Pathobiology Research
Year:2012 | Volume:14 | Issue:4|Papers Count:10

Objective: Antigen 85 complex of Mycobacterium tuberculosis includes three immunogenic proteins which are important TB vaccine candidates. The use of this protein as a component of subunit vaccines, as a part of DNA vaccines or recombinant BCG boosters, enhances their recombinant production. Recombinant production of these mycobacterial proteins located in the cell wall somehow differs from the other proteins, as they are partially apolar. Therefore, this study aims to produce recombinant Ag85C as a vaccine candidate. Methods: Ag85C gene was cloned in pJET1.2 and subsequently in pET32a (+). Both recombinant plasmids were sequenced. Expression of the recombinant protein was induced with 1mM IPTG. Recombinant Ag85C was purified through dissolving the inclusions in 8M urea buffer, absorbed to Ni-NTA resins, washed by buffers with decreasing urea concentrations, and finally eluted in aqueous solution. Western blot analysis was performed using anti-6His tag antibody, rabbit anti-M. tuberculosis polyclonal antibody, and the serum from hospitalized TB patients.Results: Ag85C successfully cloned in both plasmid vectors. The recombinant Ag85C expressed in the E. coli host and was purified with significant yield.Conclusion: Western blot results along with that of sequencing ensure accurate production of recombinant Ag85C, retaining its partial epitopes.

Objective: The use of antiretroviral drugs has proven remarkably effective in controlling the progression of human immunodeficiency virus (HIV) disease, but these benefits can be compromised by the development of drug resistance. This study aims to assess the drug resistance profile of the Pr gene in highly active antiretroviral therapy (HAART)-treated and naïve HIV-1 infected patients. Methods: A total of 30 samples from naïve and 16 samples of highly active antiretroviral therapy (HAART)-treated patients were collected and divided into two groups. After RNA extraction, RT nested PCR was performed. The final products were sequenced and then analyzed for drug-resistant mutations and subtypes.Results: No drug resistant mutations were noted in group one that have never used drug, but 40% of group two samples which are under treatment contained drug resistant mutations. According to the results, the following subtypes were seen among patients: A (50%), B (40.6%), D (6.2%), and C (3.2%).Conclusion: Transmission of drug-resistant viruses and their detection are very important epidemiologically. However our data and other studies suggest that other PIs should be replaced by LPV in the HAART regime.

Objective: Menstrual blood stromal stem cells (MBSCs) share some phenotypic and functional similarities with mesenchymal stem cells (MSCs). MSCs are shown to inhibit either the function or generation of different immune cells, including dendritic cells (DCs). However, data regarding MBSCs’ potential effects on immune system cells are elusive. Here, we examine whether MBSCs affect the generation of human monocyte-derived DCs. Methods: Menstrual blood samples were collected from apparently healthy women on the second day of their menstrual cycles. The adherent portions were subcultured to omit unwanted cells and obtain MBSCs. Magnetically-isolated peripheral blood monocytes were differentiated towards DCs through treatment with recombinant granulocyte monocyte colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) in the presence or absence of MBSCs. After five days, monocyte-derived DCs were analyzed for the expression of surface markers by flow cytometry. IL-6 level was determined in the co-culture supernatants.Results: Co-culture with MBSCs significantly down regulated the expression of DC marker (CD1a) and up regulated the expression of monocyte marker (CD14) on monocyte-derived DCs compared with the control group. IL-6 level was shown to be significantly higher in the supernatant of the monocyte-MBSC co-culture.Conclusion: Collectively, this is the first study to show the inhibitory impacts of MBSCs on the generation of DCs. IL-6 could be viewd as a potential factor mediating this effect. Regarding the known advantages over MSCs, MBSCs could be considered as a promising future candidate for immunomodulatory purposes in the clinical setting.

