Pathobiology Research
Year:2009 | Volume:12 | Issue:2|Papers Count:9

Objective: Dendritic cells (DCs) are essential for the activation and polarization of T cells during an adaptive immune response. In this research we investigated the effect of the Lymphoide DCs pulsed with heat-treated tumor lysate (HTL) as a vaccine in tumor immunotherapy.Materials and Methods: The Balb/c mice were injected subcutaneously in the right flank with Wehi- 164 fibrosarcoma cells 10 days before immunization with the DCs. Then hsp70 expression in the HTL was detected by using western blot analysis. The mice Lymphoide DCs subset were isolated by magnetic cell sorting (MACS), Then the HTL pulsed Lymphoide DCs, TL pulsed Lymphoide DCs and unpulsed Lymphoide DCs were subcutaneously injected. Tumor growth rate, survival, cytotoxic assay measured.Results: The results showed that HTL-Lymphoide DCs vaccine significantly induced the tumor growth suppression and longer survival than the other immunized mice. Immunotherapy with HTLLymphoide DCs led to a significant increase in the activity of cytotoxic T cells in the tumor tissue. Conclusion: The current study suggests that specific anti-tumor immune responses against the fibrosarcoma can be induced by HTL-Lymphoide DCs and may provide a useful therapeutic approach for cancer treatment.

Objective: The Brucella melitensis virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The product of the second gene of the virB operon, virB2, is predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in this gene, which is expected to exert polar effects on downstream genes. We researched on the evaluation of relation between virB2 mutant with immunogenicity in mouse model and intracellular replication in macrophages J774.Materials and Methods: In order to determine whether VirB2 is required for the function of the T4SS apparatus, we constructed and characterized deletion mutation of virB2 and kanamycin resistance gene replaced instead of virB2. For demonstration of intracellular replication, macrophages J774 and BALB/c mices were infected with wild type Brucella melitensis and mutated. Results: After 48 h, number of mutated Brucella severe decreased severly compaired to wild type in macrophages J774, and Brucella with virB2 deletion decreased from 1×106 CFU/spleen less than 1000 CFU/spleen during 8 weeks, also total IgG increased in both but IL-12 and IFN-g increased only in wild type. Conclusion: VirB2 was essential for intracellular replication in mouse models and J774 macrophages. The virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection and secreting IL-12 and IFN-g, but VirB2 dispensable for secretion of total IgG.

Objective: The entry of Toxoplasma gondii into the bloodstream and other tissues (such as liver, muscle, cardiac muscle, …) of intermediate hosts and its multiplication in nucleated cells may cause changes in plasma levels of various enzymes due to tissue damage. In present study the serum levels of AST, ALT, ALK/P, CPK, LDH, and ACP in rats infected experimentally with Toxoplasma gondii have been investigated.Materials and Methods: Totally, 116 uninfected rats were divided into 87 as case group and 29 as control group. The case group was infected intraperitoneally with 50000 tachyzoites. Blood specimens were taken from cases and its control once every 8 hours in the first three days and then once every three days for a period of 60 days and serum levels were measured for the mentioned enzymes. Results: During the study, the following changes were observed: AST in the first 8 hours, from the 32th hour till the 40th hour and from the 48th hour till the 56th hour; ALT in the first 8 hours and from the 48th hour till the 56th hour; ALK/P from the 24th hour till the 40th hour and from the 48th hour till the 64th hour; CPK and LDH from the 24th hour till the 40th hour and from the 48th hour till 56th hour; ACP from the 16th hour till the 48th hour. But afterward, whole offer mentioned enzyme shifted to normal levels.Conclusion: Alteration in serum enzyme levels of rats during infection with Toxoplasma gondii found is not permanent.

Objective: Sea cucumber is a traditional food and medical item and has been reported to exhibit Antioxidant, antifungal, anti tumoral and antibacterial bioactivity. The objective of this study is to describe the antibacterial activity of 4 extracts of Holoturia. Sp (sea cucumber), collected from Hengam Island of Persian Gulf. Materials and Methods: Methanol, hexane, aqueous and chloroform extracts from body wall tissue of the sea cucumber were screened for antibacterial activity against three strains of Escherichia coli Top 10 F´, TG1 and K12 using disc diffusion and broth microdilution tests methods.Result: The growth of all these strains were inhibited using concentration from 0.78 to 100 mg/ml of methanol, hexan and chloroform extracts. Among the extracts just methanol and chloroform with 100 mg/ml had bactericidal effect on TG1 and K12 strains. On the other hand, Aqueous extract had induced growth in of the all strains. Conclusion: The results suggest the possibility of applying sea cucumber as source of potential anti bacterial agents, whose compounds can be good candidates to make antibiotic products.

