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

Variations in T-helper 17 and regulatory T cells during the menstrual cycle in peripheral blood of women with recurrent spontaneous abortion

Disorders in immune system regulation may result in pregnancy abnormalities such as recurrent spontaneous abortion [RSA]. This study aims to determine the ratio of regulatory T [Treg] and T helper [Th] 17 cells in unexplained RSA [URSA] women during proliferative and secretory phases of their menstrual cycles compared to healthy non-pregnant women. In this case control study, 25 women with URSA and 35 healthy, non-pregnant women were enrolled. The percentage of Th17 and Treg cells in participants peripheral blood were determined by flow cytometry. The percentage of Th17 cells and their related cytokines in serum [IL-17A] were higher in the proliferative and secretory phases of the menstrual cycles of URSA women compared to the control women. However, a lower percentage of Treg cells and their related cytokines in serum, transforming growth factor [TGF] beta1 and interleukin [IL]-10 were detected in the proliferative but not the secretory phase of the URSA group. The ratio of Th17/CD4+ Treg was higher in the URSA group than the control group. We observed an increased ratio of Th17/CD4+ Treg during the proliferative and secretory phases in URSA women. The imbalance between Th17 and Treg cells during the proliferative phase of menstrual cycles in the URSA group may be considered a cause for spontaneous abortion

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

CD4[+] Foxp3[+] Treg and its ICOS[+] subsets in patients with myocardial infarction

Atherosclerosis is a multifactorial disorder with chronic inflammatory conditions in which immune cells play a significant role in its pathogenic process. Regulatory T cells [Treg], as a part of immune system, are involved in controlling autoimmune and inflammatory diseases. Quantitative and/or functional alteration of Tregs has been shown to play an atheroprotective role and may also promote plaque stabilization. To assess if inducible costimulatory molecule [ICOS] expression on one subtype of Treg cells with high suppressive potential correlates with the pathogenesis of atherosclerosis. Patients with myocardial infarction [MI] and/or stable angina [SA], diagnosed as atherosclerosis by angiography, and a group of individuals with normal coronary angiography [NCA] were recruited for the present study. Peripheral blood mononuclear cells [PBMCs] were prepared and the expression of ICOS, Foxp3 and CD4 molecules was tested by flowcytometry. The percentage of CD4[+] Foxp3[+] Treg cells was reduced in MI group compared to NCA and SA groups [p<0.005]. Evaluation of the two Treg subsets according to ICOS expression showed a decreased ICOS[+]/ICOS[-] Treg ratio in MI and SA groups compared to NCA individuals [p=0.002 and p=0.048, respectivly]. The present data indicate that Tregs and its ICOS[+] subsets are decreased in patients with MI or SA, suggesting a potential role for Treg in atherosclerosis progression or onset of acute coronary syndrome

effects of isoproterenol and propranolol on cytokine profile secretion by cultured tumor-infiltrating lymphocytes derived from colorectal cancer patients

Anti-tumor immunity and cytokine profiles have important roles in the development of cancer. Norepinephrine [NE] release due to sympathetic activation leads to a Th2 deviation via the beta-2 adrenergic receptor [beta -2AR] and could increase cancer progression. This study intends to determine the effects of isoproterenol [ISO; betaagonist] and propranolol [PRO; beta-antagonist] on the production of IFN-gamma, IL-4, and IL-17. Cytokine levels have been examined in tumor-infiltrating lymphocytes [TILs] and peripheral blood mononuclear cells [PBMCs] of patients with colorectal cancer [CRC]. The beta-2AR expression on lymphocyte subsets was also assessed. In this experimental study, TILs were isolated from fresh CRC tissue and patient PBMCs were obtained just prior to surgery. The cells were cultured in medium for 72 hours. Concomitantly, cells were stimulated with 10 micro g/ ml phytohemagglutinin [PHA] alone or in the presence of either 1 micro mol/L of PRO or 1 micro mol/L ISO. The concentration of cytokines in the supernatants was measured by ELISA. Three-color flow cytometry was used to determine the expression of beta-2AR on the lymphocyte subsets. Statistical analyses were performed via paired or independent t-test. Levels of IFN-gamma, IL-4 and IL-17 were elevated after PHA-stimulation of PBMCs and TILs. However, the elevation of IFN-gamma and IL-17 production by TILs in response to PHA was significantly lower than PBMCs. In the presence of ISO, the IFN-gamma/IL-4 ratio reduced in all groups, but this reduction was very low in TILs. Interestingly, the effects of PRO on cytokine production were, at least partially, comparable to those of ISO. Depressed levels of beta-2AR expression were demonstrated on CD4+IFN-gamma+ and CD4+IL-17+ lymphocytes in patients' PBMCs and TILs. This study has demonstrated the effects of ISO and PRO on cytokine production by TILs and determined beta-2AR expression on these cells. ISO failed to induce a shift toward the expected Th2 cytokine profile in CRC patients' TILs, which might be due to the downregulation of beta-2AR expression on TILs. Additionally, in this study, PRO induced a shift to a Th2 profile in PBMCs

