Search

Cholangiocarcinoma (CCA) presents a significant clinical challenge which is often identified in advanced stages, therby restricting the effectiveness of surgical interventions for most patients. The high incidence of cancer recurrence and resistance to chemotherapy further contribute to a bleak prognosis and low survival rates. To address this pressing need for effective therapeutic strategies, our study focuses on the development of an innovative cellular immunotherapy, specifically utilizing chimeric antigen receptor (CAR)-engineered natural killer (NK) cells designed to target the cMET receptor tyrosine kinase. In this investigation, we initiated the screening of a phage library displaying human single-chain variable fragment (ScFv) to identify novel ScFv molecules with specificity for cMET. Remarkably, ScFv11, ScFv72, and ScFv114 demonstrated exceptional binding affinity, confirmed by molecular docking analysis. These selected ScFvs, in addition to the well-established anti-cMET ScFvA, were integrated into a CAR cassette harboring CD28 transmembrane region-41BB-CD3ζ domains. The resulting anti-cMET CAR constructs were transduced into NK-92 cells, generating potent anti-cMET CAR-NK-92 cells. To assess the specificity and efficacy of these engineered cells, we employed KKU213A cells with high cMET expression and KKU055 cells with low cMET levels. Notably, co-culture of anti-cMET CAR-NK-92 cells with KKU213A cells resulted in significantly increased cell death, whereas no such effect was observed with KKU055 cells. In summary, our study identified cMET as a promising therapeutic target for CCA. The NK-92 cells, armed with the anti-cMET CAR molecule, have shown strong ability to kill cancer cells specifically, indicating their potential as a promising treatment for CCA in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Advanced cholangiocarcinoma (CCA) presents a clinical challenge due to limited treatment options, necessitating exploration of innovative therapeutic approaches. Bispecific T cell engager (BTE)-armed T cell therapy shows promise in hematological and solid malignancies, offering potential advantages in safety over continuous BTE infusion. In this context, we developed a novel BTE, targeting CD3 on T cells and integrin αvβ6, an antigen elevated in various epithelial malignancies, on cancer cells. The novel BTE was generated by fusing an integrin αvβ6-binding peptide (A20) to an anti-CD3 (OKT3) single-chain variable fragment (scFv) through a G 4 S peptide linker (A20/αCD3 BTE). T cells were then armed with A20/αCD3 BTE (A20/αCD3-armed T cells) and assessed for antitumor activity. Our results highlight the specific binding of A20/αCD3 BTE to CD3 on T cells and integrin αvβ6 on target cells, effectively redirecting T cells towards these targets. After co-culture, A20/αCD3-armed T cells exhibited significantly heightened cytotoxicity against integrin αvβ6-expressing target cells compared to unarmed T cells in both KKU-213A cells and A375.β6 cells. Moreover, in a five-day co-culture, A20/αCD3-armed T cells demonstrated superior cytotoxicity against KKU-213A spheroids compared to unarmed T cells. Importantly, A20/αCD3-armed T cells exhibited an increased proportion of the effector memory T cell (Tem) subset, upregulation of T cell activation markers, enhanced T cell proliferation, and increased cytolytic molecule/cytokine production, when compared to unarmed T cells in an integrin αvβ6-dependent manner. These findings support the potential of A20/αCD3-armed T cells as a novel therapeutic approach for integrin αvβ6-expressing cancers., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Crown Copyright © 2024. Published by Elsevier Masson SAS. All rights reserved.)

Multiple myeloma (MM) is an incurable hematologic malignancy characterized by the rapid proliferation of malignant plasma cells within the bone marrow. Standard therapies often fail due to patient resistance. The US FDA has approved second-generation chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (anti-BCMA-CAR2 T cells) for MM treatment. However, achieving enduring clinical responses remains a challenge in CAR T cell therapy. This study developed third-generation T cells with an anti-BCMA CAR (anti-BCMA-CAR3). The CAR incorporated a fully human scFv specific to BCMA, linked to the CD8 hinge region. The design included the CD28 transmembrane domain, two co-stimulatory domains (CD28 and 4-1BB), and the CD3ζ signaling domain (28BBζ). Lentiviral technology generated these modified T cells, which were compared against anti-BCMA-CAR2 T cells for efficacy against cancer. Anti-BCMA-CAR3 T cells exhibited significantly higher cytotoxic activity against BCMA-expressing cells (KMS-12-PE and NCI-H929) compared to anti-BCMA-CAR2 T cells. At an effector-to-target ratio of 10:1, anti-BCMA-CAR3 T cells induced lysis in 75.5 ± 3.8% of NCI-H929 cells, whereas anti-BCMA-CAR2 T cells achieved 56.7 ± 3.4% (p = 0.0023). Notably, after twelve days of cultivation, anti-BCMA-CAR3 T cells nearly eradicated BCMA-positive cells (4.1 ± 2.1%), while anti-BCMA-CAR2 T cells allowed 36.8 ± 20.1% to survive. This study highlights the superior efficacy of anti-BCMA-CAR3 T cells against both low and high BCMA-expressing MM cells, surpassing anti-BCMA-CAR2 T cells. These findings suggest potential for advancing anti-BCMA-CAR3 T cells in chimeric antigen receptor T (CAR-T) therapy for relapsed/refractory MM., (© 2024. The Author(s).)

