Harnessing exosomes in theranostic applications: advancements and insights in gastrointestinal cancer research.

Exosomes are small extracellular vesicles (30-150 nm) that are formed by endocytosis containing complex RNA as well as protein structures and are vital in intercellular communication and can be used in gene therapy and drug delivery. According to the cell sources of origin and the environmental conditions they are exposed to, these nanovesicles are very heterogeneous and dynamic in terms of content (cargo), size and membrane composition. Exosomes are released under physiological and pathological conditions and influence the pathogenesis of cancers through various mechanisms, including angiogenesis, metastasis, immune dysregulation, drug resistance, and tumor growth/development. Gastrointestinal cancer is one of the deadliest types of cancer in humans and can involve organs e.g., the esophagus and stomach, or others such as the liver, pancreas, small intestine, and colon. Early diagnosis is very important in this field because the overall survival of patients is low due to diagnosis in late stages and recurrence. Also, various therapeutic strategies have failed and there is an unmet need for the new therapeutic agents. Exosomes can become promising candidates in gastrointestinal cancers as biomarkers and therapeutic agents due to their lower immunity and passing the main physiological barriers. In this work, we provide a general overview of exosomes, their biogenesis and biological functions. In addition, we discuss the potential of exosomes to serve as biomarkers, agents in cancer treatment, drug delivery systems, and effective vaccines in immunotherapy, with an emphasis on gastrointestinal cancers.

Harnessing HEK293 cell-derived exosomes for hsa-miR-365a-3p delivery: Potential application in hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is the most frequent form of liver malignancy, and curing it is very challenging. Restoring tumor suppressor microRNAs could trigger the initiation of cellular anticancer mechanisms. Exosomes are nanosized biocarriers capable of fusing with cell membranes and delivering their cargo. The main goal of the current study was to explore the potential of human embryonic kidney cells (HEK293) cell-derived exosomes to provide an anticancer therapy based on the restoration of tumor suppressor miR-365a downregulated in HepG2 cells. To accomplish this aim, exosomes were isolated from the HEK293 cell line culture and characterized, enriched by Homo sapiens (hsa) miR-365a-3p mimics. Exosomes enabled an efficient loading and intracellular delivery of hsa-miR-365a mimics, which translated into G0/G1 cell cycle arrest, induction of oxidative stress, reduction of migration capacity, and high apoptosis rate. The findings indicate that the delivery of miR-365a-3p by HEK293-derived exosomes may act as an innovative and effective therapeutic strategy against HCC.

A Simple High Yield Technique for Isolation of Wharton's Jelly-derived Mesenchymal Stem Cell.

Background: The isolation of Mesenchymal Stem Cells (MSCs) from various tissues is possible, with the umbilical cord emerging as a competitive alternative to bone marrow. In order to fulfill the demands of cell therapy, it is essential to generate stem cells on a clinical scale while minimizing time, cost, and contamination. Here is a simple and effective protocol for isolating MSC from Wharton's Jelly (WJ-MSC) using the explant method with various supplements. Methods: Utilizing the explant method, small fragments of Wharton's jelly from the human umbilical cord were cultured in a flask. The multipotency of the isolated cells, were confirmed by their differentiation ability to osteocyte and adipocyte. Additionally, the immunophenotyping of WJ-MSCs showed positive expression of CD73, CD90, and CD105, while remaining negative for hematopoietic markers CD34 and CD45, meeting the criteria for WJ-MSC identification. Following that, to evaluate cells' proliferative capacity, various supplements, including basic Fibroblast Growth Factor (bFGF), Non-Essential amino acids (NEA), and L-Glutamine (L-Gln) were added to either alpha-Minimal Essential Medium (α-MEM) or Dulbecco's Modified Eagle's Medium-F12 (DMEM-F12), as the basic culture media. Results: WJ-MSCs isolated by the explant method were removed from the tissue after seven days and transferred to the culture medium. These cells differentiated into adipocyte and osteocyte lineages, expressing CD73, CD90, and CD105 positively and CD34 and CD45 negatively. The results revealed that addition of bFGF to α-MEM or DMEMF12 media significantly increased the proliferation of MSCs when compared to the control group. However, there were no significant differences observed when NEA or LGln were added. Conclusion: Although bFGF considerably enhances cell proliferation, our study demonstrates that MSCs can grow and expand when properly prepared Wharton's jelly tissues of the human umbilical cord.

