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.

Innate and adaptive immune cell interaction drives inflammasome activation and hepatocyte apoptosis in murine liver injury from immune checkpoint inhibitors.

Immune checkpoints (CTLA4 & PD-1) are inhibitory pathways that block aberrant immune activity and maintain self-tolerance. Tumors co-opt these checkpoints to avoid immune destruction. Immune checkpoint inhibitors (ICIs) activate immune cells and restore their tumoricidal potential, making them highly efficacious cancer therapies. However, immunotolerant organs such as the liver depend on these tolerogenic mechanisms, and their disruption with ICI use can trigger the unintended side effect of hepatotoxicity termed immune-mediated liver injury from ICIs (ILICI). Learning how to uncouple ILICI from ICI anti-tumor activity is of paramount clinical importance. We developed a murine model to recapitulate human ILICI using CTLA4+/- mice treated with either combined anti-CTLA4 + anti-PDL1 or IgG1 + IgG2. We tested two forms of antisense oligonucleotides to knockdown caspase-3 in a total liver (parenchymal and non-parenchymal cells) or in a hepatocyte-specific manner. We also employed imaging mass cytometry (IMC), a powerful multiplex modality for immunophenotyping and cell interaction analysis in our model. ICI-treated mice had significant evidence of liver injury. We detected cleaved caspase-3 (cC3), indicating apoptosis was occurring, as well as Nod-like receptor protein 3 (NLRP3) inflammasome activation, but no necroptosis. Total liver knockdown of caspase-3 worsened liver injury, and induced further inflammasome activation, and Gasdermin-D-mediated pyroptosis. Hepatocyte-specific knockdown of caspase-3 reduced liver injury and NLRP3 inflammasome activation. IMC-generated single-cell data for 77,692 cells was used to identify 22 unique phenotypic clusters. Spatial analysis revealed that cC3+ hepatocytes had significantly closer interactions with macrophages, Kupffer cells, and NLRP3hi myeloid cells than other cell types. We also observed zones of three-way interaction between cC3+ hepatocytes, CD8 + T-cells, and macrophages. Our work is the first to identify hepatocyte apoptosis and NLRP3 inflammasome activation as drivers of ILICI. Furthermore, we report that the interplay between adaptive and innate immune cells is critical to hepatocyte apoptosis and ILICI.

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.

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.

Imatinib suppresses activation of hepatic stellate cells by targeting STAT3/IL-6 pathway through miR-124.

The activation of hepatic stellate cells is the primary function of facilitating liver fibrosis. Interfering with the coordinators of different signaling pathways in activated hepatic stellate cells (aHSCs) could be a potential approach in ameliorating liver fibrosis. Regarding the illustrated anti-fibrotic effect of imatinib in liver fibrosis, we investigated the imatinib's potential role in inhibiting HSC activation through miR-124 and its interference with the STAT3/hepatic leukemia factor (HLF)/IL-6 circuit. The anti-fibrotic effect of imatinib was investigated in the LX-2 cell line and carbon tetrachloride (CCl4 )-induced Sprague-Dawley rat. The expression of IL-6, STAT3, HLF, miR-124, and α-smooth muscle actin (α-SMA) were quantified by quantitative real-time PCR (qRT-PCR) and the protein level of α-SMA and STAT3 was measured by western blot analysis both in vitro and in vivo. The LX-2 cells were subjected to immunocytochemistry (ICC) for α-SMA expression. After administering imatinib in the liver fibrosis model, histopathological examinations were done, and hepatic function serum markers were checked. Imatinib administration alleviated mentioned liver fibrosis markers. The expression of miR-124 was downregulated, while IL-6/HLF/STAT3 circuit agents were upregulated in vitro and in vivo. Notably, imatinib intervention decreased the expression of IL-6, STAT3, and HLF. Elevated expression of miR-124 suppressed the expression of STAT3 and further inhibited HSCs activation. Our results demonstrated that imatinib not only ameliorated hepatic fibrosis through tyrosine kinase inhibitor (TKI) activity but also interfered with the miR-124 and STAT3/HLF/IL-6 pathway. Considering the important role of miR-124 in regulating liver fibrosis and HSCs activation, imatinib may exert its anti-fibrotic activity through miR-124.