The treatment of breast cancer (BC) remains a formidable challenge due to the emergence of drug resistance, necessitating the exploration of innovative strategies. Chimeric antigen receptor (CAR)-T cell therapy, a groundbreaking approach in hematologic malignancies, is actively under investigation for its potential application in solid tumors, including BC. Trophoblast cell surface antigen 2 (Trop2) has emerged as a promising immunotherapeutic target in various cancers and is notably overexpressed in BC. To enhance therapeutic efficacy in BC, a fourth-generation CAR (CAR4) construct was developed. This CAR4 design incorporates an anti-Trop2 single-chain variable fragment (scFv) fused with three costimulatory domains -CD28/4-1BB/CD27, and CD3ζ. Comparative analysis with the conventional second-generation CAR (CAR2; 28ζ) revealed that anti-Trop2 CAR4 T cells exhibited heightened cytotoxicity and interferon-gamma (IFN-γ) production against Trop2-expressing MCF-7 cells. Notably, anti-Trop2 CAR4-T cells demonstrated superior long-term cytotoxic functionality and proliferative capacity. Crucially, anti-Trop2 CAR4-T cells displayed specific cytotoxicity against Trop2-positive BC cells (MDA-MB-231, HCC70, and MCF-7) in both two-dimensional (2D) and three-dimensional (3D) culture systems. Following antigen-specific killing, these cells markedly secreted interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), IFN-γ, and Granzyme B compared to non-transduced T cells. This study highlights the therapeutic potential of anti-Trop2 CAR4-T cells in adoptive T cell therapy for BC, offering significant promise for the advancement of BC treatment strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

T cell-based immunotherapy has transformed cancer treatment. Nonetheless, T cell antitumor activity can be inhibited by an immune checkpoint molecule expressed on cancer cells, program death ligand 1 (PD-L1), which interacts with the PD-1 on T cells. We generated αPD-L1 × αCD3 bispecific T-cell engager-armed T cells (BATs) to prevent PD-L1/PD-1 interaction and hence to redirect T cells to kill cancer cells. αPD-L1 × αCD3 bispecific T-cell engagers (BTEs) were produced from Chinese hamster ovary (CHO) cells to arm human primary T cells. Flow cytometry was used to investigate BTE binding to BATs. The cytotoxicity of BATs against PD-L1-expressing breast cancer (BC) cell lines was assessed in 2-dimensional (2D) and 3-dimensional (3D) culture models. The binding stability of BTE on BATs and their efficacy after cryopreservation were also examined. The CHO cell BTE expression yield was 3.34 mg/ml. The binding ability on T cells reached 91.02 ± 4.2 %. BATs specifically lysed PD-L1-expressing BC cells, with 56.4 ± 15.3 % HCC70 cells and 70.67 ± 15.6 % MDA-MB-231 cells lysed at a 10:1 effector-to-target ratio. BATs showed slight, nonsignificant lysis of PD-L1-negative BC cells, MCF-7, and T47D. Moreover, BATs significantly disrupted MDA-MB-231 3D spheroids expressing PD-L1 after 48 and 72 h of coculture. Cryopreserved BATs maintained BTE binding stability, cell viability, and anticancer activity, comparable to fresh BATs. αPD-L1 × αCD3 BATs induced the cytolysis of PD-L1-expressing BC cells in 2D and 3D coculture assays. BATs can be prepared and preserved, facilitating their use and transportation. This study demonstrates the potential of αPD-L1 × αCD3 BATs in treating cancers with positive PD-L1 expression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Carcinoma-associated fibroblasts (CAFs) play a critical role in cancer progression and immune cell modulation. In this study, it was aimed to evaluate the roles of CAFs-derived IL-6 in doxorubicin (Dox) resistance and PD-L1-mediated chimeric antigenic receptor (CAR)-T cell resistance in breast cancer (BCA)., Methods: CAF conditioned-media (CM) were collected, and the IL-6 level was measured by ELISA. CAF-CM were treated in MDA-MB-231 and HCC70 TNBC cell lines and siIL-6 receptor (IL-6R) knocked down (KD) cells to determine the effect of CAF-derived IL-6 on Dox resistance by flow cytometry and on increased PD-L1 through STAT3, AKT and ERK1/2 pathways by Western blot analysis. After pre-treating with CM, the folate receptor alpha (FRα)-CAR T cell cytotoxicity was evaluated in 2D and 3D spheroid culture assays., Results: The results showed a significant level of IL-6 in CAF-CM compared to that of normal fibroblasts (NFs). The CM with high IL-6 level significantly induced Dox resistance; and PD-L1 expression through STAT3 and AKT pathways in MDA-MB-231 and HCC70 cells. These induction effects were attenuated in siIL-6R KD cells. Moreover, the TNBC cell lines that were CM-treated with STAT3 and an AKT inhibitor had a reduced effect of IL-6 on PD-L1 expression. BCA cells with high IL-6 containing-CM treatment had resistance to cancer cell killing by FRα CAR-T cells compared to untreated cells., Conclusion: These results highlight CAF-derived IL-6 in the resistance of chemotherapy and T cell therapy. Using inhibitors of IL6-STAT3/AKT-PD-L1 axis may provide a potential benefit of Dox and CAR-T cell therapies in BCA patients., (© 2023. The Author(s).)