Exploring the role of genetic variations in NAFLD: implications for disease pathogenesis and precision medicine approaches.

Non-alcoholic fatty liver disease (NAFLD) is one of the leading causes of chronic liver diseases, affecting more than one-quarter of people worldwide. Hepatic steatosis can progress to more severe forms of NAFLD, including NASH and cirrhosis. It also may develop secondary diseases such as diabetes and cardiovascular disease. Genetic and environmental factors regulate NAFLD incidence and progression, making it a complex disease. The contribution of various environmental risk factors, such as type 2 diabetes, obesity, hyperlipidemia, diet, and sedentary lifestyle, to the exacerbation of liver injury is highly understood. Nevertheless, the underlying mechanisms of genetic variations in the NAFLD occurrence or its deterioration still need to be clarified. Hence, understanding the genetic susceptibility to NAFLD is essential for controlling the course of the disease. The current review discusses genetics' role in the pathological pathways of NAFLD, including lipid and glucose metabolism, insulin resistance, cellular stresses, and immune responses. Additionally, it explains the role of the genetic components in the induction and progression of NAFLD in lean individuals. Finally, it highlights the utility of genetic knowledge in precision medicine for the early diagnosis and treatment of NAFLD patients.

Immunomodulatory and Anti-Inflammatory Effects of Vitamin A and Tryptophan on Monocyte-Derived Dendritic Cells Stimulated with Gliadin in Celiac Disease Patients.

Celiac Disease (CeD) is an autoimmune disorder with various symptoms upon gluten exposure. Dendritic cells (DCs) play a crucial role in gliadin-induced inflammation. Vitamin A (retinol; Ret) and its metabolite, retinoic acid (RA), along with tryptophan (Trp) and its metabolite, kynurenic acid (KYNA), are known to influence the immune function of DCs and enhance their tolerogenicity. This research aims to assess the impact of gliadin on DC maturation and the potential of vitamin A and tryptophan to induce immune tolerance in DCs. The monocyte cells obtained from peripheral blood mononuclear cells (PBMCs) of celiac disease patients were differentiated into DCs in the absence or presence of Ret, RA, Trp, KYNA, and then stimulated with peptic and tryptic (PT) digested of gliadin. We used flow cytometry to analyze CD11c, CD14, HLA-DR, CD83, CD86, and CD103 expression. ELISA was carried out to measure TGF-ß, IL-10, IL-12, and TNF-α levels. qRT-PCR was used to assess the mRNA expression of retinaldehyde dehydrogenase 2 (RALDH2) and integrin αE (CD103). The mRNA and protein levels of Indoleamine 2, 3-dioxygenase (IDO) was analyzed by qRT-PCR and Western blot assays, respectively. Our findings demonstrate that PT-gliadin enhances the expression of maturation markers, i.e. CD83, CD86 and HLA-DR and promote the secretion of TNF-α and IL-12 in DCs. Interestingly, vitamin A, tryptophan, and their metabolites increase the expression of CD103, while limiting the expression of HLA-DR, CD83, and CD86. They also enhance RALDH2 and IDO expression and promote the secretion of TGF-ß and IL-10, while limiting IL-12 and TNF-α secretion. These findings suggest that vitamin A and tryptophan have beneficial effects on PT-gliadin-stimulated DCs, highlighting their potential as therapeutic agents for celiac disease. However, further research is needed to fully understand their underlying mechanisms of action in these cells.

Effect of Pioglitazone and Cetuximab on Colon Cancer Stem-like Cell (CCSLCs) Properties.

