Pathobiology Research
Year:2012 | Volume:15 | Issue:2|Papers Count:9

Objective: Leishmaniasis is one of the significant causes of morbidity and mortality in several countries. It is an important problem in endemic areas such as Iran. The goal in treatment of leishmaniasis is to reduce the disease period and leave no evidence of any remaining scars or lesions. A derivative of artemisinin is artemether. Scientists believe that the strong action of artemether against parasites is due to the presence of an endoperoxide bridge. Due to problems in the treatment of Leishmania major, in this research we have studied the effect of artemether on Leishmania major under in vitro conditions.Methods: Parasites were cultured in NNN and RPMI, after which artemether at concentrations of 5, 10, 25, 50 and 100 mg/ml were used for the promastigote assay. Apoptosis was detected by flow cytometry and DNA ladder assay.Results: The inhibitory concentration (IC50) of artemether was determined to be 25 mg/ml. The percentage of apoptotic promastigotes at 25 mg/ml of artemether was 42.28. The results of DNA fragmentation show that exposure of Leishmania major promastigote cells to 25 mg/ml of artemether lead to DNA fragmentation.Conclusion: We have proven the effect of artemether on apoptosis of Leishmania major by flow cytometry and the DNA ladder assay.

Objective: In order to overcome the limitation of systemic administration of methylene blue, this study investigated the encapsulation of methylene blue in polymeric liposomes and drug release following sonication.Methods: We encapsulated methylene blue into nanoliposomes. The dynamic light scattering (DLS) method was used to measure the size distribution of the liposomes. After loading methylene blue into these liposomes, both drug encapsulation efficiency and stability were fluorometrically determined. Biodistribution of drug was studied in vivo in a mouse model of adenocarcinoma tumor cells. The amount of drug released upon 1 MHz sonication at an intensity of 2 W/cm2 was fluorometrically verified in vitro.Results: DLS studies showed that the synthesized liposomes had an average size of 66.19±4.49 nm. Methylene blue was efficiently encapsulated in nanoparticles at an average of 65.21±3.47%. Stability of the generated liposomes decreased with time. Biodistribution study revealed that the drug content in the group that received liposomal drugs in their tumor tissue was significantly higher than in the group that received methylene blue in its free form and in the heart was inverse (P<0.05). The results indicated that a 5 min application of 1 MHz ultrasound caused a methylene blue release of approximately 51.8±8.3% from the nanoparticles.Conclusion: This study has shown that fabricated liposomes are suitable for the encapsulation and delivery of hydrophilic photosensitizers such as methylene blue. Ultrasound-triggered release was achieved by the use of a 1 MHz ultrasound.

Objective: Demyelination of CNS axons occurs under pathological conditions such as multiple sclerosis and spinal cord injuries, but can be repaired by cell therapy. Within the CNS remyelination can be achieved by transplantation of neural stem cells (NSCs). NSCs are self-renewing cells that maintain the capacity to differentiate into CNS-specific cell types and can differentiate into the three main neural phenotypes: astroglia, oligodendroglia and neurons. They may also replace or repair diseased CNS tissue.Methods: Bone marrow stromal cells (BMSCs) were aseptically isolated from the tibia and femurs of young adult Sprague Dawley rats. BMSCs were evaluated by fibronectin and CD31 markers. BMSC-derived NSCs were evaluated by nestin and NF-68. An ethidium bromide-induced demyelinated dorsal column lesion was produced in young adult rats. Transplanting NSCs derived-BMSCs into demyelinated lesions after 3 days in adult rat spinal cords was done. Three weeks after transplantation of NSCs, the spinal cords were processed to evaluate remyelination by Luxol fast blue staining.Results: After passage 3, BMSCs were evaluated and the result, showed the percentage of immunoreactive cells to fibronectin (94.7±2.65), however BMSC-derived NSCs expressed nestin (86.15±0.64) and NF-68 (84.55±0.94) which correlated with fibronectin down regulation. Histologically, the lesions showed slightly irregular elongated areas and had an average length of 1336.36±39.43 mm. Transplanted NSCs were capable of eliciting remyelination.Conclusion: These data support the conclusion that transplantation of NSCs results in functional remyelination of a dorsal column lesion and have valuable applications in the treatment of neurodegenerative diseases such as spinal cord injuries.

Objective: Titration of viruses is important to determine the quantity of virus in vaccine development, master virus seed stock preparation, viral vector studies and virus replication. In this study, we compared the CCID50% and plaque assay as a standard titration method for rotavirus (RF) and HSV-1.Methods: The MA104 and Vero cells were inoculated by RF and HSV-1 in 6- and 96-well plates. Following infection and adsorption, the optimal time for the cytopathic effect caused by the viruses was noted and the results compared.Results: The CPE (Cytopathic Effect) of RF was observed in less than 18 hours, which increased until 72 hours after inoculation. In HSV-1, the CPE was observed 24 and 72 hours after inoculation. The virus titration in the plaque assay was monitored at 96 hours post-infection for RF and at 72 hours post-infection for HSV-1. In both viruses the plaque titer method was lower than the CCID50 method, since the results indicated that 1 CCID50% was equal to 0.7 PFU.Conclusion: The plaque assay is one of the most accurate methods for viral titration. For the plaque assay, individual lesions may be isolated, which the plaques can be counted. The CCID50% method is not applicable for purification of homogenous viruses, nor is this technique reproducible.