Background Aims: Cholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA., Methods: To overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28., Results: Initially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8 + T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed., Conclusion: Taken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary, or financial interest in the products or companies described in this article., (Copyright © 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Wilms tumor 1 (WT1) is a promising target antigen for cancer immunotherapy. However, WT1 protein expression and its clinical correlation in multiple myeloma (MM) patients are still limited. We, therefore, investigated WT1 expression in 142 bone marrow and plasmacytoma samples of MM patients at different stages of the disease by immunohistochemistry. The correlations between WT1 expression and clinical parameters or treatment outcomes were evaluated. The overall positive rate of WT1 expression was 91.5%; this high prevalence was found in both bone marrow and plasmacytoma samples, regardless of the disease status. Cytoplasmic WT1 expression was correlated with high serum free light chain ratio at presentation. However, no significant association between WT1 expression and treatment outcome was observed. This study confirms the high prevalence of WT1 expression in an Asian cohort of MM, encouraging the development of immunotherapy targeting WT1 in MM patients, particularly in those with extramedullary plasmacytoma or relapsed disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Phikulsod, Sukpanichnant, Kunacheewa, Chieochansin, Junking and Yenchitsomanus.)

Adoptive T cell therapy using second-generation anti-CD19 chimeric antigen receptor T cells (anti-CD19-CAR2-T) induced complete remission in many heavily pretreated patients with B cell acute lymphoblastic leukemia (B-ALL) or diffuse large B cell lymphoma (DLBCL). However, poor clinical efficacy was observed in treating aggressive B cell lymphomas (BCL). The limited T cell function was reported by programmed cell death protein 1 ligand (PD-L1) expressed on BCL cells bound to the PD-1 receptor on T cells. To overcome this problem, we generated anti-CD19-CAR4-T cells secreting anti-PD-L1 single-chain variable fragment (scFv), namely anti-CD19-CAR5-T cells, and evaluated their functions in vitro. Both anti-CD19-CAR-T cells contain an anti-CD19 scFv derived from a monoclonal antibody, FMC63, linked to CD28/4-1BB/CD27/CD3ζ. The secreting anti-PD-L1 scFv is derived from atezolizumab. Our results showed that secreted anti-PD-L1 scFv could bind to PD-L1 and block the binding of anti-PD-L1 monoclonal antibodies on PD-L1 high tumor cells. Anti-CD19-CAR4-T and anti-CD19-CAR5-T cells efficiently killed CD19 + target tumor cells in two-dimensional (2D) and three-dimensional (3D) co-culture systems. However, anti-CD19-CAR5-T cells demonstrated superior proliferative ability. Interestingly, at a low effector (E) to target (T) ratio of 0.5:1, anti-CD19-CAR5-T cells showed higher cytotoxicity against CD19 + /PD-L1 high cells compared to that of anti-CD19-CAR4-T cells. The cytotoxicity of anti-CD19-CAR4-T cells against CD19 + /PD-L1 high could be restored