Iranian Journal of Basic Medical Sciences
Volume:13 Issue: 4, Autumn 2010

Objective (s) Recent studies on human indicate that the introduction of therapeutic use of tolerogenic dendritic cell (DC) for chronic inflammatory conditions is imminent. For the purpose of defining CGRP potency in tolerogenic DC production, we investigated the phenotype and IL-12 production of DCs generated from the monocytes of rheumatoid arthritis (RA) patients in the presence of the calcitonin gene-related peptide (CGRP), as a multifunctional neuropeptide.Materials and MethodsDCs were generated from isolated monocytes from four resistant and two early female RA patients using IL-4, GM-CSF, and CGRP at concentrations of 0, 1, and 100 nM. Then, the phenotype of neuropeptide-treated or untreated DCs was determined using flow cytometry and the IL-12 production was measured by ELISA.ResultsOur study showed that, on the last day of the culture, at a concentration of 1 nM CGRP, the mean fluorescence intensity (MFI) for CD80 increased (14.13%) and the MFIs for CD83, CD86, and HLA-DR decreased (14.57%, 5.28%, and 6.88% respectively). Moreover, at 100 nM CGRP concentration, the MFI for CD80 increased (11.10%) and the MFIs for CD83, CD86, and HLA-DR decreased (4.27%, 18.60%, and 19.75% respectively). In addition, our results indicated that the mean concentrations of IL-12 produced at 0, 1, and 100 nm CGRP concentrations measured 13.72±2.41, 11.01±1.61, and 7±1.34 pg/ml respectively. ConclusionDecreased CD83, CD86, and HLA-DR expression and reduced IL-12 production by CGRP were found in the RA patient's monocyte-derived DCs. CD83 is a well-defined DC activation marker. HLA-DR and CD86 are appropriate molecules for inducing an immune response. IL-12 promotes cell-mediated immunity. Therefore we suggest that CGRP may be used as an inducer in the production of tolerogenic DCs.

Objective(s)In recent years concerns have been raised regarding the incidence of male reproductive disorders from exposure to endocrine disruptors. So, chronic effects of di(2-ethylhexyl)phthalate were studied on histological and stereological structure of testis in adult Wistar rats. Materials and MethodsThirty two adult Wistar rats were randomly divided in four equal experiment groups; oil vehicle group and three treated groups which received 10, 100 and 500 mg/kg/day di(2-ethylhexyl)phthalate by gavage for 90 days, respectively. At the end of exposure period the volume of testes was measured by Cavellieri method, testes weight was recorded and then fixed in Bouin’s solution. Following tissue processing, 5 µm sections were stained with haematoxylin-eosin and evaluated with quantitative techniques. Seminiferous tubule diameter, germinal epithelium height, relative and total volumes of seminiferous tubules, tubular lumen and interstitial tissue were estimated.ResultsThe results showed that mean weight and volume of testis were decreased significantly (35.2% and 23.9% respectively) in rats treated with 500 mg/kg/day DEHP for 90 days. Seminiferous tubules diameter reduced, 4.4% and 13.4% in 100 and 500 mg/kg/day DEHP-treated groups, respectively. Relative volumes of tubular lumen and interstitial tissue were increased significantly in 100 (P< 0.05) and 500 (P< 0.01) mg/kg/day doses groups. Also, testosterone serum levels were significantly higher (P< 0.05) in rats exposed to 500 mg/kg/day DEHP. ConclusionPresent study indicated dose-dependent reductions of testicular parameters in adult male rats chronically exposed to di(2-ethylhexyl)phthalate.

