Evaluation of Silencing Effect of miR-133a-5p Mimic on TIM-3 Expression in AML (HL-60) Cell Line.

Acute myelogenous leukemia (AML) is a complex blood malignancy leading to immature leukemic stem cells (LSCs) proliferation. T cell immunoglobulin mucin-3 (TIM-3) is known as a biomarker of AML LSCs. Several microRNAs (miRNAs) can affect gene expression in AML. In this study, the silencing effect of miR-133a-5p on TIM-3 expression in AML cell lineage (HL-60) was investigated. It's been hypothesized that miR-133a-5p may suppress the TIM-3 expression in AML cell line. Initially, miRNA-TIM-3 prediction, enrichment, and network analysis were done. Then, miR-133a-5p mimic was transfected into HL-60 cells. The TIM-3 protein and gene expression were measured by flow cytometry analysis and real-time PCR, respectively. MTT assay was also carried out. Based on the Bioinformatics predictions, miR-133a-5p was able to silence TIM-3 expression. Also, significant pathways pertained to miR-133a-5p were obtained using enrichment analysis. According to this, miR-133a-5p was mainly engaged in the MAPK signaling pathway and Nicotine addiction pathway using the KEGG database. The TIM-3 protein expression of the transfected cells was measured as 17.15 ± 8.87% (p = 0.001). A 52.48% significant gene silencing in mRNA level was obtained in comparison to the negative control. Despite of down regulation of TIM-3, HL-60 cell viability has not been significantly changed. It has been finally confirmed that miR-133a-5p could strongly suppress TIM-3 expression in AML cell line. Presumably, down regulation of TIM-3 could affect MAPK and Nicotine addiction signaling pathways.

Evaluation of the T helper 17 cell specific genes and the innate lymphoid cells counts in the peripheral blood of patients with the common variable immunodeficiency.

BACKGROUND: Common variable immunodeficiency (CVID) is characterized by a deficiency in the immune system with a heterogeneous collection of disorders resulting in antibody deficiency and recurrent infections. T helper 17 (Th17) cells promote B-cell survival and synergize with the B-cell activating factor to induce their differentiation into the plasma cells. A sub-population of innate lymphoid cells (ILCs) also produces interleukin 17 (IL-17). This study aimed to measure the Th17 specific genes and ILCs counts in the CVID patients in comparison with control subjects. MATERIALS AND METHODS: Total messenger ribonucleic acid (mRNA) was extracted from the whole blood samples of 10 CVID patients and 10 healthy individuals. IL-17, retinoic acid receptor-related orphan receptor C2 (RORC2), IL-23R, and IL-9 gene expression were measured using the quantitative reverse transcriptase-polymerase chain reaction. Count of lineage negative/CD127(+)/CD90(+) ILCs in the blood samples was performed by the flow cytometry method. RESULTS: The transcript levels of IL-17 and RORC2 in CVID patients was strongly lower than control subjects (P = 0.049 and P = 0.046, respectively), but slight reduction in the IL-23R expression (P = 0.252) have seen in the CVID patients. Accordingly, the number of ILCs decreased significantly (P = 0.04). Interestingly, IL-9 mRNA level was more significantly in the CVID patients (P = 0.001). CONCLUSION: The results presented in this study show that the Th17 cell specific genes expression (as the determiner Th17 cells) and ILCs (another lymphoid source of IL-17) are decreased in patients with CVID and this could be an explanation for the defect of their humoral immune response. In addition, elevation of the IL-9 gene expression may shed a new light into the way toward the understanding of the mechanism of autoimmunity in the CVID patients.

Circulating endothelial cells (CECs) and E-selectin: Predictors of preeclampsia.