by adding anti-PD-L1 scFv. Our findings demonstrate the combination antitumor efficiency of anti-CD19-CAR4-T cells and anti-PD-L1 scFv against CD19 + /PD-L1 high tumors. As such, anti-CD19-CAR5-T cells should be further investigated in vivo antitumor efficiency and clinical trials as a treatment for aggressive B cell lymphoma., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Adoptive cell transfer (ACT) is a promising approach for cancer treatment. Activation of T lymphocytes by s elf-differentiated m yeloid-derived a ntigen-presenting-cells r eactive against t umor (SmartDC) resulted in specific anti-cancer function. Folate receptor alpha (FRα) is highly expressed in breast cancer (BC) cells and thus potential to be a target antigen for ACT. To explore the SmartDC technology for treatment of BC, we create SmartDC expressing FRα antigen (SmartDC-FRα) for activation of FRα-specific T lymphocytes. Human primary monocytes were transduced with lentiviruses containing tri-cistronic complementary DNA sequences encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and FRα to generate SmartDC-FRα. Autologous T lymphocytes were activated by SmartDC-FRα by coculture. The activated T lymphocytes exhibited enhanced cytotoxicity against FRα-expressing BC cell cultures. Up to 84.9 ± 6.2% of MDA-MB-231 and 89.7 ± 1.9% of MCF-7 BC cell lines were specifically lysed at an effector-to-target ratio of 20:1. The cytotoxicity of T lymphocytes activated by SmartDC-FRα was also demonstrated in three-dimensional (3D) spheroid culture of FRα-expressing BC cells marked by size reduction and spheroid disruption. This study thus portray the potential development of T lymphocytes activated by SmartDC-FRα as ACT in FRα-expressing BC treatment.

Cholangiocarcinoma (CCA) is a lethal cancer with rapid progression and poor survival. Novel and more effective therapies than those currently available are, therefore, urgently needed. Our research group previously reported the combination of gemcitabine and cytotoxic T lymphocytes to be more effective than single-agent treatment for the elimination of CCA cells. However, gemcitabine treatment of CCA cells upregulates the expression of an immune checkpoint protein (programmed death-ligand 1 [PD-L1]) that consequently inhibits the cytotoxicity of T lymphocytes. To overcome this challenge and take advantage of PD-L1 upregulation upon gemcitabine treatment, we generated recombinant PD-L1xCD3 bispecific T cell engagers (BiTEs) to simultaneously block PD-1/PD-L1 signaling and recruit T lymphocytes to eliminate CCA cells. Two recombinant PD-L1xCD3 BiTEs (mBiTE and sBiTE contain anti-PD-L1 scFv region from atezolizumab and from a published sequence, respectively) were able to specifically bind to both CD3 on T lymphocytes, and to PD-L1 overexpressed after gemcitabine treatment on CCA (KKU213A, KKU055, and KKU100) cells. mBiTE and sBiTE significantly enhanced T lymphocyte cytotoxicity against CCA cells, especially after gemcitabine treatment, and their magnitudes of cytotoxicity were positively associated with the levels of PD-L1 expression. Our findings suggest combination gemcitabine and PD-L1xCD3 BiTE as a potential alternative therapy for CCA., (© 2022. The Author(s).)

