[Comparative study on xnograft saffold and prous hydroxyapatite/tricalcium phosphate for the repair of bony defect]

The aim of this study was to compare xenograft scaffold and prous Hydroxyapatite/Tricalcium phosphate for the repair of bony defect. 5 New Zealand rabbits with the average weight of 2 kg were used. The rabbits were anesthetized with ketamine 60 mg/kg and xylazin 6 mg/kg After creating a critical-sized segmental defect [2x3 cm] in tibia bones, an implant of HA/TCP with osteoblasts in the hole of left tibia and an implant of xenograft with osteoblasts in the hole of right tibia was inserted. At the end of the second month, the animals were sacrificed. To follow up the new bone formation, x-ray images were taken at 8[th] week post-operation. To confirm the tissue repairment, the histology of repaired defects was evaluated with H and E staining after decalcification. The present study demonstrates that the critical-size segmental defect of tibia can be repaired with both synthetic HA/TCP and xenograft implants. xenograft implants showed better osteointegration compared to HA/TCP phosphate

Human calprotectin, a potent anticancer with minimal side effect

Chemotherapy by using agents such as etoposide is a common way for inhibition of tumors. This treatment is accompanied by many undesirable side effects. Calprotectin is an abundant protein in the neutrophil cytosol, it has growth-inhibitory and apoptosis-inducing activities against various cell types such as tumor cells. In this study to introduce calprotectin as a suitable substitute anticancer, its growth inhibitory effect on human gastric adenocarcinoma cell line [AGS] and human foreskin fibroblast cells [HFFF] is compared to etoposide effect on these two cell lines. Calprotectin was purified from human neutrophil by chromatography methods. AGS and HFFF cell lines were used. These cells were maintained in RPMI 1640 medium supplemented with 10% FCS in a humidified incubator [37 §C and 5% CO2]. AGS cells [10000 cells per well] were exposed to the different concentrations of calprotectin and etoposide for 24 and 48 h. MTT assay was used for evaluation of cytotoxicity. Results indicate that calprotectin has more potent anticancer activity in comparison to the etoposide but it has nearly similar inhibitory effect on the proliferation of fibroblast cells. Since calprotectin affect about 20 times more than etoposide on cancer cells, without any additional side effect, it can be concluded that it is a suitable candidate to be studied as anticancer drug

[Induction of heme oxygenase -1 by lipocalin 2 mediated by NF-kB transcription factor]

Effect of lipocalin 2 on the expression of heme oxygenase I, II and NF-kB transcription factor was the purpose of this survey. Lcn2 was cloned to pcDNA3.1 plasmid by using genetic engineering method. The recombinant vector was transfected to CHO and HEK293T to establish stable cell expressing lipocalin 2. The presence of lipocalin 2 gene in these cells was confirmed by using through RT-PCR and western blot analysis. Expression of Lcn2 was also down-regulated by siRNA in A549 cell line. Expression of heme oxygenase I, II and NF-kB transcription factor were determined in both ectopic expression Lcn2 cells and Lcn2 down regulated cells by using of RT-PCR and western blot analysis. The results showed that the expression of heme oxygenase I and those of NF-kB were higher in cells expressing recombinant lipocalin2 compared with the control cells. On the other hand, expression of heme oxygenase I and NF-kB in siRNA transfected cells was down-regulated. These findings indicate that lipocalin2 induces the expression of HO-1 and suggest Lcn2 through NF-kB induces HO-1 expression

Isolation, cloning and expression of recombinant human FVII in baculovirus expression system

Background and purpose: Factor VII is one of the important coagulation factors in extrinsic blood coagulation pathway, which can resolve the use of FVIII and FIX for hemophilia patients by activating FX. Recombinant expression of this factor can eliminate the potential problems in preparing those factors from plasma and the risk of transferring hematological diseases. Therefore, the present study intended to investigate the expression of recombinant FVII at a higher level using Gateway technology and TOPO cloning

Methods and Materials: In this experimental study, Factor VII cDNA was isolated from HepG2 cell line by PCR, and cloned to prokaryote TOPO vector by TOPO cloning reaction. The recombinant vector was extracted for bacterial colonies after screening, and was used in Gateway adapted Baculovirus DNA by LR recombination reaction. The recombinant virus was transfected onto insect cell line, and the expression of the protein was analyzed after necessary screening. Findings of the protein expression via ELISA were presented in triadic [Mean +/- SD]; the differences across the three groups were investigated using Student t-test

Results: Cloning and recombination reaction analysis by PCR determined cloning of rFVII in high accuracy [90%] in the vectors. High level expression of recombinant FVII was confirmed by SDS-PAGE, ELISA, and Western blot analysis [30g/ml]. The highest expression level was produced on the 7th day after transfection [1.960 +/- 0.076]. Determined by ELISA, this result was negatively significant in the transfected sample [P<0.001]

