increase in protein and plasmid yields of E. coli with optimized concentration of ampicillin as selection marker

Background: Escherichia coli is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in E. coli, but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid

Objectives: Alterations of protein expression and plasmid yields of E. coli in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized

Materials and Methods: The expression cassette of codon optimized EGFP for E. coli was synthesized in pUC57. The pUC57-GFP was transformed into E. coli Top10F'. The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG [0.5 mM] and incubation with 0, 100, 200 and 300 micro g.mL[-1] ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve

Results: GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 micro g.mL[-1] ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07x10[9], 3.21x10[9], 2.32x10[10] , 8.11x10[8], respectively. The plasmid yields were 55 ng.micro L[-1], 69 ng.micro L[-1], 164 ng.micro L[-1] and 41 ng.micro L[-1], respectively

Conclusion: Protein and plasmid yields of E. coli are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration [200 micro g.mL[-1]] was significantly [p < 0.01] higher than other doses

Upregulation of CD4[+]T-cell derived MiR-223 in the relapsing phase of multiple sclerosis patients

Objective: MicroRNAs [miRNA] are a class of non-coding RNAs which play key roles in post-transcriptional gene regulation. Previous studies indicate that miRNAs are dysregulated in patients with multiple sclerosis [MS]. Th17 and regulatory T [Treg] cells are two subsets of CD4[+] T-cells which have critical functions in the onset and progression of MS. The current study seeks to distinguish fluctuations in expression of CD4[+] T-cell derived miR-223 during the relapsing-remitting [RR] phase of MS [RR-MS], as well as the expressions of Th17 and Treg cell markers

Materials and Methods: this experimental study used real-time quantitative polymerase chain reaction [qRT-PCR] to evaluate CD4[+] T cell derived miR-223 expression patterns in patients that experienced either of the RR-MS phases [n=40] compared to healthy controls [n=12], along with RNA markers for Th17 and Treg cells. We conducted flow cytometry analyses of forkhead box P3 [FOXP3] and RAR-related orphan receptor ?t [ROR?t] in CD4[+] T-cells. Putative and validated targets of miR-223 were investigated in the miRWalk and miRTarBase databases, respectively

Results: miR-223 significantly upregulated in CD4[+] T-cells during the relapsing phase of RR-MS compared to the remitting phase [P=0.000] and healthy individuals [P=0.036]. Expression of ROR?t, a master transcription factor of Th17, upregulated in the relapsing phase, whereas FOXP3 upregulated in the remitting phase. Additionally, potential targets of miR-223, STAT1, FORKHEAD BOX O [FOXO1] and FOXO3 were predicted by in silico studies

Conclusion: miR-223 may have a potential role in MS progression. Therefore, suppression of miR-223 can be proposed as an appropriate approach to control progression of the relapsing phase of MS

Development of a stable cell line, overexpressing human T-cell immunoglobulin mucin 1

Background: Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 [TIM-1] glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore, targeting TIM-1 could be a potential therapeutic approach against such diseases

Objectives: In this study, we aimed to express TIM-1 protein on Human Embryonic kidney [HEK] 293T cell line in order to have an available source of the TIM-1 antigen

Materials and Methods: The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells [PBMC] and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA[TM]3.1/Hygro [+] and transformed in Escherichia coli TOP 10 F'. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR

Results: The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells

Conclusions: Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1

Innate lymphoid cells and cytokines of the novel subtypes of helper T cells in asthma

BACKGROUND: In this study, the expression of interleukin-9 (IL-9), IL-17, IL-22, and IL-25 genes that might be the potential predisposing factors for asthma as well as count of innate lymphoid cells (ILCs) as another source of inflammatory cytokines have been evaluated. OBJECTIVE: The aim of this study was to evaluate the expression of newly identified helper T cells signature cytokines and amount of ILCs. METHODS: Blood and sputum samples from 23 patients with moderate to severe asthma and 23 healthy volunteers were collected. The types of allergens to which our patients were sensitive were defined using immunoblotting method. Gene expression of studied cytokines was evaluated using quantitative transcription-polymerase chain reaction and ILCs were counted by the flow cytometry method. RESULTS: In this research, the gene expressions of IL-9, IL-17, IL-22, and IL-25 were significantly higher in asthmatics, especially in the severe form of the disease. This increase was even higher in serum samples compared with sputum samples. Counting ILCs revealed their increase in comparison with normal people. CONCLUSION: We showed the importance of IL-25, IL-22, IL-17, and IL-9 cytokines in patients with asthma as their expression levels are increased and these increase are correlated with the severity of the disease. We also showed that the increased amount of ILCs in asthmatics could confirm their potential role in the immunopathogenesis of asthma as another source of inflammatory cytokines.