Background/aim: B cell maturation antigen (BCMA) is an ideal target for adoptive T cell therapy of multiple myeloma (MM). In this study, we evaluated self-differentiated monocyte-derived dendritic cells expressing BCMA (SD-DC-BCMA) to activate T cells for killing MM cells., Materials and Methods: Lentivirus-modified SD-DC-BCMA harboring tri-cistronic cDNAs encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and BCMA was generated. Cytotoxicity of T cells activated by SD-DC-BCMA against MM cells was evaluated., Results: T cells activated by SD-DC-BCMA exhibited a dose-dependent cytotoxicity against BCMA-expressing MM cells and produced high IFN-γ levels, compared to inactivated T cells or control T cells. A significantly higher killing ability of T cells activated by SD-DC-BCMA was further demonstrated in BCMA-overexpressing cells when compared with BCMA-negative cells., Conclusion: The potency of SD-DC-BCMA to activate T cells for antigen-specific cancer killing provides a framework for therapeutic application of adoptive T cell therapy in MM., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Cholangiocarcinoma (CCA) is a lethal cancer of bile duct epithelial cells with a high mortality rate and limited therapeutic options. An effective treatment is, therefore, urgently needed to improve treatment outcomes for these patients. To develop a new therapeutic option, we engineered T cells secreting αCD133-αCD3 bispecific T-cell engager and evaluated their antitumor effects against CD133-expressing CCA cells. The cDNA encoding αCD133-αCD3 bispecific T-cell engager (αCD133-αCD3-ENG) was cloned into pCDH lentiviral construct and its expression was tested in Lenti-X 293T cells. T cells from healthy donors were then transduced with engineered lentiviruses to create T cells secreting αCD133-αCD3 engager to evaluate their antitumor activities. The average transduction efficiency into T cells was approximately 60.03±21.65%. In the co-culture system containing T cells secreting αCD133-αCD3 engager (as effector cells) and mWasabi-luciferase-expressing CCA cells (KKU-100 and KKU-213A; as target cells), the effector T cells exhibited significantly higher cytolytic activities against the target CCA cells (49.0±9.76% and 64.10±13.18%, respectively) than those observed against the untransduced T cells (10.97±10.65%; p = 0.0103 and 9.80±11.05%; p = 0.0054) at an effector-to-target ratio of 5:1. In addition, the secreted αCD133-αCD3 engager significantly redirected both transduced T cells and bystander T cells to kill the target CCA cells (up to 73.20±1.68%; p<0.05). Moreover, the transduced and bystander T cells could kill the target CCA spheroids at a rate approximately 5-fold higher than that of the no treatment control condition (p = 0.0011). Our findings demonstrate proof-of-principle that T cells secreting αCD133-αCD3 engager can be an alternative approach to treating CD133-positive CCA, and they pave the way for future in vivo study and clinical trials., Competing Interests: The authors have declared that no competing interests exist.

Second-generation anti-CD19-chimeric antigen receptor T cells (anti-CD19-CAR2 T cells) are effective for treating B-cell malignancies; however, anti-CD19-CAR2 T cells can induce human anti-mouse immune responses because anti-CD19 single-chain variable fragment (scFv) in the CAR molecules is derived from a murine FMC63 (mFMC63) monoclonal antibody. Consequently, the persistence of mFMC63-CAR2 T cells and their therapeutic efficiency in patients are decreased, which results in tumor relapse. In an attempt to remedy this shortcoming, we generated a new anti-CD19-CAR T cells containing fully human anti-CD19 scFv (Hu1E7-CAR4 T cells) to pre-clinically evaluate and compare with mFMC63-CAR4 T cells. The human anti-CD19 scFv (Hu1E7) was isolated from a human scFv phage display library and fused to the hinge region of CD8α, the transmembrane domain of CD28, three intracellular costimulatory domains (CD28, 4-1BB, and CD27), and a CD3ζ signaling domain (28BB27ζ). Compared to mFMC63-CAR2 T cells (BBζ) and mFMC63-CAR3 (BB27ζ), the mFMC63-CAR4 T cells (28BB27ζ) exerted superior anti-tumor activity against Raji (CD19 + ) target cell. The Hu1E7-CAR4 and mFMC63-CAR4 T cells demonstrated comparable cytotoxicity and proliferation. Interestingly, compared to mFMC63-CAR4 T cells, the Hu1E7-CAR4 T cells secreted lower levels of cytokines (IFN-γ and TNF-α), which may be due to the lower binding affinity of Hu1E7-CAR4 T cells. These findings demonstrated the successfulness in creation of a new CAR T cells containing a novel fully human-derived scFv specific to CD19 + cancer cells. In vivo studies are needed to further compare the anti-tumor efficacy and safety of Hu1E7-CAR4 T cells and mFMC63-CAR4 T cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wutti-in, Sujjitjoon, Sawasdee, Panya, Kongkla, Yuti, Yongpitakwattana, Thepmalee, Junking, Chieochansin, Poungvarin, Yamabhai and Yenchitsomanus.)

