids of Brucella abortus (S 19) had remarkable reactivity against all of the sera; but are not good candidates for diagnosis of brucellosis, because of structural similarities with some gram negative bacteria and remaining of anti-LPS antibodies in high levels in sera of many cases after recovery of brucellosis. In contrast, some antigenic proteins can be candidate for accurate diagnosis, and recognition of active from inactive brucellosis in human and cattle. The results of this study suggested that a group of 4-5 proteins of 44 kDa and pI 4.5-5.5 and several low molecular weight protein in human and a group of 5-6 proteins with 32-34 KDa and pI 4.5-5.7 in goat and rabbit as potential diagnostic candidates.

Dicrocoeliasis is an important livestock disease caused by digenean trematode namely Dicrocoelium dendriticum. The aim of the present study was to identify somatic and metabolic antigens of adult D.dendriticum in naturally infected sheep. Adult parasites collected from the liver of naturally infected sheep, were washed in cold phosphate buffer saline (PBS, pH=7.4) and stored at -20°c until analysis. Antigen used for the detection of antibody included somatic and metabolic of mature trematode. Somatic and excretory-secretory antigens prepared with haemogenization and incubation of adult helminths, respectively. Electrophoretic patterns of excretory secretory and somatic antigens of D.dendriticum were revealed by sodium dodecyl sulphate polyacrylamid gel electrophoresis (SDS-PAGE) and westernblotting using sera from naturally harbored sheep. In western blot analysis of antigens D.dendriticum demonstrate 1 major antigenic polypeptide 130 kDa in both somatic and metabolic antigens and 6 protein bands ranging from 25 to 60 kDa in excretory-secretory antigens which were recognized by serum of sheep naturally infected. Our findings showed that the 130 kDa molecular weight polypeptide could be used as specific antigen for the immunodiagnosis of sheep dicrocoeliasis.

Western blotting or immunoblotting commonly use for study of reaction between antigens and antibodies. Denaturation of many proteins in immunoblotting can affect greatly the reactivity of antibodies and outcome of the procedure. In this study proteins of Brucella abortus (S19) was extracted by a mild method and reaction of the extracted proteins with serum of infected human and goat and immunized rabbit compared by affinity chromatography and immunoblotting. Gamma globulin (mostly IgG) fraction of the sera was precipitated by half saturation of ammonium sulfate and linked to activated sepharose 4B. The extracted proteins were loaded on the affinity column. Attached proteins was eluted by low pH and analyzed by SDS-PAGE. Reaction of the total extract and eluted fractions with IgG fraction of sera was evaluated by Western blotting.Upon the results of affinity chromatography and immunoblotting, Brucella proteins can be classified in four groups: 1- The proteins that adsorbed to the affinity column and react with IgG in westernblotting. 2- Proteins that react with IgG in native state but no in denatured state. 3- Proteins that do not react with IgG in native state but react in denatured state. 4- Proteins that do not react with IgG in native and denatured state.

ObjectiveEpilepsy is a disorder of the brain characterized by an enduring predispositionto generate seizures. Infectious agents are mentioned in its etiology. Withidentifying and appropriate treatment of these infectious agents, preventing theirsecondary outcomes, including seizure is possible. This study was conductedto determine frequency of anti-Toxoplasma antibodies (IgG, IgM) and anti-Toxocara antibody (IgG) in epileptic patients.Materials & MethodsStudy sample consisted of 141 epileptic patients and 144 healthy people. Afterobtaining informed consents and completing demographic questionnaire, serumsamples were taken from participants. The diagnostic test of Toxoplasma IgG & IgM and Toxocara antibodies was performed under the same conditions usingELISA method in a qualified private laboratory. Samples from patients andcontrol groups with positive ELISA test in terms of anti-Toxocara antibody werealso used for confirmatory Western blot test.ResultAccording to ELISA results, 28 (19.85%) epileptic patients and 2 (1.38%) ofhealthy people had anti-Toxocara antibodies (P<001), while 39 (30.46%) of thecontrol group people and 14.18% of patients had anti-Toxoplsma antibodies(P=0.001).ConclusionFrequency of anti-Toxoplasma gondii is lower in epileptic than healthy individualsand this result is contrary to investigations that have reported higher levels ofthis antibody in such patient groups. ELISA results for Toxocara showed thatthe frequency of anti-Toxocara antibody in epileptic patients might empower theprobability that this parasite may cause central nervous system damage. Westernblotting has high specificity and is a proper confirmative method for diagnosisof toxocariasis.