Objective: Influenza virus A (H1N1) is an important subtype of the influenza respiratory viruses, which has important worldwide implications. Hemagglutinin (HA), an important viral antigen, is responsible for binding to human cell receptors leading to an onset of the disease process. Considering the critical role of viral attachment, this study focuses on the extraction and cloning of HA and its large subunit HA1 genes to generate recombinant baculovirus shuttle vectors (bacmid) in order to produce recombinant proteins in insect cells. Methods: Human influenza virus A/New Caledonia 99/20/(H1N1) was propagated in MDCK cell culture. Total viral RNA was extracted using easy-red solution. The full-length HA genome and HA1 fragment were amplified by RT-PCR using specific primers, cloned into a pGEM®-TEasy vector, and then subcloned into a pFastBac HT plasmid. Finally, recombinant bacmids that contained the genes of interest were produced in E. coli DH10BacTM cells.Results: Expected PCR products of HA genes were evaluated through gel electrophoresis and restriction enzyme analysis. Recombinant pGEM®-TEasy vectors and pFastBac HT donor plasmids were confirmed by PCR, digestion, and sequencing. Construction of recombinant bacmid DNA was verified by using blue-white colony screening, overnight electrophoresis, and PCR analysis that used either pUC/M13 or gene-specific primers. Conclusion: In this study, we have successfully constructed recombinant Bacmid DNA that encoded the full-length HA genome and its HA1 subunit. We intend to transfect sf9 insect cells with these constructs to generate recombinant baculovirus and produce large amounts of desired proteins for future studies.

Objective: Infection with herpes simplex virus type 1 induces viral latency in neuron trigeminal ganglions. The late associated transcript (LAT) is uniquely expressed in infected neural cells, however no coding protein associated with these transcripts has been identified in infected cells. It has been shown that six microRNAs transcribed from LAT have the capabilities to affect the cell signaling pathways, thus interfering in pathways such as those of cell differentiation and proliferation. Transforming growth factor beta (TGF-b) pathway is a critical pathway among cell signaling circuits. The Smad4 protein, as an important member of the TGF-b signaling pathway, mediates the connection between membrane receptors, cytoplasmic kinases, and nuclear transcription factors.Methods: This study bioinformatically and experimentally evaluated LAT microRNA expression and assessed microRNA targeting of Smad4 transcripts in human neuroblastoma cells by using real-time PCR.Results: Analysis of two different software results showed that the Smad4 gene was targeted by LAT-derived microRNAs at multiple sites. Over-expression of LAT microRNAs in BE2(c) cells caused reduction in Smad4 transcripts.Conclusion: The results of bioinformatical analysis with relative quantification of Smad4 transcripts and its downstream-related genes such as cyclinD, CDK2, and Myc showed that the LAT transcript could control Smad4 expression.

Objective: Animal studies show that vaccination with epitope-based peptides results in protective immunity. However, immunodominance should be regarded as a major challenge in this area. Considering the advantages of epitopic-vaccines against hepatitis C virus (HCV) infection, herein, we compared the occurrence of immunodominance following mice immunization with three different HCV epitopic-peptide formulations.Methods: We synthesized four CD8+ epitopic-peptides (C1, E6, N, E4) that were derived from HCV-antigens. A polytope-peptide (C1E6NE4) spanning fusion of epitopes was designed based on immunoinformatics analyses for optimum proteasomal cleavage. BALB/c mice received three subcutaneous injections that contained 10 mg of peptide (minimal epitopes, or mixture of four epitopes or long-polytope) formulated with CpG (50 mg) and Montanide-ISA720 (70%) adjuvants in the tail-base at three-week intervals. Considering the H2-Dd (BALB/c)-restriction of C1 and E4-epitopes, three weeks after the last injection splenocytes from vaccinated animals were subjected to IFNg/IL4 ELISpot assays in the presence of C1 and E4-peptides.Results: All vaccinated animals promoted Th1-oriented responses as evidenced by detection of IFNg-secreting cells and a low-level of IL4 secretion. Mice injected with minimal CTL-epitopes provoked stronger responses, however, due to the higher affinity of E4-epitope for H2-Dd, frequency of E4-specific cells was considerably higher than C1-specific ones, showing some level of immunodominance. Interestingly, animals vaccinated with polytope-peptide developed high-quality balanced responses against both C1 and E4-epitopes, however at a lower intensity.Conclusion: These results supported the superiority of polytope-peptides over minimal epitopes, yet emphasized the key role of polytope design and optimization to avoid epitope dominancy.