Long-term medication with dexamethasone - a synthetic glucocorticoid (GC) drug - results in hyperglycemia, or steroid-induced diabetes. Although recent studies revealed that dexamethasone directly induces pancreatic β-cell apoptosis, its molecular mechanisms remain unclear. In our initial analysis of mRNA transcripts, we discovered the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway may be involved in dexamethasone-induced pancreatic β-cell apoptosis. In the present study, a mechanism of dexamethasone-induced pancreatic β-cell apoptosis through the TRAIL pathway was investigated in cultured cells and isolated mouse islets. INS-1 cells were cultured with and without dexamethasone in the presence or absence of a glucocorticoid receptor (GR) inhibitor, RU486. We found that dexamethasone induced pancreatic β-cell apoptosis in association with the upregulation of TNSF10 (TRAIL) mRNA and protein expression. Moreover, dexamethasone upregulated the TRAIL death receptor (DR5) protein but suppressed the decoy receptor (DcR1) protein. Similar findings were observed in mouse isolated islets: dexamethasone increased TRAIL and DR5 compared to that of control mice. Furthermore, dexamethasone stimulated pro-apoptotic signaling including superoxide production, caspase-8, -9, and -3 activities, NF-κB, and Bax but repressed the anti-apoptotic protein, Bcl-2. All these effects were inhibited by the GR-inhibitor, RU486. Furthermore, knock-down DR5 decreased dexamethasone-induced caspase 3 activity. Caspase-8 and caspase-9 inhibitors protected pancreatic β-cells from dexamethasone-induced apoptosis. Taken together, dexamethasone induced pancreatic β-cell apoptosis by binding to the GR and inducing DR5 and TRAIL pathway.

Multiple myeloma (MM) patients considered to be at high cytogenetic risk commonly fail to respond to standard treatment. A thorough understanding of the molecular mechanism of MM development is, therefore, needed. We endeavored to explore the transcriptional signature among different subgroups of newly diagnosed MM using gene chip-based expression microarray. Bone marrow samples of 15 newly diagnosed Thai MM patients were included. The chromosomal translocation t(4;14) was the most frequently identified genetic alteration in the high-risk subgroup. Cluster analysis from expression profiling demonstrated that high-risk MM have a distinctly different expression pattern compared to standard-risk patients. The most significant differentially expressed gene was UCHL1 . Functional enrichment analysis by Gene Set Enrichment Analysis, FUNRICH, and Gene Ontology Panther pathway revealed the gene sets involved in cell cycle control to be enriched in the t(4;14) high-risk group. Interestingly, among the well-established downstream targets of UCHL1 , only CCND2 was significantly expressed in the t(4;14) high-risk group. Suppression of UCHL1 protein level by LDN-5744 inhibitor could arrest the cell cycle in G1 phase in cell lines. These findings shed light on the molecular mechanism of UCHL1 in t(4;14) high-risk MM and support the evidence that alteration of the UCHL1 pathway may play a role in the pathogenesis of high-risk MM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kamseng, Siriboonpiputtana, Puavilai, Chuncharunee, Paisooksantivatana, Chareonsirisuthigul, Junking, Chiraphapphaiboon, Yenchitsomanus and Rerkamnuaychoke.)

Current treatments for cholangiocarcinoma (CCA) are largely unsuccessful due to late diagnosis at advanced stage, leading to high mortality rate. Consequently, improved therapeutic approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy is a newly potential therapy that can recognize specific surface antigen without major histocompatibility complex (MHC) restriction. Mucin 1 (MUC1) is an attractive candidate antigen as it is highly expressed and associated with poor prognosis and survival in CCA. We, therefore, set forth to create the fourth-generation CAR (CAR4) construct containing anti-MUC1-single-chain variable fragment (scFv) and three co-stimulatory domains (CD28, CD137, and CD27) linked to CD3ζ and evaluate anti-MUC1-CAR4 T cells in CCA models. Compared to untransduced T cells, anti-MUC1-CAR4 T cells produced increased levels of TNF-α, IFN-γ and granzyme B when exposed to MUC1-expressing KKU-100 and KKU-213A CCA cells (all p < 0.05). Anti-MUC1-CAR4 T cells demonstrated specific killing activity against KKU-100 (45.88 ± 7.45%, p < 0.05) and KKU-213A cells (66.03 ± 3.14%, p < 0.001) at an effector to target ratio of 5:1, but demonstrated negligible cytolytic activity against immortal cholangiocytes. Furthermore, the anti-MUC1-CAR4 T cells could effectively disrupt KKU-213A spheroids. These activities of anti-MUC1-CAR4 T cells supports the development of this approach as an adoptive T cell therapeutic strategy for CCA.