BACKGROUND: Circulating endothelial cells (CECs) and E-selectin are known as sensitive and specific markers of endothelial dysfunction. This study investigated whether CECs and E-selectin are surrogate biomarkers of preeclampsia and if measurement of CECs and E-selectin, early in the third trimester, could be a means of predicting preeclampsia. METHODS: In this prospective, descriptive-analytic study, rollover test was performed on 523 pregnant women during 28-30 weeks of gestation. CECs were measured by anti-CD 146-driven immunomagnetic isolation in women with positive rollover test. They were followed up prospectively until delivery without any active intervention. Women with and without preeclampsia were determined. The number of CECs and level of E-selectin were compared in the two studied groups. RESULTS: From the 47 pregnant women with positive rollover test who were selected and followed up, 22 individuals were diagnosed with preeclampsia while the remainder were normotensive. Mean CEC numbers was significantly higher in preeclamptic women than normal pregnancies (24.7 cells/mL vs. 13 cells/mL). The best cut-off point for CEC numbers was 6.5 with a sensitivity of 78.9% and a specificity of 69.1%. The level of E-selectin was significantly higher in mothers with preeclampsia (p < 0.05). CONCLUSIONS: Higher levels of CECs and E-selectin in women with positive rollover test who developed preeclampsia prior to onset of the complication were predictive of preeclampsia. However, larger studies are needed to confirm these findings.

Clinical and Laboratory Parameters of Autoinflammatory Disorders in Single Tertiary Care Center.

Autoinflammatory diseases (AIDs) are disorders with an inborn error of innate immunity, characterized by recurrent episodes of fever and inflammatory attacks. The spectrum of AIDs is expanding, but there are no standardized clinical criteria for the diagnosis of the patients. This study aims at establishing the first autoinflammatory registry of an Iranian population focusing on the clinical and laboratory features that may help clinicians for a better understanding and diagnosis of these disorders. Clinical and laboratory characteristics of patients who were clinically and or genetically diagnosed with AIDs were collected during 15 years. The updated version of classification criteria from the Eurofever Registry was used for the clinical diagnosis. Twenty-eight patients (16 males and 12 females) with the mean±SD age of 28.03±14.49 years (from 2 to 68 years) were entered this study. About 29% were genetically diagnosed. Familial Mediterranean fever (FMF) was the most common diagnosis of the patients. Fever duration episodes were between 1-10 days. Some of the clinical manifestations from the most to the least common were as follows: arthralgia and arthritis (80%), myalgia (76%), coughs and shortness of breath (68%), fatigue (60%), abdominal pain (56%), increased erythrocyte sedimentation rate(ESR) (48%), and splenomegaly (24%). Here, we presented the most common clinical manifestations of Iranian AIDs who have registered in our AID registry which would be a useful guide for managing the same patients and also designing the future studies.

The assessment of feto-maternal hemorrhage in an artificial model using anti-D and anti-fetal hemoglobin antibody by flow cytometry.

BACKGROUND: When fetal red cells enter the maternal circulation from placenta, an event would be happened that is described as feto-maternal hemorrhage (FMH). This life-threatening condition could be detected by using RBC antigens (surface antigens and intracellular antigens). Therefore, the measurement of fetal RBC in an artificial model would be useful to calculate FMH and consequently the dosage of Rhogam for prophylaxis. The aim of the present study was to evaluate FMH in an artificial mixture model. METHODS: A series of 40 artificial specimens were prepared consisting of Rh(D) negative adult blood (non-immunized) spiked with varying amounts of Rh(D) positive cord blood from mothers between 20-30 years old in Shahid Beheshti Hospital, Tehran, Iran. Monoclonal anti-D and anti-HbF (fetal hemoglobin) were used for detection of fetal RBC in artificial mixture sample modeling. RESULTS: This study showed that the percentage of fetal cells in artificial sample for anti-D antigen is in ranges of 0.28%-0.32% for a 0.25% dilution mixture, and 1.3%-2.05% for the mixture with dilution 2%. In addition, the ranges of data for anti-HbF staining was obtained 0.2%-0.34% for the 0.25% dilution sample, and the ranges of 1.04-1.8% for the 2% dilution. The regression analysis indicated that the correlation of anti-D assessment with expected standard method was r2 = 0.9672 and anti-HbF assessment was r2 = 0.8842. CONCLUSION: Although both molecule targets could be used for detection of fetal RBC, in this model, anti-D staining was more accurate than anti-HbF staining. However, since anti-D can not be utilized for low-density or weak phenotype and other incompatibility, the anti-HbF labeling could be used for all FMH.

Evaluation of the effect of TIM-3 suppression by miR-498 and its effect on apoptosis and proliferation rate of HL-60 cell line.

