Expression of miR-let7b and miR-19b in progressive familial intrahepatic cholestasis (PFIC) children / Expresión de miR-let7b y miR-19b en niños con colestasis intrahepática familiar progresiva (PFIC)

Background MicroRNAs (miRNAs) are a group of small non-coding RNAs that bind to the target mRNA and regulate gene expression. Recently circulating microRNAs were investigated as markers of diseases and therapeutic targets. Although various studies analyze the miRNA expression in liver disease, these studies on PFIC are few. Progressive familial intrahepatic cholestasis (PFIC) is a rare liver disease with autosomal recessive inheritance. Most children with PFIC progress to cirrhosis and liver failure and consequently need to have a liver transplant. The aim of this study is the investigation of the miR-19b and miR-let7b expression levels in Iranian PFIC children. Methods 25 PFIC patients, 25 healthy children and 25 Biliary Atresia patients were considered as case and two control groups respectively. Blood samples were obtained and Liver function tests (LFTs) were measured. After RNA extraction and cDNA synthesis, quantitative PCR was performed using specific primers for miR-19b and miR-let7b. The U6 gene is used as an internal control. Results qPCR on PFIC patients’ samples demonstrated that the miR-19b and the miR-let7b expression were significantly decreased in patients compared to the control groups, with a p-value<0.0001 and p-value=0.0006 receptively. Conclusion In conclusion, circulating micro-RNA like miR-19b and miR-let7b have a potential opportunity to be a non-invasive diagnostic marker or therapeutic target for PFIC in the future (AU)

Antecedentes Los microRNA (miRNA) son un grupo de pequeños RNA no codificantes que se unen al ARNm diana y regulan la expresión génica. Recientemente se han investigado los microRNA circulantes como marcadores de enfermedades y dianas terapéuticas. Aunque varios estudios analizan la expresión de miRNA en enfermedades hepáticas, estos estudios sobre PFIC son escasos. La colestasis intrahepática familiar progresiva (PFIC) es una enfermedad hepática rara con herencia autosómica recesiva. La mayoría de los niños con PFIC progresan a cirrosis e insuficiencia hepática y, en consecuencia, requieren de un trasplante de hígado. El objetivo de este trabajo es la investigación de los niveles de expresión de miR-19b y miR-17b en niños iraníes con PFIC. Métodos Se consideraron 25 pacientes con PFIC, 25 niños sanos y 25 pacientes con atresia biliar como grupos de casos y controles. Se obtuvieron muestras de sangre y se midieron las pruebas de función hepática (LFT). Después de la extracción de RNA y la síntesis de cDNA, se realizó PCR cuantitativa usando cebadores específicos para miR-19b y miR-17b. El gen U6 se utiliza como control interno. Resultados La qPCR en muestras de pacientes con PFIC demostró que la expresión de miR-19b y miR-17b disminuyó significativamente en los pacientes en comparación con dos grupos de control, con un valor de p<0,0001 y un valor de p=0,0006, receptivamente. Conclusión En conclusión, los micro-RNA circulantes, como miR-19b y miR-let7b, tienen una oportunidad potencial de ser un marcador de diagnóstico no invasivo o un objetivo terapéutico para PFIC en el futuro (AU)

Expression of miR-let7b and miR-19b in progressive familial intrahepatic cholestasis (PFIC) children.