Objective(s)Sickle cell disease is a genetic disorder characterized by chronic haemolytic anaemia. Haemoglobin S containing red blood cells may be susceptible to oxidative stress due to imbalance between production of reactive oxygen species and the countering effect of the various antioxidants present in the body.Materials and MethodsWe evaluated some antioxidant enzymes which include glutathione peroxidase, superoxide dismutase, and catalase. We also determined malondialdehyde, C- reactive protein and fibrinogen using commercial kits in 144 adult sickle cell disease patients (68 males and 76 females) in steady state and 80 apparently healthy age/sex matched controls; 40 sickle cell trait (20 males/20 females) and 40 normal haemoglobin (20 males/20 females).ResultsThe result showed that serum glutathione peroxidase, superoxide dismutase and catalase were lower in sickle cell disease patients compared with controls. Malondialdehyde, C-reactive protein and fibrinogen were significantly increased in sickle cell disease patients compared to the controls in both sexes. Malondialdehyde correlated negatively with superoxide dismutase (P< 0.01), glutathione peroxidase (P< 0.05), and catalase (P< 0.05) and positively (P< 0.05) with C - reactive protein and fibrinogen. ConclussionThis study shows that malondialdehyde correlated negatively with antioxidant enzymes and positively with acute phase proteins in sickle cell anaemia patients in steady state.

Objective(s)Rapid tests for detection of Streptococcus agalactiae or Group B Streptococci (GBS) at the onset of labor are needed to permit early intrapartum antibiotic prophylaxis. This study aimed to evaluate the PCR assays targeting the 16S ribosomal RNA gene (16S rDNA) for detection of the GBS in comparison with a specific culture method. Materials and MethodsTwo swabs were used to obtain vaginal specimens from the 330 pregnant women attended delivery room at Hedayat hospital, Tehran, Iran. One swab was analyzed by direct plating onto selective GBS agar medium (ISLAM) and theother swab was used for a PCR assay, which amplified the 16S rDNA of S. agalactiae. Comparative study between the selective culture and the PCR assay was done among the 330 tested women.ResultsThe GBS colonization rate based on the culture results was 20.6% (68/330). Both culture and PCR methods were positive for 56 and negative for 253 women. The culture method was positive and PCR was negative in 12 women. The culture was negative and the PCR positive for 9 women. Sensitivity of the PCR assay was 82.3% and specificity was 96.5%. The positive predictive value was 86.15% and negative predictive value was 95.4%. ConclusionISLAM diagnostic procedure and PCR are rapid and reliable analyzing methods, which might be useful for accurate diagnosis of GBS colonization in pregnant women at the time of delivery.

Objective(s)Tuberculosis (TB) is the leading cause of mortality among the infectious diseases, especially in developing countries. One of the main goals in tuberculosis research is to identify antigens which have the ability of inducing cellular and/or humoral immunity in order to use them in diagnostic reagents or vaccine design. The aim of this study was to clone and express the TB10.4 protein in Escherichia coli expression system.Materials and MethodsDNA was extracted from Mycobacterium tuberculosis H37Rv. Gene specific primers were designed using Gene Runner software according to sanger sequence database. Gene tb10.4 fragment was amplified by PCR method and purified tb10.4 gene was cloned into pET 102/D vector. Plasmid containing pET102/D-10.4 was transformed into competence E. coli TOP10. A positive transformant was chosen and plasmids DNA was isolated and subsequently transformed into competence E. coli BL21(DE3). The bacterium was induced by IPTG and its lysates were loaded directly onto SDS-PAGE. Purified recombinant protein was achieved using metal affinity chromatography (Ni-nitrilotriacetic acid).ResultsTB10.4 molecule was successfully cloned, expressed, and purified. An approximately 26.4 kDa exogenous protein was observed on the SDS-PAGE. The recombinant protein was confirmed by DNA sequencing of correct insert.ConclusionThe success of expressing the TB10.4 protein could serve as a basis for further studies on the usefulness of the gene and its expression product in the development of subunit vaccine and diagnostic method.

Objective(s)Nonenzymatic glycosylation (glycation) occurs in many macromolecules in aging and diabetes due to exposure of biomolecules to high level of glucose. Glycation can changes function, activities and structure of many biomolecules. Considering this important role of transferrin (Trf) in iron transport and antioxidant activity in plasma this study was carried out to investigate the effect of glycation in these processes.Materials and MethodsIn this study, human apo-Trf (5 mg/ml in sodium phosphate buffer pH= 7.4) was treated with different concentrations of glucose in different period of times (10 days and 20 days). Rate of glycation was measured using thiobarbituric acid method. The effect of glycation on iron binding antioxidant capacity of apo-Trf was investigated using two methods (RBC hemolysis and fluorescent). ResultsResult showed that rate of glycation of apo-Trf was increased with increase in glucose concentration and time of incubation (P< 0.05). Lower iron binding antioxidant capacity was observed for glycted Trf as compared to native Trf (P< 0.05). ConclusionImpairment of antioxidant capacity of glycated Trf can suggest a relationship between glycation of Trf and oxidative stress that occurs due to hyperglycemia in diabetic patients.