BACKGROUND: One of the main reasons for cancer resistance to chemotherapy is the presence of cancer stem cells (CSCs) in cancer tissues. It is also believed that CSCs are the unique originators of all tumor cells. On the other hand, the Epithelial-Mesenchymal Transition pathway (EMT) can act as the main agent of metastasis. Therefore, it is possible that targeting CSCs as well as the EMT pathway could help in cancer therapy. Considering that CSCs constitute only a small percentage of the total tumor mass, enrichment before study is necessary. In our previous study, CSCs were enriched in the human colon cancer cell line HT29 by induction of EMT. These CSC-enriched HT29 cells with mesenchymal morphology were named "HT29-shE". In the present study, these cells were used to investigate the effect of pioglitazone (Pio) and Cetuximab (Cet) in order to find CSC and EMT targeting agents. METHOD: The viability and IC50 rate of cells treated with different concentrations of Pio and Cet were evaluated using the MTT test. EMT and CSC markers and cell morphology were assessed in Pio and Cet treated and untreated HT29-shE cells using flow cytometry, realtime PCR, immunocytochemistry, and microscopic monitoring. RESULTS: The findings showed that Pio and Cet at concentrations of 250 µM and 40 µg/ml, respectively, decrease cell viability by 50%. Also, they were able to reduce the expression of CSC markers (CD133 and CD44) in the CSC enriched HT29 cell line. Furthermore, Pio and Cet could efficiently reduce the expression of vimentin as a mesenchymal marker and significantly upregulate the expression of E-cadherin as an epidermal marker of EMT and its reverse mesenchymal-- to-epithelial transition (MET). In addition, the mesenchymal morphology of HT29-shE changed into epithelial morphology after Cet treatment. CONCLUSION: Pio and Cet could inhibit EMT and reduce CSC markers in the EMT induced/CSC enriched cell line. We expect that focus on finding EMT/CSC-targeting agents like these drugs can be helpful for cancer treatment.

Evaluation of long non-coding RNAs EGOT, NRAV, NRIR and mRNAs ISG15 and IFITM3 expressions in COVID-19 patients.

Individuals with Coronavirus Disease 2019 (COVID-19) may show no symptoms to moderate or severe complications. This variation may be due to differences in the strength of the immune response, including a delayed interferon (IFN) response in asymptomatic patients and higher IFN levels in severe patients. Some long non-coding RNAs (lncRNAs), as regulators of the IFN pathway, may contribute to the emergence of different COVID-19 symptoms. This study aimed to comparatively investigate the relationship between lncRNAs (eosinophil granule ontogeny transcript (EGOT), negative regulator of antiviral response (NRAV), and negative regulator of interferon response (NRIR)), alongside interferon-stimulated genes (ISGs) like ISG-15 and interferon-induced transmembrane protein 3 (IFITM3) in COVID-19 patients with asymptomatic, moderate, and severe symptoms. Buffy coat samples were collected from 17 asymptomatic, 23 moderate, 22 severe patients, and 44 healthy controls. Quantitative real-time PCR was utilized to determine the expression levels. In a comparison between COVID-19 patients and healthy individuals, higher expression levels of EGOT and NRAV were observed in severe and moderate patients. NRIR expression was increased across all patient groups. Meanwhile, ISG15 expression decreased in all patient groups, and the moderate group showed a significant decrease in IFITM3 expression. Comparing COVID-19 patient groups, EGOT expression was significantly higher in moderate COVID-19 patients compared to asymptomatic patients. NRAV was higher in moderate and severe patients compared to asymptomatic. NRIR levels did not differ significantly between the COVID-19 patient groups. ISG15 was higher in moderate and severe patients compared to asymptomatic. IFITM3 expression was significantly higher in severe patients compared to the moderate group. In severe COVID-19 patients, EGOT expression was positively correlated with NRAV levels. EGOT and NRAV showed a significant positive correlation in asymptomatic patients, and both were positively correlated with IFITM3 expression. This study suggests that EGOT, NRAV, NRIR, ISG15, and IFITM3 may serve as diagnostic biomarkers for COVID-19. The lncRNA NRAV may be a good biomarker in a prognostic panel between asymptomatic and severe patients in combination with other high-sensitivity biomarkers. EGOT, NRAV, and ISG15 could also be considered as specific biomarkers in a prognostic panel comparing asymptomatic and moderate patients with other high-sensitivity biomarkers.

Production of a Ribosome-Displayed Mouse scFv Antibody Against CD133, Analysis of Its Molecular Docking, and Molecular Dynamic Simulations of Their Interactions.