Therapeutic potential of targeting regulatory mechanisms of hepatic stellate cell activation in liver fibrosis.

Hepatic fibrosis is a manifestation of different etiologies of liver disease with the involvement of multiple mediators in complex network interactions. Activated hepatic stellate cells (aHSCs) are the central driver of hepatic fibrosis, given their potential to induce connective tissue formation and extracellular matrix (ECM) protein accumulation. Therefore, identifying the cellular and molecular pathways involved in the activation of HSCs is crucial in gaining mechanistic and therapeutic perspectives to more effectively target the disease. In addition to a comprehensive summary of our current understanding of the role of HSCs in liver fibrosis, we also discuss here the proposed therapeutic strategies based on targeting HSCs.

Soluble total antigen derived from Toxoplasma gondii RH strain prevents apoptosis, but induces anti-apoptosis in human monocyte cell line.

Apoptosis plays crucial role in the pathogenesis of toxoplasmosis, as it limits further development of the disease. The current study aimed to investigate the effects of different concentrations of soluble total antigen (STAg) of Toxoplasma gondii (Nicolle et Manceaux, 1908) on the apoptotic and anti-apoptotic pathways. PMA-activated THP-1 cell line was sensed by T. gondii STAg and the expression patterns of caspase-3, -7, -8, -9, Bax, Bcl-2, and Mcl-1 genes were evaluated. The results showed statistically significant concentration-dependent overexpression of both Bcl-2 (P-value < 0.0001) and Mcl-1 (P-value = 0.0147). The cas-7 showed overexpression in all concentrations (P-value < 0.0001). The cas-3 was suppressed in concentrations 100, 80, and 40 µg, but statistically significant downregulated in concentrations 10 and 20 µg. The Bax was suppressed in concentrations 100 to 20 µg, while it slightly downregulated 1.42 fold (P-value = 0.0029) in concentration 10 µg. The expression of cas-8 and -9 was suppressed in all concentrations. Our results indicated that T. gondii STAg downregulated and suppressed apoptotic and upregulated anti-apoptotic pathways. The upregulation of cas-7 in this study may indicate the role of T. gondii STAg in activation of inflammatory responses.

The Protective Effects of Live and Pasteurized Akkermansia muciniphila and Its Extracellular Vesicles against HFD/CCl4-Induced Liver Injury.

Akkermansia muciniphila, as a member of the gut microbiota, has been proposed as a next-generation probiotic. Liver fibrosis is the main determinant of liver dysfunction and mortality in patients with chronic liver disease. In this study, we aimed to determine the beneficial effects of live and pasteurized A. muciniphila and its extracellular vesicles (EVs) on the prevention of liver fibrosis. The response of hepatic stellate cells (HSCs) to live and pasteurized A. muciniphila and its EVs was examined in quiescent, lipopolysaccharide (LPS)-activated LX-2 cells. Liver fibrosis was induced in 8-week-old C57BL/6 mice, using a high-fat diet (HFD) and carbon tetrachloride (CCl4) administration for 4 weeks. The mice were concomitantly treated via oral gavage with three forms of bacteria. The relative expression of different fibrosis and inflammatory markers was assessed in the tissues. Histological markers, serum biochemical parameters, and cytokine production were also analyzed, and their correlations with the relative abundance of targeted fecal bacteria were examined. All A. muciniphila preparations exhibited protective effects against HSC activation; however, EVs showed the greatest activity in HSC regression. Oral gavage with A. muciniphila ameliorated the serum biochemical and inflammatory cytokines and improved liver and colon histopathological damages. The relative expression of fibrosis and inflammatory biomarkers was substantially attenuated in the tissues of all treated mice. The composition of targeted stool bacteria in the live A. muciniphila group was clearly different from that in the fibrosis group. This study indicated that A. muciniphila and its derivatives could successfully protect against HFD/CCl4-induced liver injury. However, further studies are needed to prove the beneficial effects of A. muciniphila on the liver. IMPORTANCE Akkermansia muciniphila, as a member of the gut microbiota, has been proposed as a next-generation probiotic. Liver fibrosis is the main determinant of liver dysfunction and mortality in patients with chronic liver disease. In this study, we aimed to determine the beneficial effects of live and pasteurized A. muciniphila and its extracellular vesicles (EVs) on the prevention of liver fibrosis. The results of the present study indicated that oral administration of live and pasteurized A. muciniphila and its EVs could normalize the fecal targeted bacteria composition, improve the intestinal permeability, modulate inflammatory responses, and subsequently prevent liver injury in HFD/CCl4-administered mice. Following the improvement of intestinal and liver histopathology, HFD/CCl4-induced kidney damage and adipose tissue inflammation were also ameliorated by different A. muciniphila treatments.