Objective(s)Bupropion is an atypical antidepressant that is widely used in smoke cessation under FDA approval. The study of synaptic effects of bupropion can help to finding out its mechanism(s) for stopping nicotine dependence. In this study the effects of perinatal bupropion on the population spike (PS)amplitude of neonates were investigated. Materials and Methods Hippocampal slices were prepared from 18-25 days old rat pups. The experimental groups included control and bupropion-treated. Bupropion (40 mg/Kg, i.p.) was applied daily in perinatal period as pre-treatment. Due to the studying acute effects, bupropion was also added to the perfusion medium (10, 50, 200 μM for 30 min). The evoked PS was recorded from pyramidal layer of CA1 area, following stimulation of Schaffer collaterals. ResultsA concentration of 10 μM bupropion had no significant effects on the PS amplitude. The 50 μM concentration of bupropion reduced the amplitude of responses in 50% of the studied cases. At a concentration of 200 μM, the recorded PS amplitudes were reduced in all slices (n= 22). Amplitude was completely abolished in 8 out of the 22 slices. The decrease of the PS amplitude was found to be more in the non-pre-treated slices than in the pre-treated slices when both were perfused with 200 μM bupropion.Conclusion The results showed the perinatal exposure to bupropion and its acute effects while indicating that at concentrations of 50 and 200 μM bupropion reduced the PS amplitude. It was also found that there was evidence of synaptic adaptation in comparison of bupropion-treated and non-treated slices whereas they were both perfused with 200 µM.

Objective(s)The liver has major role in the organism homeostasis, interactions with other systems, synthesis and metabolism of bile production, drug detoxification and hormone inactivation. Cholestasis can be defined as an impairment of the bile flow which can lead to hepatocytes necrosis and finally cirrhosis. Some studies reported a gastric acid secretion reduction in cirrhotic subjects, while others reported normal production gastric acid secretion. Our aim was to evaluate the effects of cholestasis and cirrhosis on gastric acid and pepsin secretions and its possible mechanism in rat.Materials and MethodsMale Wistar rats were randomly divided into five groups (n= 8): control, cholestasis, sham cholestasis, cirrhosis and sham cirrhosis. Laparatomy was done under general anesthesia and then bile duct ligation (BDL) was performed. After 2 and 4 weeks in cholestasis and cirrhosis groups respectively, gastric content was collected by wash-out technique. Basal and stimulated acid and pepsin secretions were measured by using titration and the Anson method respectively in all groups. In order to measure stimulated acid and pepsin secretions, pentagastrin (25 µg/kg, i.p.) was used. Nitric Oxide (NO) metabolites of gastric tissue were determined by Griess microassy method.ResultsAcid and pepsin secretions were significantly reduced in cholestatic and cirrhotic rats in comparison with control and sham groups (P< 0.01). NO metabolite of gastric tissue was significantly increased in cholestatic and cirrhotic rats (P< 0.01).ConclusionReducing of gastric acid and pepsin output in cholestatic and cirrhotic rats may be due to increasing in NO content of gastric tissue.

Objective(s) The aim of this study was to characterize the hepatitis B virus surface protein genotypes and sequence variations among hepatitis B virus surface antigen (HBsAg) positive chronic patients in Hormozgan province, south of Iran. Materials and Methods A total of 8 patients enrolled in this study. The surface gene was amplified and directly sequenced. Genotypes and nucleotide/amino acid substitutions were identified compared to the sequences obtained from the database. Results All strains belongedto genotype D. Overall 77 “muta­tions” occurred at 45 nucleotide positions, of them, 44 (57.14%) were silent (no amino acid altering) and 33 (42.86%) were missense (amino acid changing). A number of 24 (80%) out of 30 amino acid changes occurred in different immune epitopes within surface protein, of which, 9 (30%) in B cell epitopes in 7 residues (2 occurred in “a” determinant region); 8 (42.1%) in T helper epitopes in 7 residues and 7 (10%) in 4 residues inside CTL epitopes. Conclusion Hepatitis B virus genome containing mutated immune epitopes no longer could be recognized by specific T-cells of the host immune surveillance and did not enhance anti-HBs production. This could led to the progression of chronicity of hepatitis B virus infection.