BACKGROUND: MicroRNAs (miRNAs) are a group of small non-coding RNAs that bind to the target mRNA and regulate gene expression. Recently circulating microRNAs were investigated as markers of diseases and therapeutic targets. Although various studies analyze the miRNA expression in liver disease, these studies on PFIC are few. Progressive familial intrahepatic cholestasis (PFIC) is a rare liver disease with autosomal recessive inheritance. Most children with PFIC progress to cirrhosis and liver failure and consequently need to have a liver transplant. The aim of this study is the investigation of the miR-19b and miR-let7b expression levels in Iranian PFIC children. METHODS: 25 PFIC patients, 25 healthy children and 25 Biliary Atresia patients were considered as case and two control groups respectively. Blood samples were obtained and Liver function tests (LFTs) were measured. After RNA extraction and cDNA synthesis, quantitative PCR was performed using specific primers for miR-19b and miR-let7b. The U6 gene is used as an internal control. RESULTS: qPCR on PFIC patients' samples demonstrated that the miR-19b and the miR-let7b expression were significantly decreased in patients compared to the control groups, with a p-value<0.0001 and p-value=0.0006 receptively. CONCLUSION: In conclusion, circulating micro-RNA like miR-19b and miR-let7b have a potential opportunity to be a non-invasive diagnostic marker or therapeutic target for PFIC in the future.

Circulating NKG2C + NK cell expressing CD107a/LAMP-1 subsets at the onset of CMV reactivation in seropositive kidney transplant recipients.

Cytomegalovirus (CMV) infection contributes to morbidity and mortality among kidney transplant recipients. Natural killer (NK) cells can battle against CMV in kidney transplant recipients (KTRs). This study aimed to analyze the association between CMV reactivation and the proportion of NK cell subsets and their activity. In a cross-sectional study, ten CMV reactivated KTRs, and ten non- CMV reactivated ones were recruited. Ten matched healthy controls were also included in this cohort. The presence of anti-CMV-IgG Ab in both KTR subgroups from seronegative donors and healthy controls was determined. The frequency of distinct subsets of memory-like NK cells was analyzed through NKG2C, NKG2A, and CD57 using flow cytometry. The activity of NK cells was evaluated after stimulation via coculture with K562 cell line and then assessment of the frequency of CD107a and granzyme B. The mRNA levels of transcription factors, including T-bet, EAT, and inflammatory proteins, including IFN-γ and perforin contributing to NK cell activation, were also evaluated. Results showed a significantly lower frequency of NKG2C + NKG2A-CD57+ NK cell population in CMV-reactivated KTRs compared to non-reactivated ones (P-value:0.003). NKG2C+ NK cells expressing CD107a/LAMP-1 significantly was increased in CMV-reactivated KTRs compared to non-reactivated ones (P-value: 0.0002). The mRNA level of IFN-γ had a significant increase in the CMV-reactivated KTRs vs. nonreactive ones (P-value: 0.004). Finally, evaluation of the NK cells' cytotoxicity and activity through assessment of CD107a/LAMP-1 expression and IFN-γ secretion may be helpful for the identification of the risk of CMV reactivation in KTRs.

Effects of labeling human mesenchymal stem cells with superparamagnetic zinc-nickel ferrite nanoparticles on cellular characteristics and adipogenesis/osteogenesis differentiation.

OBJECTIVE: An attractive cell source for stem cell-based therapy are WJ-MSCs. Hence, tracking WJ-MSCs using non-invasive imaging procedures (such as MRI) and contrast agents (Zn0.5Ni0.5Fe2O4, NFNPs) are required to evaluate cell distribution, migration, and differentiation. RESULTS: Results showed that the bare and dextrin-coated NFNPs were internalized inside the WJ-MSCs and had no effect on the cell viability, proliferation, apoptosis, karyotyping, and morphology of WJ-MSCs up to 125 µg/mL. Besides, treated WJ-MSCs were differentiated into osteo/adipocyte-like cells. The expression of RUNX 2, SPP 1 (P < 0.05), and OCN (P > 0.05) genes in the WJ-MSCs treated with dextrin-coated NFNPs was higher than the untreated WJ-MSCs; and the expression of CFD, LPL, and PPAR-γ genes was reduced in WJ-MSCs treated with both NFNPs in comparison with the untreated WJ-MSCs (P > 0.05). CONCLUSION: Overall, results showed that dextrin-coated NFNPs had no adverse effect on the cellular characteristics, proliferation, and differentiation of WJ-MSCs, and suggesting their potential clinical efficacy.