Microbiological survey and occurrence of bacterial foodborne pathogens in raw and ready-to-eat green leafy vegetables marketed in Tehran, Iran.

Fresh leafy (FL) and ready-to-eat (RTE) vegetables are recognized as an important source of foodborne disease outbreaks worldwide. Currently, there are no data available for the prevalnce of bacterial foodborne pathogens (FBPs) in raw vegetables consumed in Iran. Here, we evalated the presence of common bacterial FBPs among 366 samples of raw vegetables including 274 FL and 92 RTE collected from 21 districts of Tehran. The presence of FBPs were screened using conventional microbiological culture methods and real-time PCR assays. Overall, a higher rate of bacterial contamination was detected in FL compared to RTE samples using both detection methods. The results obtained by microbiological methods showed that Staphylococcus aureus (134/366, 36.6%), followed by Escherichia coli (85/366, 23.2%) and Clostridium perfringens (66/366, 18%) were detetcted as the most prevalent pathogens in this study. Vibrio cholerae was not detected in any of the samples either by microbiological methods or by the real-time PCR assays. There was a noticeable reduction in the proportion of Campylobacter positive samples using conventional microbiological methods (3.5%) compared to the real-time PCR assay (20.7%). The proportion of FL and RTE positive samples obtained by conventional microbiological methods was significantly different (P < 0.05) for C. perfringens, Campylobacter spp. and S. aureus. The proportion of positive samples in FL and RTE vegetables obtained by the real-time PCR assays was significantly different (P < 0.05) for C. perfringens, S. aureus, Helicobacter pylori and STEC/EHEC, the last one was found more frequently in RTE than in FL samples. Our findings indicated a contamination of FL and RTE vegetables in Iran with a range of well-known and emerging FBPs. Positivity and the distribution of bacterial species from the current data indicated different contamination sources, and overall a lack of effective decontamination steps during the production chain. Moreover, further information about the quality of the water, the hygiene measures implemented during the processing, storage and marketing are required to better identify the critical points and define the proper measures.

Investigation of Clarithromycin Resistance-Associated Mutations and Virulence Genotypes of Helicobacter pylori Isolated from Iranian Population: A Cross-Sectional Study.

Antibiotic resistance has brought into question the efficiency of clarithromycin which is a vital component of eradication therapy for Helicobacter pylori infection. The point mutations within the 23S rRNA sequence of H. pylori isolates which contribute to clarithromycin resistance have yet to be fully characterized. This study was aimed to detect clarithromycin resistance-associated mutations and assess the prevalence of key virulence factors of H. pylori among Iranian patients. Amplification of 16S rRNA and glmM genes were done to identify H. pylori. Minimal inhibitory concentration (MIC) of clarithromycin in 82 H. pylori clinical isolates was determined by agar dilution method. Subsequently, various virulence markers including cagA, vacA, sabA, babA, and dupA of H. pylori were identified by PCR. PCR-sequencing was applied to detect point mutations in the 23S rRNA gene. Based on MIC values, 43.9% of H. pylori isolates showed resistance to clarithromycin. The babA and cagA genes were detected in 92.7% and 82.9% of isolates, assigned to be higher than other virulence factors. No significant relationship was found between the H. pylori virulence genotypes and clarithromycin susceptibility (P > 0.05). Analyzing the 23S rRNA sequences revealed A2143G (4/48, 8.3%) and A2142G (3/48, 6.2%) as the most prevalent mutations in clarithromycin-resistant isolates. Additionally, several novel mutations including G2220T, C2248T, A2624C, G2287A, T2188C, G2710C, C2248T, G2269A, and G2224T were also detected among either resistant or susceptible isolates. Our findings revealed the presence of several point mutations in the 23S rRNA gene of H. pylori isolates which may be associated with resistance to clarithromycin.