Conclusion: Findings of the analysis of the recombinant protein expression by Baculovirus expression system indicated its production in a larger scale than similar eukaryote and prokaryote expression systems

Introduction of an indirect haemagglutination test as a rapid diagnostic method in comparison with ELISA using antigen B for diagnosis of human hydatid disease

Early diagnosis of human hydatid disease by detecting the specific antibodies in patients' sera is considered as an important step in treatment of infection. But the diagnostic efficiencies of assays greatly depend on the characteristics of antigen that is used and various conditions in performance. In present study, we tried to st and ardize an indirect haemagglutination test, using antigen B for diagnosis of hydatid disease. Sera from 80 patients with surgically confirmed hydatidosis and 40 sera from healthy donors were examined. To detect the cross-reactant antibodies, 53 sera from patients with other parasitic infectious and diseases were applied in this study. IHA was performed with sheep RBC that was sensitized by various concentrations of crude antigen and antigen B. The best results were obtained by IHA with applying antigen B [10 micro g/ml] for 40 min at 37°C or 60 min at room temperature. Diagnostic value of antigen B [sensitivity 93.75%, specificity 100% and efficiency 97.12%] was significantly higher than related value of crude antigen [sensitivity 65%, specificity 100% and efficiency 83.81%] in IHA under the optimum condition. Sensitivity and specificity of ELISA using crude antigen [10 micro g/ml] were obtained 80% and 94.62%, respectively. Corresponding values of ELISA using antigen B were also obtained as 72.5% and 98.92%, respectively. It is suggested that the IHA, as a serological assay, is a valuable method with high diagnostic efficiency for serodiagnosis of hydatid disease, when is performed by purified antigen B. It is a rapid diagnostic assay with any needs neither for expensive instruments nor expert personnel so is useful for seroepidemiological studies and field trial in endemic areas

Uptake of autologous and allogenic tumor cell antigenic by dendritic cells

Dendritic cells [DCs] are professional antigen presenting cells [APCs], and there is considerable interest in their application as a cellular adjuvant for cancer immunotherapy. Previous studies indirectly demonstrated that DCs were able to take up tumor lysate [crude soluble tumor antigens] and also cross-present tumor associated antigens [TAA] which elicits anti-tumor immune response. To provide direct evidence that demonstrates the uptake of tumor lysate by DCs and to find out whether this capability is restricted to allogenic or autologous tumor lysate preparation. DCs were generated from magnetic bead-isolated monocytes of B-CLL patients as well as healthy donors. Proteins of tumor lysate were conjugated with FITC. Their uptake by autologous as well as allogenic DCs was analyzed using FACS flowcytometry system. In both autologous and allogenic experiments, green fluorescence intensity [FL1] of immature DCs incubated with FITClabeled tumor lysate was clearly higher than unpulsed counterparts, which were considered as background. Immature DCs are able to efficiently take up FITClabeled tumor lysate of autologous as well as allogenic sources. This finding confirms the results of previous studies, which have demonstrated that tumor lysate-pulsed DCs were able to elicit cytotoxic anti-tumor response and concluded that DCs could take up tumor lysate

Unresponsiveness to recombinant hepatitis B vaccine in healthy Iranian neonates: Association with HLA antigens

Hepatitis B is an important infectious disease. Since several years ago, mass vaccination against this viral infection has become as part of routine vaccination schedule of Iran. However, some healthy neonates, children and adults fail to generate a protective antibody response after vaccination. To investigate distribution of HLA class-I and class-II antigens in healthy Iranian neonates vaccinated with recombinant hepatitis B vaccine. HLA-typing was performed on peripheral blood lymphocytes isolated from 25 responder and 23 nonresponder [anti-HBs < 10 IU/L] healthy neonates, using the standard microlymphocytotoxicity method. Anti-HBs antibody was quantitated by sandwich ELISA. The frequency of HLA-DR7 [p<0.01], DQ2 [p<0.02] and DR13 [p<0.05] was significantly higher in the nonresponder neonates compared to the responder group. The DR1 and DQ3 antigens were over-represented [p<0.05] in the responder vaccinees, implying positive association with the anti-HBs antibody response. Statistical analysis revealed increased frequencies of B7-DR7-DR53-DQ2 and DR13-DR52-DQ2 haplotypes in the nonresponder neonates [p<0.05]. Conclusions: We found a significant association between lack of antibody response to recombinant hepatitis B vaccine and expression of certain HLA class- II antigens in healthy Iranian neonates