Effect of Cytomegalovirus Recombinant Phosphoprotein 150 (pp150) on Maturation and Function of Murine Dendritic Cells: an In-Vitro Study.

BACKGROUND: Tegument protein pp150 of cytomegaloviruses (CMVs) plays a vital role in all stages of viral life cycle, representing the most important tegument protein candidate for HCMV treatment. However, the exact role of pp150 in immune regulation is yet to be elucidated. OBJECTIVE: To examine the effects of pp150 on the maturity and function of murine dendritic cells (DCs). METHODS: Maturity status (CD40, CD86, and MHC-II expression) and phagocytic capacity of DCs (dextran uptake assay) were characterized. Gene expression profiles of ROR-γ, GATA-3, T-bet, and FOXP-3 as well as the protein expression of INF-γ (Th1), IL-4 (Th2), IL-35 (Treg), IL-17A (Th17), IL-22, TNF-α, IL-6, and IL-2 were evaluated in T cells co-cultured with DCs. RESULTS: A significant increase in CD40, CD86, and CCR7 expression and a reduction in the phagocytosis rate were observed in pp150-stimulated DCs compared with unstimulated DCs. T cells co-cultured with stimulated DCs showed higher expressions of ROR-γ, IL-6, IL-2, IL-17A, IL-22, and TNF-α. CONCLUSION: Despite improvements in maturity status, pp150-stimulated DCs did not seem to be able to induce Th1 or Th2 immunity. In fact, Th17 and its mediators, IL-17A and IL-22, might be the main inflammatory factors involved in pp150-stimulated DC's mechanism of action. However, it is necessary to conduct further investigations to corroborate these observations.

A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments.

BACKGROUND: Despite the ease of conventional splicing by overlap-extension (SOEing) PCR technique in theory, when splicing more than two fragments, and especially if one of the complementary sequences is A-T rich, the attachment of the fragments would be challenging. A new rapid and highly efficient SOEing PCR assay was developed for simultaneous splicing of multiple DNA fragments and induction of site-directed mutagenesis in a single tube. METHODS: The method was adapted for splicing human beta-globin UTRs to OCT4, SOX2, KLF4, C-MYC, LIN28A, and destabilized GFP for the construction of chimeric DNA fragments for in vitro transcription. In addition, the native Kozak sequence of beta-globin (K1) was replaced by the strongest Kozak sequence (K2) using site-directed mutagenesis to enhance the expression of target genes. RESULTS: ChimericGFPd2/K1, GFPd2/K2, OCT4, and KLF4 were created by the optimized conventional SOEing PCR. The single tube method was able to create the chimeric SOX2, C-MYC, and LIN28A in high quality and quantity in comparison with the conventional SOEing PCR. Moreover, using single tube SOEing PCR, the reaction time and materials that are required in the conventional SOEing PCR were significantly reduced. Fluorescent microscopy and flow cytometry examinations indicated highly efficient translation of K2 sequence in comparison with the K1sequence. CONCLUSION: Single tube SOEing PCR is a valuable method to construct more multiple fragments with high yield. The method can successfully be applied for construction of various kinds of complex chimeric genes.

Mesenchymal stem cells can induce regulatory T cells via modulating miR-126a but not miR-10a.