The pentaspan transmembrane glycoprotein CD133, prominin-1, is expressed in cancer stem cells in many tumors and is promising as a novel target for the delivery of cytotoxic drugs to cancer-initiating cells. In this study, we prepared a mouse library of single-chain variable fragment (scFv) antibodies using mRNAs isolated from mice immunized with the third extracellular domain of a recombinant CD133 (D-EC3). First, the scFvs were directly exposed to D-EC3 to select a new specific scFv with high affinity against CD133 using the ribosome display method. Then, the selected scFv was characterized by the indirect enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (ICC), and in silico analyses included molecular docking and molecular dynamics simulations. Based on ELISA results, scFv 2 had a higher affinity for recombinant CD133, and it was considered for further analysis. Next, the immunocytochemistry and flow cytometry experiments confirmed that the obtained scFv could bind to the CD133 expressing HT-29 cells. Furthermore, the results of in silico analysis verified the ability of the scFv 2 antibody to bind and detect the D-EC3 antigen through key residues employed in antigen-antibody interactions. Our results suggest that ribosome display could be applied as a rapid and valid method for isolation of scFv with high affinity and specificity. Also, studying the mechanism of interaction between CD133's scFv and D-EC3 with two approaches of experimental and in silico analysis has potential importance for the design and development of antibody with improved properties.

Low incidence of microsatellite instability in gastric cancers and its association with the clinicopathological characteristics: a comparative study.

Gastric cancer is a complex heterogeneous disease with different molecular subtypes that have clinical implications. It is characterized by high mortality rates and limited effective therapies. Microsatellite instability (MSI) has been recognized as a subgroup with a good prognosis based on TCGA and ACRG categorizations. Besides its prognostic and predictive value, gastric cancers with high MSI exhibit different clinical behaviors. The prevalence of high MSI has been assessed in gastric cancer worldwide, especially in East Asia, but there is a lack of such information in the Middle East. Therefore, this study aimed to investigate the incidence and status of MSI in Iranian gastric cancer patients using 53 samples collected from 2015 to 2020 at Taleghani Hospital Medical Center. DNA from tumoral and normal tissues were extracted and assessed through multiplex-PCR based on five mononucleotide repeats panel. Clinicopathological variables, including age, sex, Lauren classification, lymph node involvement, TNM stage, differentiation, localization, and tumor size, were also analyzed. With 2 males and 2 females, high microsatellite instability represented a small subgroup of almost 7.5% of the samples with a median age of 60.5 years. High microsatellite instability phenotypes were significantly associated with patients aged 68 years and older (p­value of 0.0015) and lower lymph node involvement (p­value of 0.0004). Microsatellite instability was also more frequent in females, with distal gastric location, bigger tumor size, and in the intestinal type of gastric cancer rather than the diffuse type.

Obeticholic acid-loaded exosomes attenuate liver fibrosis through dual targeting of the FXR signaling pathway and ECM remodeling.

End-stage of liver fibrosis as a precancerous state could lead to cirrhosis and hepatocellular carcinoma which liver transplantation is the only effective treatment. Previous studies have indicated that farnesoid X receptor (FXR) agonists, such as obeticholic acid (OCA) protect against hepatic injuries. However, free OCA administration results in side effects in clinical trials that could be alleviated by applying bio carriers such as MSC-derived exosomes (Exo) with the potential to mimic the biological regenerative effect of their parent cells, as proposed in this study. Loading OCA into the Exo was conducted via water bath sonication. Ex vivo bio distribution studies validated the Exo-loaded OCA more permanently accumulated in the liver. Using CCL4-induced liver fibrosis, we proposed whether Exo isolated from human Warton's Jelly mesenchymal stem cells loaded with a minimal dosage of OCA can facilitate liver recovery. Notably, Exo-loaded OCA exerted additive anti-fibrotic efficacy on histopathological features in CCL4-induced fibrotic mice. Compared to baseline, Exo-mediated delivery OCA results in marked improvements in the fibrotic-related indicators as well as serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations. Accordingly, the synergistic impact of Exo-loaded OCA as a promising approach is associated with the inactivation of hepatic stellate cells (HSCs), extracellular matrix (ECM) remodeling, and Fxr-Cyp7a1 cascade on CCL4-induced liver fibrosis mice. In conclusion, our data confirmed the additive protective effects of Exo-loaded OCA in fibrotic mice, which suggests a valuable therapeutic strategy to combat liver fibrosis. Furthermore, the use of Exo for accurate drug delivery to the liver tissue can be inspiring.