Objective: One of the major issues in bone tissue engineering is the design and fabrication of bioactive, bioresorbable porous 3D scaffolds capable of maintaining their structure and integrity over a predictable period of time. One such approach is the fabrication of composite scaffolds.Methods: In this study we present fabrication and characterization of novel silk/bioglass-composite scaffolds. Regenerated fibroin was constructed from mulberry silk cocoons and calcium silicophosphate bioactive glass was made by sol-gel processing. For fabrication of a homogenous composite, grained bioglass particles were modified with 3-aminopropyltriethoxysilane coating. Fibroin/bioglass composite scaffolds were fabricated by the freeze-dry technique at different concentrations.Results: Silk protein extract was evaluated by FTIR and XRD methods. FTIR spectrum showed sharp amide peaks at 1655 cm-1 and 1530 cm-1 wave lengths, which confirmed the existence of fibroin. XPS analysis demonstrated that the amino groups were established on the surface of the glass powder. The fabricated 3D scaffolds were morphologically analyzed by scanning electron microscopy, which showed uniformly dispersed bioglass particles in all structures. Scaffolds were seeded with human mesenchymal stem cells for 21 days.Conclusion: Considering the cytocompatibility of the scaffolds and osteogenic differentiation during three weeks, it could be concluded that the appropriate combination of structural and biological properties make the silk/bioglass composite scaffold a probable choice for potential use in bone tissue engineering.

Objective: In an attempt to develop safer and more effective gene therapy approaches as a realistic treatment for various forms of cancer, researchers are increasingly using tumor-specific promoters (TSP) to drive the expression of the gene of interest and eradicate cancer cells. In this study, for the first time we introduce the Oct-4 promoter as a cancer-specific promoter with a high efficacy.Methods: We cloned Oct-4 promoter and enhancers into a pGl3 control reporter vector and analyzed the expression of luciferase as a reporter gene in an AGS gastric cell line. Next, we used a suicide-inducing vector that included an Oct-4 promoter and the TK gene in the presence of the non-toxic prodrug, ganciclovir, to eradicate cancer cells. Cells were treated with PI and connexin V. FACS analysis was conducted to assess the effect of the system on cell cycle and apoptosis induction.Results: Under the activity of the Oct-4 promoter, luciferase expression was three-fold higher than the SV40 promoter. The HSV/TK/GCV system activated by the Oct-4 promoter and enhancers induced apoptosis (86.17%) in the AGS cell line. We verified that this system induced S-phase/G2-phase cell cycle arrest in the AGS cell line.Conclusion: These data indicate that the Oct-4 promoter is active in the AGS cell line. Oct-4 gene is expressed in a wide variety of tumors but not in normal cells. Thus, Oct-4 appears to be a promising tumor-specific promoter for numerous tumors.

Objective: Understanding gene expression variations by using RNA transcript analysis methods during hepatic viral protein interactions with the IFN pathway in hepatic cell lines has recently gained importance. One of the most powerful techniques in gene expression quantification is quantitative real-time RT-PCR. Reference genes used as normalizer in this method may be affected across various experimental conditions or treatments. Hence, in the present study, the influence of IFN-a treatment on the mRNA levels of common reference genes including ACTB, GAPDH, TBP, HPRT1 and HMBS was evaluated in Huh-7 or HepG2 cell lines.Methods: Cells were treated with different concentrations of IFN-a. Then, using geNorm and NormFinder programs, we evaluated the expression stabilities of the above prominent reference genes in three sample groups that included each hepatic cell line and the total data sets.Results: HPRT-1 and GAPDH were the most stable reference genes in the Huh-7 cell line, whereas ATCB, HMBS and GAPDH were the most stable in the HepG2 cell line. TBP was one of the least stable reference genes in the three studied groups.Conclusion: This investigation will provide appropriate reference genes for standardization of quantitative real-time PCR data in an IFN-a stimulated model of hepatocyte cell lines.

Objective: Dermatophytosis is one of the most common pandemic fungal infections that is a major health problem in cities and villages. This study aims to evaluate PCR sensitivity and accuracy in the detection of nail dermatophytosis compared to conventional direct and culture detection methods, and performs an assessment of Trichophyton rubrum in patients suspected of having nail dermatophytosis.Methods: This experiment was a descriptive-experimental study carried out on 71 nail samples obtained from patients with suspected nail dermatophytosis. All clinical samples of nails or chips were divided into three sections and each section underwent direct examination, culture and molecular tests. In the molecular test, we used fungal rRNA universal primers (ITS1 and ITS4) and Trichophyton rubrum-specific primers.Results: In this study, for the first time in Iran and based on a modified protocol, DNA was directly extracted from tissues of infected nails in less than five hours. Additionally a comparison of the results obtained from routine laboratory methods such as direct examination and culture with PCR verified the high sensitivity and accuracy of PCR compared to the other studied methods.Conclusion: PCR, as a rapid, accurate method, can be a good replacement for conventional culture and direct examination.