Among the different immunosuppressive properties attributed to mesenchymal stem cells (MSCs), one relies on their ability to induce regulatory T cells (iTregs) from conventional T cells under particular inflammatory context. Stable Foxp3 expression plays a major role in the phenotypic and functional stability of iTregs. However, the mechanism behind Foxp3 induction in iTregs by MSCs remains unknown. Here, we assessed the possible effect of MSCs on miR-126a and miR-10a expression in iTregs and, consequently on Foxp3 stability, a regulatory pathway that has not yet been explored. We first demonstrated that in vitro MSC-iTreg generation was directly associated with strong modifications of miR-126a. We next infused high doses of MSCs in a murine model of allogeneic skin transplantation (C57BL/6 into Balb/c). This treatment significantly prolonged skin allograft survival compared to PBS treated mice. When splenocytes from grafted mice were collected, we observed that the expression of Foxp3 gene was elevated at day 5 and 10 post-graft merely in MSCs treated mice. Moreover, Foxp3 expression was not associated with modified miR-10a expression comparable to in vitro experiments. Thus, our data identify a solid mechanism where MSCs induce conversion of conventional T cells to iTregs through strong modifications of miR-126a. Although miR-10a expression level remains unchanged in vitro and in vivo, we observed expression of this miR in MSC-DC condition.

Effect of Mesenchymal Stem Cells on ILT3 Expression in the Splenocytes of Skin Graft Recipient Mice.

BACKGROUND: Mesenchymal stem cells (MSCs) are considered as effective therapeutic cells in transplantation due to their immunomodulatory activities. However, precise mechanism of MSCs immunomodulatory activity is not completely understood. OBJECTIVES: To study the role of Immunoglobulin-like transcripts-3 (ILT3) immunomodulatory receptor in immune tolerance induced by MSCs in skin transplantation model and induction of tolerogenic dendritic cells (Tol-DCs) by MSCs through up-regulation of ILT3. METHODS: C57BL/6 skin grafts were transplanted to the back of BALB/c mice. Recipient mice received MSCs on days 0, 1 and 2 post transplantation. On days 2, 5 and 10 post skin transplantation, ILT3 and forkhead box P3 (FOXP3) expression in the spleens of MSCs treated mice were evaluated. Furthermore, MSCs and DCs were co-cultured in cell culture plates and transwell systems. Then, the expressions of ILT3 mRNA and protein in MSC-treated DCs were evaluated. Additionally, MSC-treated DCs were co-cultured with allogeneic T-cells and FOXP3 expression in T-cells was evaluated. RESULTS: The expression of ILT3 and FOXP3 were higher in the splenocytes of MSCs-treated mice early post-transplantation. Furthermore, we observed that MSC-treated DCs can increase FOXP3 expression in T-cells. But, we could not find any differences in ILT3 expression between MSC-treated DCs and untreated ones. CONCLUSION: One of the mechanisms underlying MSCs immunomodulatory function could be up-regulating ILT3 expression in splenocytes. But our results did not support the hypothesis that MSCs induce Tolergenic DCs by up-regulation of ILT3.

Hepatocyte isolation from unused/rejected livers for transplantation: initial step toward hepatocyte transplantation, the first experience from iran.

BACKGROUND: Hepatocyte transplantation is being used in patients with liver-based metabolic disorders and acute liver failure. Hepatocytes can be isolated from unused/rejected livers under sterile conditions. OBJECTIVES: The quality of the hepatocytes is very important and the main and initial step in hepatocyte transplantation is hepatocyte isolation. In this study we tried to set up the methods of hepatocyte isolation in order to use the high quality cells in acute liver failure or congenital metabolic disorders. MATERIALS AND METHODS: In this study, during a year, hepatocytes were isolated from 7 unused/rejected livers among more than 300 harvested livers in Shiraz University of Medical Sciences. The two step collagenase perfusion method was used under GMP (Good manufacturing practice) for hepatocyte isolation. RESULTS: Highly quality hepatocytes with high viability and low contamination were isolated. The mean viability was 71.8% ± 21.7. In the first 4 cases microbial contamination by Staphylococci, Diphtheroid and Klebsiella was detected, however the last 3 cases were free of any micro organisms. After 5 weeks of cryopreservation in -140°C, the cell viability was still acceptable. CONCLUSIONS: Hepatocyte isolation can be performed as the main and initial step for cell transplantation from unused/rejected liver. It is the first experience in Iran.