Co-solute assistance in refolding of recombinant proteins

Prokaryotic expression system is the most widely used host for the production of recombinant proteins but inclusion body formation is a major bottleneck in the production of recombinant proteins in prokaryotic cells, especially in Escherichia coli. In vitro refolding of inclusion body into the the proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct refolding. Co-solutes play a major role in refolding process and can be classified according to their function as, aggregation suppressors and folding enhancers. This study presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial process.Key words: Refolding, protein aggregation, low-molecular-weight additives, arginine

CD8+ T Cells as a Source of IFN-γ Production in Human Cutaneous Leishmaniasis

Cutaneous leishmaniasis (CL) is usually a self-healing skin lesion caused by different species of Leishmania parasite. Resistance and susceptibility of mice to Leishmania major infection is associated with two types of CD4+ T lymphocytes development: Th1 type response with production of cytokine IFN-γ is associated with resistance, whereas Th2 type response with production of cytokines IL-4 and IL-5 is associated with susceptibility. A clear Th1/Th2 dichotomy similar to murine model is not defined in human leishmaniasis and we need as much information as possible to define marker(s) of protection. We purified CD4+/CD8+ T cells, stimulated them with Leishmania antigens and analysed gene and protein expression of Th1/Th2 cytokines in volunteers with a history of self-healing CL who are presumed to be protected against further Leishmania infection. We have seen significant upregulation of IFN-γ gene expression and high IFN-γ production in the Leishmania stimulated CD4+ T cells and CD8+ T cells. We concluded that both antigen-specific IFN-γ producing CD4+ Th1 cells and IFN-γ producing CD8+ T cells contribute to the long term protection in individuals with a history of CL. This proves the importance of CD8+ T cells as a source of IFN-γ in Th1-like immune responses

Advantage of using a home-made elisa kit for detection of Helicobacter pylori infection over commercially imported kits

Purpose: To evaluate a home-made ELISA kit for detection of Helicobacter pylori (Hp) infection and comparison of its immunologic criteria with those of foreign commercial kits. Methods: A home-made IgG ELISA kit was developed using soluble antigenic fractions of Hp proteins. Confirmed sera were tested and serological criteria were evaluated through assessment of 199 serum samples. Results: The accuracy, sensitivity and specificity values of home-made kit were 92, 92 and 90.4%, respectively. These immunologic criteria for Trinity kit were 95.2, 95.2 and 95% in comparison with IBL kit (91.3, 92.2 and 88.5%), BIOHIT kit (72.4, 41.6 and 94.1%) and HelicoBlot2.1 (94.2, 93.4 and 100%). Kappa agreement assessment demonstrated that two of the imported ELISA kits had fair to moderate agreement with the home-made kit while the other one had a poor agreement value. Conclusions: Apart from comparable values between the home-made kit and the most efficient imported kit (Trinity) there was significant cost benefit. Therefore, we recommend the home-made kit as a suitable substitution for detection of Hp infection in the Iranian population

Pre-Symptomatic Human Cytomegalovirus Disease Diagnosis in Renal Transplant Recipients by the Virus DNA PCR

Human cytomegalovirus (HCMV, CMV) is a major infectious complication of renal transplantation. The objective of this survey was to optimize and establish a polymerase chain reaction (PCR) technique for rapid and early detection of CMV disease in renal transplant recipients. In a cross sectional study, a total of eighty-one EDTA-blood samples were collected as simple nonrandomized (sequential) weekly from thirty-seven renal transplant recipients during a 1-6 months period after their transplantation in Kidney Transplant Center of Shaheed Labbafinejad Hospital of Tehran. Peripheral blood leukocytes (PBLs) were isolated and DNA was extracted. HCMV DNA in PBLs was detected by PCR using a conserved set of primers. Amplified fragment was confirmed by restriction fragment length polymorphism (RFLP) and sequencing. Correlation between PCR results and patients’ data was analyzed. Twelve patients from thirty-seven renal transplant recipients had positive samples containing HCMV DNA in PBLs (32.4%), whereas, five of them showed symptomatic CMV disease (13.5%) and seven of them did not show symptomatic CMV disease, but had some signs of pre-symptomatic CMV disease. Twenty-five patients had negative PCR results, and all of them did not have symptomatic CMV disease. Considering type one error (α = 0.05), a nonparametric Fisher’s exact test showed a good correlation between two variables of positive PCR results and symptomatic CMV disease in renal transplant recipients (P=0.002). In conclusion, establishing methods for early detection of HCMV DNA, even prior to showing symptomatic CMV disease, has been shown to be an effective way for starting antiviral therapy, prior to patients’ experience of symptomatic CMV disease

Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

Background: Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines espe­cially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression.Methods: L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 μg of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR–tansfected promastigotes. West­ern blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits.Results: The PTR1 protein was not expressed in pcDNA-rPTR– tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR– tansfected promastigotes.Conclusion: This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis

PCL/PVA nanofibrous scaffold improve insulin-producing cells generation from human induced pluripotent stem cells

Pancreatic differentiation of stem cells will aid treatment of patients with type I diabetes mellitus (T1DM). Synthetic biopolymers utilization provided extracellular matrix (ECM) and desired attributes in vitro to enhance conditions for stem cells proliferation, attachment and differentiation. A mixture of polycaprolactone and polyvinyl alcohol (PCL/PVA)-based scaffold, could establish an in vitro three-dimensional (3D) culture model. The objective of this study was investigation of the human induced pluripotent stem cells (hiPSCs) differentiation capacity to insulin-producing cells (IPCs) in 3D culture were compared with conventional culture (2D) groups evaluated at the mRNA and protein levels by quantitative PCR and immunofluorescence assay, respectively. The functionality of differentiated IPCs was assessed by C-peptide and insulin release in response to glucose stimulation test. Real-Time PCR results showed that iPSCs-IPCs expressed pancreas-specific transcription factors (Insulin, Pdx1, Glucagon, Glut2 and Ngn3). The expressions of these transcription factors in PCL/PVA scaffold were higher than 2D groups. In addition to IPCs specific markers were detected by immunochemistry. These cells in both groups secreted insulin and C-peptide in a glucose challenge test by ELISA showing in vitro maturation. The results of current study demonstrated that enhanced differentiation of IPCs from hiPSCs could be result of PCL/PVA nanofibrous scaffolds. In conclusion, this research could provide a new approach to beta-like cells replacement therapies and pancreatic tissue engineering for T1DM in the futur