Objective: Utility of PCR-RFLP and species-specific PCR as novel and fast methods for identification and discrimination of causative agents of relapsing fever, Borrelia persica and B. microtii in infected blood were investigated. Materials and Methods: Genomic DNA of B.persica and B.microtii species were extracted from the highly infected blood samples. Two fragments of GlpQ and 16SrDNA genes were amplified using specific primers and then the PCR products were sequenced. Based on sequence variation between the two species, species-specific primers as well as restriction enzymes were respectively designed and selected for discrimination of these species.Results: The results showed that using PCR technique we could easily amplify and detect the Borrelia species within the infected blood samples. Two different profiles of RFLP were produced when GlpQ PCR products of B.persica and B.microtii treated by SspI, TaqI, DraI, HinfI, and EcoRV restriction enzymes. Also when 16SrDNA was digested with TaqI enzyme we could discriminate between these two species. Based on GlpQ sequence variation, a set of primer 795r-BMGLPF produced specific band of 451 bp for B.microtii and a set of primer 128f-BPGLPR produced specific band of 252 bp for B.persica which could discriminate the both species well.Conclusion: In this study the discrimination of the two species of B.persica and B.microtii was investigated by PCR-RFLP and species-specific PCR methods for the first time. Both methods could easily distinguish the species from each other. Due to accuracy and speed of the molecular methods, they could be replaced with the classic methods. These fast and accurate diagnostic methods could be recommended for diagnosis laboratories in Iran and middle-east countries where both B.persica and B.microtii are prevalent.

Objective: Diabetes mellitus is accompanied with higher incidence of cardiovascular disorders. There is some evidence on antidiabetic and cardiovascular improving potential of Crataegus spp (CS). Thus, the endothelium-dependent effect of oral administration of CS branchlet for 6 weeks on contractile and relaxatory response of thoracic aorta from diabetic rats was investigated.Materials and Methods: Male Wistar rats were divided into control, CS-treated control, diabetic, CStreated diabetic, and glibenclamide-treated diabetic groups. Treated groups received CS-mixed pelleted food at a weight ratio of 6.25%. Body weight and serum glucose level was measured before the study and at weeks 3 and 6. At the end of study, contractile reactivity of thoracic aortic rings to KCl and phenylephrine and relaxatory response to acetylcholine and sodium nitroprusside was determined using isolated tissue setup.Results: Serum glucose level significantly decreased in CS-treated diabetic group (p<0.01) versus untreated diabetics. In addition, endothelium-intact CS-treated diabetic group showed a significantly lower contraction to KCl and phenylephrine (p<0.05) as compared to diabetic group and endothelium removal abolished this response. Meanwhile, relaxation response of endothelium-intact rings to acetylcholine was significantly higher in CS-treated diabetic group as compared to diabetics (p<0.05). In addition, there were no significant changes amongst the groups regarding relaxatory response to sodium nitroprusside.Conclusion: Chronic oral administration of CS through affecting endothelial-related agents could decrease contractile response and enhance relaxatory response in aortic tissue of diabetic rat and this may be beneficial in prevention of long-term vascular complications of diabetes.

Objective: The greatest challenge in cancer gene therapy is to achieve the high specificity and efficiency in targeting of cancer cells. Because the goal of cancer gene therapy is to eradicate cancer cells, many therapeutic genes could be detrimental if unintentionally expressed in normal cells. Using promoter of the genes which are expressed specifically in cancer cells or have much more expression in cancer cells than normal cells, is very noticeable tool in cancer gene therapy (CGT). In this study we were searching for cancer specific promoter which could highly express therapeutic gene. Materials and methods: In order to apply a cancer specific promoter for creating a CGT construct, a promoter which has 34% similarity to Survivin core promoter was amplified from human genome by using Nested-PCR. Survivin is a member of anti-apoptotic gene and its over-expression was observed in up to 70% of breast cancers. This gene fragment contains two transcriptional binding sites which were similar to Survivin promoter according to the evaluation of Promoter Scan, EPD, Transfac, Compel and TRRD program. These binding sites were recognized by STAT1 and E2F transcription factors. This promoter was cloned into pCDNA3.1/Hygro+ plasmid in along with hypoxia and estrogen modules and pro-apoptotic gene tBid.Results: Semi-quantitative RT-PCR results of transfected cancer cells showed that this gene fragment (Survivin like promoter) have relatively same potential as CMV promoter to direct tBid gene expression.Conclusion: Utilization of chimeric promoter containing Survivin like promoter could be a promising tool in killing cancer cells naturally by inducing apoptosis. This construct is highly effective in transcriptional targeting of tBid in comparison to control construct.

Objective: Escherichia coli O157:H7 is a gram-negative rod-shaped bacterium. E coli O157:H7 is an enterohemorrhagic (EHEC) strain of the bacterium Escherichia coli and a cause of foodborne illness. Infection often leads to bloody diarrhea by producing a toxin called Shiga toxin, which damages the intestines, and occasionally leads to kidney failure, especially in young children and elderly people. A 2241 bp fepA gene of E.coli O157:H7 codes for production of a ferric enterobactin binding membrane protein that is essential for iron uptake by the bacterium. Inhibition of iron uptake can protect invasion of host by the bacterium. In this study we attempted to evaluate immunogenicity of the membrane protein, FepA.Materials and Methods: In order to produce recombinant FepA protein, the genomic, fepA gene of 2241 bp long was amplified by PCR from E coli O157:H7. The PCR product was ligated to pET28a. The recombinant protein was then expressed in E. coli BL21DE3 by IPTG induction. SDS-PAGE analysis was carried out and the recombinant protein was purified by Ni-NTA affinity chromatography. The purified recombinant protein was injected to Balb/C mice in order to induce immunity. Antibody titer was determined by ELISA. Results: The recombinant protein of 85 KD was produced and purified. Immunogenicity of the recombinant protein was determined by injecting Balb/C mice. The antibody produced therein could efficiently recognize and bind ferric enterobactin binding protein, thus heaving mice tolerance of 106LD50. Conclusion: With a view to the significant recognition by the antibody of ferric enterobactin binding protein, the notion of its application in restriction of enterobacteriacea propagation could be feasible.