Different methods of detaching adherent cells and their effects on the cell surface expression of Fas receptor and Fas ligand.

In cell culture environment, some cells adhere firmly to the culture plates and may be vulnerable to cell detachment during passage. Therefore, it is important to harvest cells with a proper detaching method to maintain the viability of cells after detachment. Trypsinization is frequently used for cellular dissociation and detachment. However, most surface proteins and the extracellular matrix are degraded by enzymatic digestion. A mild cell detachment buffer, accutase, is recommended for the replacement of trypsin to dissociate adherent cells and thereby avoid cellular damage. In this study, we demonstrated that use of accutase for cellular detachment may compromise some surface proteins. Compared with ethylenediaminetetraacetic acid (EDTA)-based nonenzymatic cell dissociation buffers, accutase was associated with significant decreases in the surface Fas ligands and Fas receptors. Moreover, we found that accutase may be able to cleave surface Fas ligands into pieces. Our results also illustrated that surface proteins required 20 h to recover after accutase treatment. We demonstrated that using accutase to dissociate adherent cells compromised the expression of Fas ligands and Fas receptors on the cell surface. These findings indicate that it is important to choose suitable cell detachment buffers and allow cells to recover after detachment before experiments.

The role of S100A9 in the interaction between pancreatic ductal adenocarcinoma cells and stromal cells.

BACKGROUND: A major feature of the microenvironment in pancreatic ductal adenocarcinoma (PDAC) is the significant amount of extracellular matrix produced by pancreatic stellate cells (PSCs), which have been reported to enhance the invasiveness of pancreatic cancer cells and negatively impact the prognosis. METHODS: We analyzed the data from two publicly available microarray datasets deposited in the Gene Expression Omnibus and found candidate genes that were differentially expressed in PDAC cells with metastatic potential and PDAC cells cocultured with PSCs. We studied the interaction between PDAC cells and PSCs in vitro and verified our finding with the survival data of patients with PDAC from the website of The Human Protein Atlas. RESULTS: We found that PSCs stimulated PDAC cells to secrete S100A9, which attracted circulatory monocytes into cancer tissue and enhanced the expression of programmed death-ligand 1 (PD-L1) on macrophages. When analyzing the correlation of S100A9 and PD-L1 expression with the clinical outcomes of patients with PDAC, we ascertained that high expression of S100A9 and PD-L1 was associated with poor survival in patients with PDAC. CONCLUSIONS: PSCs stimulated PDAC cells to secrete S100A9, which acts as a chemoattractant to attract circulatory monocytes into cancer microenvironment and induces expression of PD-L1 on macrophages. High expression of S100A9 and PD-L1 was associated with worse overall survival in a cohort of patients with PDAC.

The influence of a caveolin-1 mutant on the function of P-glycoprotein.

The genetic heterogeneity in cancer cells has an increased chance in the acquisition of new mutant such as drug-resistant phenotype in cancer cells. The phenotype of drug resistance in cancer cells could be evaluated by the number or function of drug transporters on cell membranes, which would lead to decreased intracellular anti-cancer drugs concentration. Caveolae are flask-shaped invaginations on cell membrane that function in membrane trafficking, endocytosis, and as a compartment where receptors and signaling proteins are concentrated. Caveolin-1 (CAV1) is the principal structural protein of caveolae and closely correlates with multidrug resistance in cancer cells. In a systematic study of the ubiquitin-modified proteome, lysine 176 of CAV1 was identified as a potential post-translational modification site for ubiquitination. In this article, we identified a mutation at lysine 176 to arginine (K176R) on CAV1 would interfere with the biogenesis of caveolae and broke the interaction of CAV1 with P-glycoprotein. Functional assays further revealed that K176R mutant of CAV1 in cancer cells increased the transport activity of P-glycoprotein and decreased the killing ability of anti-cancer drugs in non-small-cell lung cancer cell lines.

Electrolysed-reduced water dialysate improves T-cell damage in end-stage renal disease patients with chronic haemodialysis.