Objective(s)In recent years highly selective COX-2inhibitors were withdrawn from the market because of an increased risk of cardiovascular complications. In this study we were looking for potent compounds with moderate selectivity for cox-2. So, four analogues of 4, 5-diaryl-2-(2-alkylthio-5-imidazolyl) imidazole derivatives were synthesized and their anti-inflammatory and anti-nociceptive activities were evaluated on male BALB/c mice (25-30 g). Molecular modeling and in vitro COX-1 and COX-2 isozyme inhibition studies were also performed. Materials and Methods2-(2-Alkylthio-5-imidazolyl)-4,5-diphenylimidazole compounds were obtained by the reaction of benzyl with 2-alkylthio-1-benzylimidazole-5-carbaldehyde, in the presence of ammonium acetate. Spectroscopic data and elemental analysis of compounds were obtained and their structures elucidated. Anti-nociception effects were examined using writhing test in mice. The effect of the analogues (7.5, 30, 52.5 and 75 mg/kg) against acute inflammation were studied using xylene-induced ear edema test in mice. Celecoxib (75 mg/kg) was used as positive control.ResultsAll four analogues exhibited anti-nociceptive activity against acetic acid induced writhing, but did not show significant analgesic effect (P< 0.05) compared with celecoxib. It was shown that analogues injected 30 min before xylene application reduced the weight of edematic ears. All analogues were found to have less selectivity for COX-2 in comparison to celecoxib. ConclusionInjected doses of synthesised analogues possesses favorite anti-nociceptive effect and also has anti-inflammatory effects, but comparing with celecoxib this effect is not significantly different. On the other hand selectivity index for analogues is less than celecoxib and so we expect less cardiovascular side effects for these compounds.

Objective(s)In addition to antihypertensive effects, amlodipine may exhibit cardiovascular protective effects in heart tissue. The aim of this study was to evaluate the effects of amlodipine and/or high cholesterol diet on blood, heart tissue concentration and mRNA expression of endothelin-1 (ET-1) in male New Zealand white rabbits.Materials and MethodsA total of 40 male New Zealand rabbits were divided into four groups: the normal control group, normal group receiving amlodipine, high-cholesterol diet group and high-cholesterol diet with amlodipine group. After 8 weeks, all the animals anesthetized and blood or tissues samples were collected.ResultsAfter 8 weeks of a high cholesterol diet, the group with such a diet had a significantly higher ratio of left ventricle(LV) weight to body weight than the control group (P= 0.0001). After treatment with amlodipine for 8 weeks, ET-1 level was reduced considerably in comparison with the control (P= 0.01) and high-cholesterol diet groupes (P= 0.01). Amlodipine consumption caused significant reduction (P= 0.01) in the level of ET-1 in heart tissues of high-cholesterol diet group but it had no remarkable effect on the reduction of heart tissue ET-1 in amlodipine group compared with the control group. ConclusionThe present study demonstrates that ventricular prepro-ET-1 mRNA quantitatively increases in the high-cholesterol diet rabbits which results in development of ventricular hypertrophy. It seems that the treatment with amlodipine retards the progression of LV hypertrophy through attenuation of ET-1 levels independent of lipid changes.

Objective(s)Recent studies revealed the neuroprotective effects of epigallocatechin gallate (EGCG) on a variety of neural injury. The purpose of this study was to determine the effects of EGCG on the tissue lipid peroxidation after spinal cord injury (SCI).Materials and MethodsRats were randomly divided into four groups of 7 rats each as follows: sham-operated group, trauma group, and EGCG-treatment groups (50 mg/kg, i.p., immediately and 1 hr after SCI). The rats were euthanized 24 hr after injury and then, spinal cord samples were taken for determination of malodialdehyde levels, as an indicator of lipid peroxidation. ResultsThe results showed that MDA levels were significantly decreased in EGCG-treatment groups. ConclusionOn the basis of these findings, we propose that EGCG may be effective in protection of spinal cord tissue from injury