Introduction: Mood disorders such as depression and anxiety disorders have been affecting a relatively high proportion of the world's population. Neuroplasticity hypothesis of depression proposes that lack of brain-derived neurotrophic factor (BDNF) can cause structural changes in the brain. The extract of Ginkgo biloba (Gb) leaves can restore much of the damage in the nervous system. We examined the antidepressant role of Gb extract (EGb 761) on BDNF expression modulation in the hippocampus of rats subjected to repeated restraint stress (RRS). Methods: Adult male rats were randomly divided into 10 groups: control, controlvehicle treated, stress, stress-vehicle treated, as well as three control and three experimental groups pretreated with EGb (15, 30, 60mg/kg, IP daily) for 21 days. They underwent restraint stress on a daily basis, 6 hours for 21 consecutive days. Weight changes, locomotor activity and forced swim test (FST) were employed to assess depressive-like symptoms. The serum corticosterone level was also measured by ELISA. Hippocampal BDNF DNA methylation and protein expression were assayed by methylation sensitive restriction enzymes (Real Time PCR) and Westernblotting respectively in all groups. Results: Pre-treatment with 30 and 60 mg/kg/day of Gb extract significantly attenuated depressive-like effects in the body weight, FST and serum corticosterone level in RSS rats compared to control groups. Further, it inhibited chronic stress-induced alterations in the hippocampal BDNF DNA methylation and protein expression. Conclusion: These findings suggest that Gb can induce an antidepressant role through its modulation effect on the hippocampal BDNF expression.

Introduction: Toxoplasma gondii is a protozoan parasite which is globally prevalent in human and animals. Toxoplasma infection is commonly asymptomatic, but can cause serious medical problems in immunocompromised individuals and in fetus. Recombinant antigens of the parasite may be helpful in diagnosing the infection more precisely. The goal of this study was to construct and evaluate the functionality of a prokaryotic expression plasmid pGEX-P35, harboring P35 surface antigen gene of T. gondii and to perform preliminary studies on its ability to detect T. gondii specific antibodies.Material and Methods: A 450 bp fragment of the P35 gene was amplified and inserted into pGEM-T plasmid, sequenced, cut, and then inserted into pGEX-4T-1 plasmid to produce the recombinant plasmid pGEX-P35. In order to confirm that the plasmid construct was capable of expressing P35 in bacterial cells, it was transformed into BL21 strain of E. coli and expressed. The resultant recombinant protein was purified and subjected to SDS-PAGE and Western-blot analysis.Results: A 450 band of PCR product was visualized on 1% agarose gel. Comparison of resultant DNA sequence with GenBank databases showed 100% identity with AF01275. Restriction enzyme analysis confirmed subcloning and correction of orientation. SDS-PAGE analysis showed a 42 kDa band of purified expressed protein. Western-blot analysis using mouse antibody against RH strain of T. gondii showed that the recombinant P35 antigen could be recognized by specific antibodies.Conclusion: Purified and specific recombinant antigens obtained by molecular biology techniques are attractive alternatives for detection of serum antibodies. We amplified and cloned a fragment from P35 gene of Toxoplasma gondii encoding P35 tachyzoite-specific surface antigen. Sequenced fragment was accepted by GenBank with an accession number of DQ092625.  This confirms previous results from other countries estimating just 1% divergence at the level of DNA sequence between lineages isolated from different geographical areas. As the recombinant P35 (rP35) antigen is recognized by specific antibodies, it is suggested to evaluate the (rP35) for diagnosis of clinical Toxoplasma gondii infections.

Transforming growth factor-β (TGF-β ) induces pro-inflammatory cytokines expression including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α ) and these cytokines are associated with the development of atherosclerosis. Curcumin has anti-atherogenic effects and anti-inflammatory properties in the vascular wall, but the relative mechanisms are almost unknown. In the present study, we investigate the effect of curcumin on modulating the proinflammatory action of TGF-β in human vascular smooth muscle cells (VSMCs) and its molecular mechanisms. Cultured VSMCs were seeded into several groups: a control group (untreated group), a group treated with TGF-β , and several groups treated with TGF-β plus inhibitors. The cells were pretreated with diphenyleneiodonium chloride, DPI, (20 μ M), curcumin (5, 10 and 20 μ M) and NAcetyl-L-Cysteine, NAC, (10 mM) and then TGF-β (5 ng/mL) was added to the culture medium. The mRNA levels of IL-6 and TNF-α were detected by quantitative Real-Time Polymerase Chain Reaction. For monitoring the Smad2 linker region phosphorylation (pSmad2L), the westernblotting technique was applied and reactive oxygen species (ROS) generation was measured by utilizing 2′ , 7′-dichlorofluorescein diacetate-based assay. TGF-β increased the mRNA expression of IL-6 (p=0. 02 and p=0. 001) and TNF-α (p =0. 014 and p=0. 001) in a time-dependent manner, ROS production (p=0. 03) and Smad2L phosphorylation (p=0. 015). Pre-treatment with curcumin, DPI and NAC inhibited TGF-β – induced IL-6 (p=0. 04) and TNF-α (p=0. 001) mRNA expression, Smad2L phosphorylation (p=0. 02) and ROS production (0. 03). Pharmacological inhibition by Curcumin blocks TGF-β – induced ROS production, Smad2L phosphorylation, and IL-6 and TNF-α mRNA expression in human VSMCs.