A crosstalk between epigenetic modulations and non-alcoholic fatty liver disease progression.

Non-alcoholic fatty liver disease (NAFLD) has recently emerged as a major public health concern worldwide due to its rapidly rising prevalence and its potential to progress into end-stage liver disease. While the precise pathophysiology underlying NAFLD remains incompletely understood, it is strongly associated with various environmental triggers and other metabolic disorders. Epigenetics examines changes in gene expression that are not caused by alterations in the DNA sequence itself. There is accumulating evidence that epigenetics plays a key role in linking environmental cues to the onset and progression of NAFLD. Our understanding of how epigenetic mechanisms contribute to NAFLD pathophysiology has expanded considerably in recent years as research on the epigenetics of NAFLD has developed. This review summarizes recent insights into major epigenetic processes that have been implicated in NAFLD pathogenesis including DNA methylation, histone acetylation, and microRNAs that have emerged as promising targets for further investigation. Elucidating epigenetic mechanisms in NAFLD may uncover novel diagnostic biomarkers and therapeutic targets for this disease. However, many questions have remained unanswered regarding how epigenetics promotes NAFLD onset and progression. Additional studies are needed to further characterize the epigenetic landscape of NAFLD and validate the potential of epigenetic markers as clinical tools. Nevertheless, an enhanced understanding of the epigenetic underpinnings of NAFLD promises to provide key insights into disease mechanisms and pave the way for novel prognostic and therapeutic approaches.

Chemo-immune cell therapy by intratumoral injection of adoptive NK cells with capecitabine in gastric cancer xenograft model.

Introduction: Gastric cancer is one of the most commonly known malignancies and is the fifth cancer-related death globally. Whereas natural killer (NK) cells play a critical role in tumor elimination; therefore, adoptive NK cell therapy has become a promising approach in cancer cytotherapy. Hence, this study investigated the chemo-immune cell therapy in MKN-45 derived xenograft gastric cancer model. Methods: Three groups of animals have received the following treatments separately: activated NK cells, capecitabine, the combination of capecitabine and activated NK cells, and one was considered as the control group. Morphometric properties of tumor samples were evaluated at the end of the study. NK cells infiltration was evaluated by immunohistochemistry (IHC) of hCD56. Mitotic count and treatment response was assessed by hematoxylin and eosin (H&E) staining. The proliferation ratio to apoptosis was determined by IHC assessment of Ki67 and caspase 3. Results: The results indicated that the NK cell therapy could effectively decrease the mitotic count in pathology assessment, but the tumor was not completely eradicated. In combination with metronomic chemotherapy (MC) of capecitabine, NK cell therapy demonstrated a significant difference in tumor morphometric properties compared to the control group. The proliferation ratio to apoptosis was also in line with pathology data. Conclusion: Although NK cell therapy could effectively decrease the mitotic count in vivo, the obtained findings indicated lesser potency than MC despite ex vivo activation. In order to enhance NK cell therapy effectiveness, suppressive features of the tumor microenvironment and inhibitory immune checkpoints blockade should be considered.

Characterisation of extracellular vesicles isolated from hydatid cyst fluid and evaluation of immunomodulatory effects on human monocytes.