Positive and negative regulation of Th17 cell differentiation: evaluating the impact of RORC2

Th17 cells are known to be involved in some types of inflammations and autoimmune disorders. RORC2 is the key transcription factor coordinating Th17 cell differentiation. Thus, blocking RORC2 may be useful in suppressing Th17-dependent inflammatory processes. The aim was to silence RORC2 by specific siRNAs in naive T cells differentiating to Th17. Time-dependent expression of RORC2 as well as IL-17 and IL-23R were considered before and after RORC2 silencing. In this experimental study, naive CD4+ T cells were isolated from human cord blood samples. Cytokines TGFbeta plus IL-6 and IL-23 were used to polarize the naive T cells to Th17 cells in X-VIVO 15 serum free medium. A mixture of three siRNAs specific for RORC2 was applied for blocking its expression. RORC2, IL-17 and IL-23R mRNA and protein levels were measured using qRT-PCR, ELISA and flow cytometry techniques. Pearson correlation and one-way ANOVA were used for statistical analyses. Significant correlations were obtained in time-dependent analysis of IL-17 and IL-23R expression in relation with RORC2 [R=0.87 and 0.89 respectively, p<0.05]. Silencing of RORC2 was accompanied with almost complete suppression of IL-17 [99.3%; p<0.05] and significant decrease in IL-23R gene expression [77.2%, p<0.05]. Our results showed that RORC2 is the main and the primary trigger for upregulation of IL-17 and IL-23R genes in human Th17 cell differentiation. Moreover, we show that day 3 could be considered as the key day in the Th17 differentiation process

RORC2 gene silencing in human Th17 cells by siRNA: design and evaluation of highly efficient siRNA

RNA interference-based gene silencing has recently been applied as an efficient tool for functional gene analysis. RORC2 is the key transcription factor orchestrating Th17 cells differentiation, the cells that are known as the pathogenic elements in various autoimmune diseases. The aim of this study was to design efficient siRNAs specific for RORC2 and to evaluate different criteria affecting their functionality. Three siRNA duplexes specific for RORC2 mRNA were designed. Th17 cells were produced from IL-6 and IL-1 treated cord blood CD4[+] T cells. The T cells were transfected with three different designed siRNAs against RORC2 and the expression of RORC2 gene was measured using quantitative real time PCR. Different levels of RORC2 down regulation were observed in the presence of each of the designed siRNAs. Efficient siRNA with 91.1% silencing activity met the majority of the established bioinformatics criteria while the one with 46.6% silencing activity had more deviations from these criteria. The more bioinformatics criteria are considered, the more functionality were observed for silencing RORC2. However, the importance of the type of criteria per se should not be neglected. Although all recommended criteria are important for designing siRNA but their value is not the same

Determination of HLA-B27 subtypes in Iranian patients with ankylosing spondylitis

The human leukocyte antigen-B27 is one of the class I molecules of the major histocompatibility complex which is strongly associated with ankylosing spondylitis [AS]. The strength of the disease association with B27 varies markedly among racial and ethnic populations. It is an allele family, which constitutes about 31 subtypes, with a considerable geographic and ethnic difference in distribution. It is important to know whether certain subtypes show any preferential association with AS. Because there is no report regarding HLA-B27 subtypes in Iranian patients with AS, main purpose of the present study was to assess the frequency of subtypes of human leukocyte antigen [HLA]-B27 in patients with ankylosing spondylitis in Iranian population One hundred and nineteen AS patients [82 HLA-B27 positive and 37 HLA-B27 negative] were selected for this study. HLA-B27 positive patients were by polymerase chain reaction amplification with sequence-specific primers [PCR-SSP] for B 27 subtyping. The results of present study revealed that only two subtypes were detected in Iranian patients, including B 2705 [52 patients, 63.4%] and B 2702 [30 patients, 36.6%]. Our results showed a restricted number of HLA-B27 subtypes associated with AS in Iran and an elevated frequency of the B 2705 allele in these patients similar to other Euro-Caucasoid [Aryan] groups in the world