[Comparative study of IL17 in normal and symptomatic dental pulps by immunohistochemistry technique]

IL17 is a pro-inflammatory cytokine which is often produced from CD4+ Th17 cells and uncertainty exists about its protective or destructive function. One of destructive functions of IL17 is through acting on osteoclasts. The aim of this study was to evaluate IL17 protein expression in tissue obtained from normal and symptomatic dental pulps. Healthy dental pulp samples and irreversible dental pulp samples were obtained from 20 third molars and 20 carious molars respectively. After tissue processing, pulp samples were immunostained with IL17 antibodies. Thereafter, distribution and staining intensity of IL17 protein was evaluated by the SID score and findings were analyzed using the Mann-Whitney U test. Analysing SID score with Mann-whitney test showed significant difference in IL17 [P=0.002] between symptomatic dental pulp tissues [2.35 +/- 1.23] and healthy samples [1.15 +/- 0.93]. The results indicated that significantly a greater level of IL17 is found in pulps of symptomatic teeth than those of normal teeth. Therefore IL17 may be suggested as a pathologic marker of inflammatory function in irreversible pulpitis

Expression of chemokine receptors on Th1/Th2 CD4[+] lymphocytes in patients with multiple sclerosis

Th1 cells preferentially express CXCR3, CCR5 and CCR6, while CCR3 and CCR4 are predominantly expressed by Th2 cell subsets. Multiple Sclerosis [MS] is a TH-1 cell-dependant chronic inflammatory disease of the central nervous system, and immunomudolatory cytokines could alter the chemokine expression pattern of these lymphocyte subsets. This study was performed to measure chemokine receptor expression on CD4 T cells for evaluation of Th1/ Th2 dominantly in IFN-beta treated patients. Flowcytometry was used to detect chemokine receptor expression on CD4 T cell population in PBMCs obtained from MS and healthy control groups. Twenty six MS patients participated in this study before and after IFN-beta therapy and the same number of healthy individuals were included. The percentage of lymphocytes was 41.28 +/- 10.30 in the blood of MS group compared with 36.88% +/- 5.51% in the control group [p=0.017]. The CD4[+] CXCR3[+] cells were 18.86% +/- 8.46% in healthy group, 30.78% +/- 9.8% in pre-treated MS patients and 21.06% +/- 9.23% in post-treated group [p<0.001]. The CD4[+]CCR4[+] cell subsets were 27.35% +/- 10.15% in healthy group; 28.17% +/- 8.9% in pre-treated group and 34.20% +/- 8.96% in the post-IFN-beta treatment group. The subset of CD4[+]CCR4[+] was found to be dominant after IFN-beta therapy in comparison with the control group [p<0.001]. CD4[+]CCR5[+] percentage was 1.24% +/- 0.92% in the healthy people, 1.23% =/- 0.71% in the MS patients and 0.76% +/- 0.49% in post-treatment status [p=0.003]. CD4[+]CCR3[+] cell subsets were 0.62% +/- 0.67% in control group, 0.28% +/- 0.26% in the MS patients [p=0.022] and 0.39% +/- 0.54% in IFN-beta treated patients [p=0.334]. An association was found for CXCR3 expression in pre- and post- treatment status [r=0.840, p<0.001] as well as for CCR4[+] expression [r=0.712, p<0.001] in the same groups. The Th1 response was dominant in pre-treatment states, and then the chemokine receptor expression of Th1/Th2 cell subsets could be used for monitoring and the evaluation of the MS disease status