Hydatidosis is a disease caused by the larval stage of Echinococcus granulosus, which involves several organs of intermediate hosts. Evidence suggests a communication between hydatid cyst (HC) and hosts via extracellular vesicles. However, a little is known about the communication between EVs derived from HC fluid (HCF) and host cells. In the current study, EVs were isolated using differential centrifugation from sheep HCF and characterized by western blot, electron microscope and size distribution analysis. The uptake of EVs by human monocyte cell line (THP-1) was evaluated. The effects of EVs on the expression levels of pro- and anti-inflammatory cytokines were investigated using quantitative real-time PCR (RT-PCR), 3 and 24 h after incubation. Moreover, the cytokine level of IL-10 was evaluated in supernatant of THP-1 cell line at 3 and 24 h. EVs were successfully isolated and showed spherical shape with size distribution at 130.6 nm. After 3 h, the expression levels of pro-inflammatory cytokine genes (IL1Β, IL15 and IL8) were upregulated, while after 24 h, the expression levels of pro-inflammatory cytokines were decreased and IL13 gene expression showed upregulation. A statistically significant increase was seen in the levels of IL-10 after 24 h. The main mechanism of the communication between EVs derived from HCF and their host remains unclear; however, time-dependent anti-inflammatory effects in our study suggest that HC may modulate the immune responses via EVs.

Helicobacter pylori-derived outer membrane vesicles suppress liver autophagy: A novel mechanism for H. pylori-mediated hepatic disorder.

BACKGROUND: Helicobacter pylori outer membrane vesicles (OMVs) are nano-sized structures, which have been recently suggested to play a crucial role in H. pylori pathogenesis. There are growing evidence indicating the relationship of H. pylori infection with extra-gastroduodenal diseases, especially liver-related disorders. This study was aimed to investigate the effects of H. pylori-derived OMVs on autophagy in hepatic stellate cells (HSCs). MATERIAL AND METHODS: A selection of five clinical strains of H. pylori with different virulence genotypes were included. The OMVs were isolated by ultracentrifugation and characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The protein concentration of OMVs was measured by BCA assay. MTT assay was used to determine the viability of LX-2 cells (human HSCs) treated with OMVs. The expression level of MTOR, AKT, PI3K, BECN1, ATG16 and LC3B genes was assessed in OMVs-treated LX-2 cells using quantitative real-time PCR. Moreover, immunocytochemistry was performed to evaluate the protein expression of MTOR and LC3B autophagy markers. RESULTS: H. pylori strains produced round shape nano-vesicles ranging from 50 to 500 nm. Treatment of HSCs with H. pylori-derived OMVs at concentration of 10 µg/mL for 24 h significantly elevated the expression of autophagy inhibitory markers (PI3K, AKT, and MTOR) and suppressed the mRNA expression level of autophagy core proteins (BECN1, ATG16 and LC3B). Immunocytochemistry also presented a substantial reduction in the concentration of LC3B autophagy core protein, and a marked elevation in the amount of MTOR autophagy inhibitory protein. CONCLUSION: This study revealed that H. pylori-derived OMVs could potentially suppress autophagy flux in HSCs as a novel mechanism for H. pylori-mediated liver autophagy impairment and liver disease development. Further studies are required to elucidate the exact role of OMV-carried contents in liver autophagy, and liver-associated disorders.

Combination of pioglitazone and dendritic cell to optimize efficacy of immune cell therapy in CT26 tumor models.

Introduction: The maturation faith of dendritic cells is restrained by the inflammatory environment and cytokines, such as interleukin-6 and its downstream component. Therefore, introducing the suitable antigen to dendritic cells is crucial. However, reducing the severity of the suppressive tumor microenvironment is indispensable. The present study examined the combination therapy of lymphocyte antigen 6 family member E (LY6E) pulsed mature dendritic cells (LPMDCs) and pioglitazone against colorectal cancer (CRC) to elevate the effectiveness of cancer treatment through probable role of pioglitazone on inhibiting IL-6/STAT3 pathway. Methods: Dendritic cells were generated from murine bone marrow and were pulsed with lymphocyte antigen 6 family member E peptide to assess antigen-specific T-cell proliferation and cytotoxicity assay with Annexin/PI. The effect of pioglitazone on interleukin (IL)-6/STAT3 was evaluated in vitro by real-time polymerase chain reaction (PCR). Afterward, the CRC model was established by subcutaneous injection of CT26, mouse colon carcinoma cell line, in female mice. After treatment, tumor, spleen, and lymph nodes samples were removed for histopathological, ELISA, and real-time PCR analysis. Results: In vitro results revealed the potential of lysate-pulsed dendritic cells in the proliferation of double-positive CD3-8 splenocytes and inducing immunogenic cell death responses, whereas pioglitazone declined the expression of IL-6/STAT3 in colorectal cell lines. In animal models, the recipient of LPMDCs combined with pioglitazone demonstrated high tumor-infiltrating lymphocytes. Elevating the IL-12 and interferon-gamma (IFN-γ) levels and prolonged survival in lysate-pulsed dendritic cell and combination groups were observed. Conclusion: Pioglitazone could efficiently ameliorate the immunosuppressive feature of the tumor microenvironment, mainly through IL-6. Accordingly, applying this drug combined with LPMDCs provoked substantial CD8 positive responses in tumor-challenged animal models.