Objective: This study investigated the effects of adipose-derived mesenchymal stem cells (MSCs) and conditioned medium injection on cell infiltration in the brains of an experimental C57BL/6 mice model of autoimmune encephalomyelitis (EAE).Methods: EAE was induced with myelin oligodendrocyte glycoprotein (35-55 peptides) in 20 mice. MSCs were obtained from the adipose tissue of C57BL/6 mice and cultured with Eagle’s minimum essential medium/alpha medium (DMEM) after removal of non-adherent cells. After 2 to 3 passages, 15 days after induction of EAE, 4 groups (n=5) of C57BL/6 mice were treated as follows: i) MSCs were injected intraperitoneally, ii) MSCs were injected intravenously, iii) conditioned medium was administrated intraperitoneally, and iv) the control group received no treatment. After day 60, the mice brains were removed and the effect of adipose-tissue MSCs and administration of conditioned medium was investigated.Results: Leukocyte infiltration and clinical scores were significantly reduced in animals that received MSC and conditioned medium compared to untreated animals. Body weight increased significantly in the treated groups compared to the control group. Percentage of survival also increased in the animals that received MSCs and conditioned medium as compared to the control group.Conclusion: MSCs had immunomodulatory and neurogenerative functions which reduced leukocyte infiltration and improved clinical scores in the EAE animals that received MSC and conditioned medium compared to untreated animals with EAE.

Objective: Bacillus cereus (B. cereus) is a gram-positive, spore-forming bacteria widely distributed in the environment. This bacteria is an opportunistic human pathogen that can cause diarrheal and emetic types of food poisoning. The diarrhea type of food poisoning can be caused by hemolysin BL (HBL), non-hemolytic (NHE), and cytotoxin K enterotoxins. Rice is commonly contaminated with B. cereus. The objective of this study is to detect enterotoxigenic genes of the NHE complex and assess their incidence in B. cereus isolates in rice samples from Zanjan, Iran.Methods: We randomly purchased 10 different rice samples from food stores and cultured them in PEMPA. Following biochemical testing, the bacterial colonies were identified by PCR. B. cereus isolates were checked for the NHE complex genes by specific primers using multiplex PCR.Results: Results showed that rice samples were contaminated with B. cereus. The NHE complex genes were found in 8 bacterial samples.Conclusion: B. cereus is able to tolerate high temperatures; in cooked rice the spores can undergo germination by reheating. The results of this study have shown that NHE multiplex PCR is a prompt, reliable method for the differentiation between non-enterotoxigenic and enterotoxigenic isolates of B. cereus. Despite its common dietary role, rice in Iran has rarely been investigated from a microbiological point of view. Enhancing awareness about virulence and prevalence of genes involved in food poisoning would be effective in the prevention of food poisoning.

Objective: Traditional methods for storing microbial DNA samples use the low-temperatures of 4oC, -20oC or -80oC. The DBC Card is a new method of storing microbial DNA at room temperature. This study evaluates the stability and determine the best storing method of Bacterial DNA on the DBC card.Methods: In this study, we used a pair of primers from the conserved domain of 16srRNA designed to identify Escherichia coli. Four different types of samples we prepared: i) bacterial suspension, ii) bacterial DNA extracted by the phenol-chloroform method, iii) bacterial lysate with lysis buffer and iv) bacterial DNA produced by the boiling method. All four samples were spotted on separate DBC cards and dried at room temperature. After periods of 3, 5 and 7 months, Escherichia coli samples were checked for DNA stability with two molecular techniques, conventional PCR with 1, 2 and 3 disks as a source of bacterial DNA (1 mm diameter) and Real-Time PCR.Results: Data showed that DNA stability was maintained after 7 months on a DBC disk, even using only one disk as a DNA source. The bacterial suspension was the best method for long-term storage of Escherichia coli DNA on the DBC Card.Conclusion: In traditional methods for storing sample, DNA quality reduces after freezing and thawing. However in the DBC method, DNA quality was maintained for a long duration. Advantages of the DBC method are easy sample handing, low cost, faster extraction, and reduced individual and environmental contamination.