study on stress, depression and NK cytotoxic potential in women with recurrent spontaneous abortion

It has been recognized that acute and chronic stress has an impact on the immune system. Acute stress may have a stimulating effect on the immune system, while in the case of chronic stress specially depression, the immune system could be down-regulated. However, an association between depression and a higher number of circulating white blood cells with increased activity has been reported. Elevation in immune cell numbers and alteration in cytokine profiles are documented for women suffering sporadic spontaneous abortion with a high stress score. In spite of these contradictory results and to make a new approach in immunological [NK activity] as well as psychological parameters [stress/depression] in women suffering from recurrent spontaneous abortion [RSA] the present study was planned. Forty-five women with a history of RSA and a matched control group were participated in this study. A questionnaire for life events known as life change units [LCU] and the Beck Depression Inventory [BDI] outlines were used and the socio-psychological events were recorded after visiting and interview. Fresh peripheral blood lymphocytes were taken as a source of NK activity and K562 cell line were used as NK sensitive target. The experiments were performed and the cells were analyzed with a flow-cytometer. The stress and the depression scores were determined 245 +/- 83.6 and 27.6 +/- 8.8 for women with RSA and 224 +/- 79.6 and 19.4 +/- 7.1 for non-RSA group respectively. There was an association between life stress scores and depression scores with r=0.65 and P=0.000 for RSA women. A correlation with r=-0.34 and P=0.02 was found between depression scores and NK cytotoxicity. The Pearson correlation test showed a lack of relationship between high stress score and NK activity with the r=0.011 and P=0.95, but r=-0.30 and P=0.072 was obtained for high depression scores and NK cytotoxicity. Therefore, it could be suggested that in the case of women with a history of recurrent spontaneous abortion, modulation for immunological parameters [i.e immunotherapy] concurrently with managing psychological aspects [stress/depression] could be modified for the benefit of the patients

assessment of NK cytotoxicity and CD56+/CD16+ phenotype by flow cytometry in PBL isolated from women with recurrent spontaneous abortion

Human peripheral blood NK cells constitutively express CD56 and CD16 antigens. Peripheral blood NK cells seem to be strongly related with decidual NK cells, and may reflect the decidual NK cell functional status. There are varied reports concerning the relationship between NK cell cytotoxicity in women with recurrent spontaneous abortion. To study NK activity in women with history of RSA by using a non-radioactive cytotoxicity assay. Peripheral blood lymphocytes from RSA and healthy multiparous women were obtained. Lymphocytes were isolated and mixed with K562 NK-sensitive cell line. A non-radioactive method for NK cell cytotoxicity assessment was utilized. Dead K562 cell populations were detected by FACS Calibur flow cytometry, and the data obtained was analysed on cell-Quest software. The proportion of CD16+/CD56+ cells was then calculated. The proportion of NK cells were 9.21% +/- 3.06 and 13.48% +/- 4.09 in healthy women and RSA, respectively. The percentage of cytotoxicity was determined to be 19.3% +/- 3.9 in healthy group and 27.1% +/- 6.5 in RSA group with an effector:target ratio of 50:1. The data shows an increase in PBLs potential for in vitro cytotoxicity assay in RSA individuals. The analyses indicate that there is a weak positive correlation between NK cytotoxicity potential and the percentage of NK cells in PBL population. The present study demonstrates that the percentage of CD56+/CD16+ cells increases in individuals with recurrent spontaneous abortion. We conclude that NK cytotoxicity as well as NK number is partially involved in RSA