BACKGROUND: T-cell damage by increased oxidative stress in end-stage renal disease (ESRD) patients undergoing chronic haemodialysis (HD) led to the increased T-cell apoptosis and the alteration of surface markers and Th1/Th2 ratio in CD4(+) T lymphocytes. Antioxidant electrolysed-reduced water (ERW) was used as the dialysate in ESRD patients undergoing chronic HD to test for improved oxidative stress-related T-cell apoptosis, alterations of surface markers and intracellular cytokine profile. METHODS: We evaluated apoptosis formation by annexin V, CD25-related surface markers, and cytokine ratio of Th1/Th2 in CD4(+) T lymphocytes and Tc1/Tc2 in CD8(+) T lymphocytes of 42 ESRD patients haemodialysed with ERW for 1 year. RESULTS: In comparison to 12 healthy individuals, the ESRD patients had more T-cell apoptosis and less CD3(+), CD4(+) and CD8(+) T cells and CD25/CD69/CD94/CD3(+) phenotypes at baseline. Lower intracellular IL-2 and IFN-gamma levels in the Th1/CD4(+) and Tc1/CD8(+) cells and higher intracellular IL-4, IL-6 and IL-10 levels in the Th2/CD4(+) and Tc2/CD8(+) cells were also noted in the ESRD patients. After a 1-year ERW treatment, the patients had a decrease in T-cell apoptosis and increases in CD3(+), CD4(+) and CD8(+) cell numbers and CD25/CD69/CD94/CD3(+) phenotypes in the T cells. The intracellular IL-2 and IFN-gamma levels in the Th1/Tc1 cells significantly (P < 0.05) increased and the intracellular IL-4, IL-6 and IL-10 levels in the Th2/Tc2 cells decreased. Furthermore, the Th1/Th2 and Tc1/Tc2 cytokine ratios were improved toward a normal status. CONCLUSION: One-year ERW treatment effectively ameliorated T-cell apoptosis, altered CD25-related surface markers and intracellular cytokine profile in the HD patients.

The role of B7 ligands (CD80 and CD86) in CD152-mediated allograft tolerance: a crosscheck hypothesis.

BACKGROUND: The regulatory mechanism by which the B7 ligands (CD80 and CD86) direct the CD28/CD152 costimulatory pathways is unclear. This study investigated the role of CD80 and CD86 in a CD152-mediated allograft tolerance model. METHODS: A low-responding cardiac transplant model (BALB/c-->B10.A) with possible long-term acceptance was used. Immunocytochemical and flow cytometric analyses of the graft-infiltrating cells were conducted to characterize this transplant model. The influence of anti-CD80 and anti-CD86 treatments on the proliferation and interleukin (IL)-2 productions of the tolerated splenocytes (SC) was analyzed. The role of CD80 and CD86 in the induction and maintenance of the graft acceptance in this transplant model were also tested. RESULTS: B10.A mice could accept the BALA/c cardiac allografts (11/22), and an anti-CD152 antibody blocked the graft acceptance (10/10). Immunocytochemical and flow cytometric analyses showed that CD152+ cells were predominant among the CD4+ cells infiltrating the 100-day grafts of the B10.A recipients (B10.A-100). Either anti-CD80 or anti-CD86 treatment significantly enhanced polyclonal proliferation and IL-2 production of the B10.A-100 SC. Blockade of either CD80 or CD86 prohibited the tolerance transmitted by adoptive transfer, and anti-CD80 or anti-CD86 plus skin grafting undermined the established allograft tolerance. CONCLUSIONS: Both CD80 and CD86 were essential for the induction and maintenance of the CD152-mediated allograft tolerance.

Co-delivery of GM-CSF gene enhances the immune responses of hepatitis C viral core protein-expressing DNA vaccine: role of dendritic cells.

Hepatitis C virus (HCV) infection has become a critical public health problem worldwide. In Taiwan, it has been estimated that more than 300,000 people, 2% of the general population, have HCV infection. It has been well documented that direct delivery of gene intramuscularly can generate both humoral and cellular immunity, which more closely simulates the conditions of infection. In this study, female Balb/c mice immunized with HCV core plasmid DNA with or without adjuvant GM-CSF cytokine gene could induce both cellular immune response and HCV core-specific antibody titers after injection. Furthermore, the mice immunized with HCV core plus GM-CSF genes showed higher antibody titer and cytotoxic T cell activity compared to those of mice immunized with HCV core gene only (P < 0.05). To explore the effect of GM-CSF gene, the mice were immunized with reporter gene and cytokine gene plasmid. Increased levels of reporter protein and infiltrating cells around muscle tissue were noted. Moreover, the protein could be detected in inguinal node 24 hr after injection, especially in mice immunized with HCV/core plasmid plus GM-CSF gene. It was also observed that reporter protein expressing CD11c(+) dendritic cells could be seen in the inguinal node. These data suggest that the GM-CSF gene did enhance HCV core specific immune response when co-immunized with HCV core DNA plasmid. Although more studies are needed, dendritic cells that appeared around the naked DNA injection area and that local lymph nodes might play a critical role in the immune response induced by naked DNA immunization.