Cholangiocarcinoma (CCA) is a lethal bile duct cancer that responds poorly to current standard treatments. A new therapeutic approach is, therefore, urgently needed. Adoptive T cell transfer using chimeric antigen receptor (CAR) T cells is a new therapeutic modality with demonstrated efficacy in hematologic malignancies. However, its efficacy against solid tumors is modest, and further intensive investigation continues. An important factor that influences the success of CAR T cell therapy is the selection of a target antigen that is highly expressed on cancer cells, but markedly less so in normal cells. Integrin αvβ6 is upregulated in several solid tumors, but is minimally expressed in normal epithelial cells, which suggests integrin αvβ6 as an attractive target antigen for CAR T cell immunotherapy in CCA. We investigated integrin αvβ6 expression in pathological tissue samples from patients with liver fluke-associated CCA. We then created CAR T cells targeting integrin αvβ6 and evaluated their anti-tumor activities against CCA cells. We found overexpression of the integrin αvβ6 protein in 23 of 30 (73.3%) CCA patient tissue samples. Significant association between high integrin αvβ6 expression and short survival time ( p = 0.043) was also observed. Lentiviral constructs were engineered to encode CARs containing an integrin αvβ6-binding peptide (A20) derived from foot-and-mouth disease virus fused with a second-generation CD28/CD3ζ signaling domain (A20-2G CAR) or with a fourth-generation CD28/4-1BB/CD27/CD3ζ signaling domain (A20-4G CAR). The A20-2G and A20-4G CARs were highly expressed in primary human T cells transduced with the engineered lentiviruses, and they exhibited high levels of cytotoxicity against integrin αvβ6-positive CCA cells ( p < 0.05). Interestingly, the A20-2G and A20-4G CAR T cells displayed anti-tumor function against integrin αvβ6-positive CCA tumor spheroids ( p < 0.05). Upon specific antigen recognition, A20-4G CAR T cells produced a slightly lower level of IFN-γ, but exhibited higher proliferation than A20-2G CAR T cells. Thus, the A20-4G CAR T cells with lower level of cytokine production, but with higher proliferation represents a promising potential adoptive T cell therapy for integrin αvβ6-positive CCA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Phanthaphol, Somboonpatarakun, Suwanchiwasiri, Chieochansin, Sujjitjoon, Wongkham, Maher, Junking and Yenchitsomanus.)

Purpose: Treatment of breast cancer (BC) by standard methods is effective in the early stage, but ineffective in the advanced stage of disease. To develop an adoptive T cell therapy for advanced and severe BC, we generated fourth-generation chimeric antigen receptor (CAR) T cells targeting folate receptor alpha antigen (FRα) expressed on BC cells, and preclinically evaluated their anti-BC activities., Methods: The fourth-generation FRα-CAR T cells containing extracellular FRα-specific single-chain variable fragment (scFv) and three intracellular costimulatory domains (CD28, 4-1BB, and CD27) linked to CD3ζ were generated using a lentiviral system, and then were evaluated for their anti-BC activities in two-dimensional and three-dimensional (spheroid) cultures., Results: When our fourth-generation FRα-CAR T cells were cocultured with FRα-expressing MDA-MB-231 BC cell line at an effector to target ratio of 20:1, these CAR T cells specifically lysed 88.7 ± 10.6% of the target cells. Interestingly, the cytotoxic lysis of FRα-CAR T cells was more pronounced in target cells with higher surface FRα expression. This specific cytotoxicity of the CAR T cells was not observed when cocultured with FRα-negative MCF10A normal breast-like cell line at the same ratio (34.3 ± 4.7%). When they were cocultured with MDA-MD-231 spheroid, the FRα-CAR T cells exhibited antitumor activity marked with spheroid size reduction and breakage., Conclusion: This proof-of-concept study thus shows the feasibility of using these fourth-generation FRα-CAR T cells for adoptive T cell therapy in BC.