BACKGROUND: Acute Myeloid Leukemia (AML) is a Cancer of hematopoietic stem cells with a rapid progression. TIM-3 is expressed on leukemic stem cells (LSCs) in most types of AML and might have a positive effect on maintenance of malignant phenotype. MicroRNAs play important roles in either cancer progression or suppression. In this study were evaluated, the inhibitory effect of miR-498 on TIM-3 expression and its impact on proliferation and survival of HL-60 cell line. METHODS: Firstly, the probable inhibitory effect of miR-498 on TIM-3 expression was predicted. HL-60 cells were cultured and expression of TIM-3 was induced on the cells using phorbol miristate acetate. The cells were transfected with miR-498 and expression level of TIM-3 were measured using with q-RT-PCR and flow cytometry methods. In addition, the effect of suppression of TIM-3 expression in HL-60 cell line was analyzed with apoptosis and cell proliferation assays. RESULTS: Bioinformatics analyses predicted that miR-498 has high ability to silence TIM-3 gene expression. Our experiments confirmed that miR-498 was able to strongly silence TIM-3 expression (68% silencing) in HL-60 cell line (P < 0.002). Also, the cells with suppressed expression of TIM-3 had a lower proliferation and higher apoptosis rates. CONCLUSION: Based on our results, the miR-498 can effectively suppress TIM-3 expression in the AML cell line. TIM-3 suppression, in turn, inhibits malignant cell proliferation and induces its apoptosis. Collectively, suppression of TIM-3 by miR-498 can be considered as a potential powerful way for treatment of AML.

The +4259A>C polymorphism of TIM-3 but not -1637C>T polymorphism of TIM-1 is associated with Multiple sclerosis in Isfahan population.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) which initiated and mediated by autoreactive T helper1 cells directed against myelin antigens. One of T cell surface receptors is T cell immunoglobulin and mucin domain (TIM) family which its importance in immunology is recently discovered. These molecules have important immunological function by regulation of T effector cells. METHODS: In the present study, we analyzed the frequency of +4259A>C polymorphism in TIM-3 and -1637C>T polymorphism in TIM-1 gene in MS patients and healthy controls using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) method. RESULTS: We found that the polymorphism +4259A>C in exon 3 of the TIM-3 gene is associated with susceptibility to the MS (P = 0.029, OR (95%CI) = 1.841) but the other polymorphism, -1637T>C, in the promoter region of TIM-1 is not (p= 0.064). CONCLUSION: Our findings suggest that +4259A>C polymorphism in TIM-3 gene may be one of the important genetic factors associated with the MS susceptibility among Iranian populations.

Stimulation of Camel Polyclonal Antibody against Human T cell Immunoglobulin and Mucin 3.

Background: T cell Immunoglobulin, Mucin (TIM)-3, is a type I transmembrane glycoprotein belonging to TIM family. This receptor expresses on T helper type 1 (Th1) cells that binds to galectin-9 (Gal9); inducing an inhibitory signal. As a result, apoptosis of Th1 cells occurs and cytotoxicity of CD8 T cells becomes evident in vitro. Therefore, this immunomodulatory molecule may be used as a novel target for clinical purposes. The production of camel polyclonal antibodies against TIM-3-expressing cell line was the purpose of this study. Objectives: In this study, we aimed to use HEK 293 cells expressing human TIM-3 to obtain camel polyclonal antibody against TIM-3 by immunization. Materials and Methods: A pre-synthesized human TIM-3cDNA was inserted into pcDNA3.1 plasmid and the new construct was transfected in HEK cell. TIM-3 expression was confirmed by qRT-PCR and flow cytometry. A camel (6 months old) was immunized with the lysate prepared from rTIM-3 expressing HEK cells 4 times. The anti-TIM-3 antibody level was evaluated using ELISA method. Results: TIM-3 was successfully cloned in HEK cells with 88% success rate. High level of anti-TIM-3 antibody was detected in the serum of the camel immunized with the recombinant cell lysate, after final injection. Conclusions: Our rhTIM-3 cell display system can be useful for future diagnostic or therapeutic approaches.

Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3).