Pyrosequencing analysis for rapid and accurate detection of clarithromycin resistance-associated mutations in Iranian Helicobacter pylori isolates.

BACKGROUND: Treatment of Helicobacter pylori (H. pylori) infection has become challenging following the development of primary antibiotic resistance. A primary therapeutic regimen for H. pylori eradication includes clarithromycin; however, the presence of point mutations within the 23S rRNA sequence of H. pylori contributes to clarithromycin resistance and eradication failure. Thus, we aimed to develop a rapid and precise method to determine clarithromycin resistance-related point mutations using the pyrosequencing method. METHODS AND RESULTS: H. pylori was isolated from 82 gastric biopsy samples and minimal inhibitory concentration (MIC) was evaluated using the agar dilution method. Clarithromycin resistance-associated point mutations were detected by Sanger sequencing, from which 11 isolates were chosen for pyrosequencing. Our results demonstrated a 43.9% (36/82) prevalence in resistance to clarithromycin. The A2143G mutation was detected in 8.3% (4/48) of H. pylori isolates followed by A2142G (6.2%), C2195T (4.1%), T2182C (4.1%), and C2288T (2%). Although the C2195T mutation was only detected by Sanger sequencing, the overall results from pyrosequencing and Sanger sequencing platforms were comparable. CONCLUSIONS: Pyrosequencing could be used as a rapid and practical platform in clinical laboratories to determine the susceptibility profile of H. pylori isolates. This might pave the way for efficient H. pylori eradication upon detection.

Diagnostic potential and pathogenic performance of circulating miR-146b, miR-194, and miR-214 in liver fibrosis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins. Due to the lack of an accurate test for an early diagnosis of liver fibrosis and the invasiveness of the liver biopsy procedure, there is an urgent need for effective non-invasive biomarkers for screening the patients. we aimed to evaluate the diagnostic performance of circulating miRNAs (miR-146b, -194, -214) and their related mechanisms in the pathogenesis of liver fibrosis. The expression levels of miR-146b, -194, and -214 were quantified in whole blood samples from NAFLD patients using real-time PCR. The competing endogenous RNA (ceRNA) network was constructed and a gene set enrichment analysis (GSEA) was performed for HSC activation-related genes. Also, the transcription factor (TF)-miR co-regulatory network and the survival plot for three miRNAs and core genes were illustrated. The qPCR results showed that the relative expression of miR-146b and miR-214 significantly increased in NAFLD patients, while miR-194 showed significant down-regulation. The ceRNA network analysis implicated NEAT1 and XIST as sponge candidates for these miRNAs. The GSEA results identified 15 core genes involved in HSC activation, primarily enriched in NF-κB activation and autophagy pathways. STAT3, TCF3, RELA, and RUNX1 were considered potential transcription factors connected to miRNAs in the TF-miR network. Our study elucidated three candidate circulating miRNAs differentially expressed in NAFLD that could serve as a promising non-invasive diagnostic tool for early detection strategies. Also, NF-κB activation, autophagy, and negative regulation of the apoptotic process are the main potential underlying mechanisms regulated by these miRNAs in liver fibrosis pathogenesis.

Therapeutic implications of targeting autophagy and TGF-ß crosstalk for the treatment of liver fibrosis.

Liver fibrosis is characterized by the excessive deposition and accumulation of extracellular matrix components, mainly collagens, and occurs in response to a broad spectrum of triggers with different etiologies. Under stress conditions, autophagy serves as a highly conserved homeostatic system for cell survival and is importantly involved in various biological processes. Transforming growth factor-ß1 (TGF-ß1) has emerged as a central cytokine in hepatic stellate cell (HSC) activation and is the main mediator of liver fibrosis. A growing body of evidence from preclinical and clinical studies suggests that TGF-ß1 regulates autophagy, a process that affects various essential (patho)physiological aspects related to liver fibrosis. This review comprehensively highlights recent advances in our understanding of cellular and molecular mechanisms of autophagy, its regulation by TGF-ß, and the implication of autophagy in the pathogenesis of progressive liver disorders. Moreover, we evaluated crosstalk between autophagy and TGF-ß1 signalling and discussed whether simultaneous inhibition of these pathways could represent a novel approach to improve the efficacy of anti-fibrotic therapy in the treatment of liver fibrosis.

Fibroblast growth factor 2 reduces endoplasmic reticulum stress and apoptosis in in-vitro Non-Alcoholic Fatty Liver Disease model.

PURPOSE: Non-Alcoholic fatty liver disease is characterized by the accumulation of excess fat in the liver, chronic inflammation, and cell death, ranging from simple steatosis to fibrosis, and finally leads to cirrhosis and hepatocellular carcinoma. The effect of Fibroblast growth factor 2 on apoptosis and ER stress inhibition has been investigated in many studies. In this study, we aimed to investigate the effect of FGF2 on the NAFLD in-vitro model in the HepG2 cell line. METHODS: The in-vitro NAFLD model was first induced on the HepG2 cell line using oleic acid and palmitic acid for 24 h and evaluated by ORO staining and Real-time PCR. The cell line was then treated with various concentrations of fibroblast growth factor 2 for 24 h, total RNA was extracted and cDNA was consequently synthesized. Real-time PCR and flow cytometry was applied to evaluate gene expression and apoptosis rate, respectively. RESULTS: It was shown that fibroblast growth factor 2 ameliorated apoptosis in the NAFLD in-vitro model by reducing the expression of genes involved in the intrinsic apoptosis pathway, including caspase 3 and 9. Moreover, endoplasmic reticulum stress was decreased following upregulating the protective ER-stress genes, including SOD1 and PPARα. CONCLUSIONS: FGF2 significantly reduced ER stress and intrinsic apoptosis pathway. Our data suggest that FGF2 treatment could be a potential therapeutic strategy for NAFLD.

Human Wharton's jelly mesenchymal stem cells derived-exosomes enriched by miR-124 promote an anti-fibrotic response in an experimental model of liver fibrosis.

BACKGROUND: Liver fibrosis is a significant challenge to global health that results in organ failure through inflammation and the release of fibrotic biomarkers. Due to the lack of effective treatments for liver fibrosis, anti-fibrotic and anti-inflammatory therapies are being developed. Since there has been an association between aberrant expression of miR-124 and liver disease progression, we investigated whether delivery of miR-124 through human Wharton's jelly mesenchymal stem cells derived-exosomes (hWJMSC-Exo) can improve liver fibrosis. METHODS: We established a 6-week carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis, then we administered hWJMSC-Exo and miR-124-3p-enriched exosomes (ExomiR-124) for three weeks. The extent of fibrosis and inflammation was assessed by histology, biochemistry, Real-time PCR, immunohistochemistry, and Enzyme-linked immunoassays (ELISA). The inflammatory status of the spleen was also investigated using flow cytometry. RESULTS: Based on the gene and protein expression measurement of IL-6, IL-17, TGF-ß, STAT3, α-SMA, and COL1, In vivo administration of Exo and ExomiR-124 effectively reduce collagen accumulation and inhibition of inflammation. Regarding histopathology findings, the therapeutic effect of ExomiR-124 against liver fibrosis was significantly greater than hWJMSC-Exo. In addition, we found that Exo and ExomiR-124 was capable of phenotype switching of splenic monocytes from inflammatory Ly6Chi to restorative Ly6Clo. CONCLUSIONS: MSC-derived exosomes demonstrated anti-inflammatory effect via different aspects. Aside from the therapeutic approach, enrichment of exosomes as a nanocarrier by miR-124 revealed the down-regulation of STAT3, which plays a crucial role in liver fibrosis. The anti-inflammatory and anti-fibrotic properties of ExomiR-124 could be a promising option in liver fibrosis combination therapies.