OBJECTIVES: As T-cell immunoglobulin and mucin domain 3 (TIM-3) is an immune regulatory molecule; its blocking or stimulating could alter the pattern of immune response towards a desired condition. Based on the unique features of nanobodies, we aimed to construct an anti-TIM-3 nanobody as an appropriate tool for manipulating immune responses for future therapeutic purposes. MATERIALS AND METHODS: We immunized a camel with TIM-3 antigen and then, synthesized a VHH phage: mid library from its B cell's transcriptome using nested PCR. Library selection against TIM-3antigen was performed in three rounds of panning. Using phage-ELISA, the most reactive colonies were selected for sub-cloning in soluble protein expression vectors. The Nanobody was purified and confirmed with a nickel-nitrilotriacetic acid (Ni-NTA) column, SDS-PAGE and Western blotting. A flowcytometric analysis was performed to analyze the binding and biologic activities of theTIM-3 specific nanobody with TIM-3 expressing HL-60 and HEK cell lines. RESULTS: Specific 15kD band representing for nanobody was observed on the gel and confirmed with Western blotting. The nanobody showed significant specific immune-reactivity against TIM-3 with a relatively high binding affinity. The nanobody significantly suppressed the proliferation of TIM-3 expressing HL-60 cell line. CONCLUSION: Finally, we successfully prepared a functional anti-humanTIM-3 specific nanobody with a high affinity and an anti-proliferative activity on an AML cell line in vitro.

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™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.

Investigating of microsatellites instability in patients with hereditary non-polyposis colorectal cancer in Isfahan.

BACKGROUND: Microsatellites or simple sequence repeats are repeating sequences of deoxyribonucleic acid (DNA). Mutation in mismatch repair (MMR) genes can cause microsatellites instability (MSI) in some tumors. In familial disorder of hereditary non-polyposis colorectal cancer (HNPCC), there is a defect in the mechanism of MMR and clearly defective MMR cause unstable microsatellites. This study has been conducted for investigating the instability of microsatellites in alleles of BAT-26 of MSH2 gene in patients with HNPCC in Isfahan, which is an important prognostic biomarker for the prediction of the treatment outcome. MATERIALS AND METHODS: DNA extraction from forty HNPCC patients peripheral blood samples were performed by using the DNA extraction kit. The polymerase chain reaction (PCR) reaction to amplify BAT-26 was performed. The PCR products were studied by electrophoresis on agarose gel. RESULTS: The size of specific band was 121 bp out of 40 HNPCC samples and based on the above method, it was shown that 12 cases (30%) demonstrated MSI. Chi-square test showed this difference is statistically significant (P < 0.05). CONCLUSIONS: The present study was conducted to evaluate the MSI in HNPCC patients. It was determined that the polymorphisms in BAT-26 of MSH2 gene could detect MSI with high sensitivity. Previous reports as well as our results have shown that the use of BAT-26 alone would be sufficient to identify HNPCC-associated MSH2 gene. Identifying MSI in these genes as a marker for prognosis, according to the present study and other researches is important to predict the treatment outcomes.

Genetic defects and the role of helper T-cells in the pathogenesis of common variable immunodeficiency.

Common variable immunodeficfiiency (CVID) is a primary immunodeficiency syndrome representing a heterogeneous set of disorders resulting mostly in antibody deficiency and recurrent infections. However, inflammatory and autoimmune disorders and some kinds of malignancies are frequently reported as a part of the syndrome. Although it is one of the most widespread primary immunodeficiency, only recently some genetic defects in CVID have been identified. Mutations have been detected in inducible T-cell costimulator (ICOS), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), B-cell activating factor-receptor (BAFF-R), B-cell receptor complex (CD19, CD21 and CD81) and CD20. On the other hand, recent studies have shown a decrease in T-helper-17 cells frequency and their characteristic cytokines in CVID patients and this emphasis on the vital role of the T-cells in immunopathogenesis of the CVID. Furthermore, in the context of autoimmune diseases accompanying CVID, interleukin 9 has recently attracted a plenty of considerations. However, the list of defects is expanding as exact immunologic pathways and genetic disorders in CVID are not yet defined. In this review, we have a special focus on the immunopathogenesis of CVID, recent advances in understanding the underlying etiology and genetics for patients.

Positive and Negative Regulation of Th17 Cell Differentiation: Evaluating The Impact of RORC2.

OBJECTIVE: 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 naïve 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. MATERIALS AND METHODS: In this experimental study, naïve CD4(+)T cells were isolated from human cord blood samples. Cytokines TGFß plus IL-6 and IL-23 were used to polarize the naïve 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. RESULTS: 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). CONCLUSION: 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.

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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.