Current treatment of cholangiocarcinoma (CCA) - a lethal bile duct cancer - is ineffective because the disease is usually diagnosed at late and advanced stage. Thus, a novel therapeutic modality is urgently required. Fourth-generation chimeric antigen receptor (CAR4) T cells was created to target CD133, a well-known cancer stem cell marker, that is highly expressed and associates with cancer progression. The anti-CD133-CAR4 T cells showed high efficacy against CD133-expressing CCA cells. Tumour cell lysis occurred in a dose- and CD133 antigen-dependent manner, and significantly higher, up to 57.59% ± 9.62 at effector to target ratio of 5:1 in a CCA cell line - KKU-213A cells, compared to mock control (p = 0.008). Similarly, significant IFN-γ (p = 0.011) and TNF-α (p = 0.002) upregulation was observed upon tumour treatment. The effectiveness of our anti-CD133-CAR4 T cells will be beneficial not only for CD133-expressing CCA, but also for other CD133-expressing tumours. This study may guide future in vivo study and clinical trials., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cholangiocarcinoma (CCA) is an aggressive tumor that is associated with high rates of recurrence and mortality. This is due, in part, to the fact that CCA cells and their microenvironment secrete immunosuppressive cytokines, transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), that inhibit dendritic cell (DC) functions, which, in turn, results in the decreased anti-tumor activity of T-cells. We hypothesized that the TGF-β receptor and IL-10 blockade on dendritic cells would improve DC function, thereby allowing improved activation of T cells against CCA cells. To test our hypothesis, we generated self-differentiated DCs (SD-DCs) via transduction of human peripheral blood monocytes with lentivirus expressing IL-4 and GM-CSF. SD-DCs were transduced with a second lentivirus containing short-hairpin RNAs (shRNAs) to knock-down TGF-βRII and IL-10RA mRNAs. Immunoblot confirmed the reduced expression levels of TGF-β and IL-10 receptors in both SD-DCs that were transduced with a single and/or combination of lentiviruses containing shRNAs. SD-DCs were thereafter pulsed with tumor antigens extracted from CCA cell lines in an effort to activate DC function. MHC class II (HLA-DR) and co-stimulatory molecules (CD40 and CD86) on SD-DCs were upregulated to levels comparable to those on DCs generated by the conventional method. Suppression of TGF-β and IL-10 receptors on SD-DCs influenced the effector T-cells to produce IFN-γ, which enhanced their ability to kill CCA cells. The preparation of adoptive effector T-cells holds the potential of becoming a novel therapy for cellular immunotherapy in CCA.

Background: Cholangiocarcinoma (CCA) is a fatal cancer with high resistance to anticancer drugs.  The development of new drugs or compounds to be used alone or in combination with currently available chemotherapeutic agents to improve the treatment of CCA is needed. Compound A (CpdA), which is a small plant-derived glucocorticoid receptor modulator, strongly inhibited the growth and survival of several cancers.  However, the effect of CpdA on cholangiocarcinoma has not been elucidated. The aim of this study was to investigate the effect of CpdA on CCA., Methods: Cytotoxicity of CpdA was tested in primary cells including peripheral blood mononuclear cells (PBMCs), fibroblasts, and human umbilical vein endothelial cells (HUVECs), as well as on CCA cell lines (KKU-100, KKU-055, and KKU-213) was examined. Cell cycle distribution and IL-6 expression was assessed by flow cytometry and real-time polymerase chain reaction, respectively.  The effect of combination CpdA and cisplatin was evaluated by cell viability assay., Results: CpdA significantly inhibited cell cycle at G1 phase in CCA cell lines, and reduced IL-6 mRNA expression.  However, combination CpdA and cisplatin did not enhance the inhibitory effect. TGFβR-II expression was increased in CCA cells after the combination treatment., Conclusions: These results indicate the potential of CpdA for CCA treatment. However, combination treatment with CpdA and cisplatin increased CCA cell survival. The molecular mechanism is likely attributable to promotes cell survival via the TGFβR-II signaling pathway. The combination of CpdA with other anticancer drugs for CCA treatment should be further examined.

Cholangiocarcinoma (CCA) can resist chemotherapy resulting in treatment failure. Gemcitabine, a chemotherapeutic drug, can sensitize cancer cells to become susceptible to cytotoxic T-lymphocytes (CTLs). We, therefore, hypothesized that a combination of gemcitabine and CTLs would be more effective for CCA treatment than individual therapy. To test this hypothesis, we conducted an in vitro study using gemcitabine combined with CTLs to treat gemcitabine-resistant CCA (KKU-213) cells. KKU-213 cells were pretreated with gemcitabine and tested for killing by CTLs activated by dendritic cells that were prepared by three different methods, including: (i) monocyte-derived dendritic cells pulsed with cancer cell lysate (Mo-DC + Lys), (ii) self-differentiated dendritic cells pulsed with cancer-cell lysate (SD-DC + Lys), and (iii) SD-DC presenting tumor-associated antigen PRKAR1A (SD-DC-PR). KKU-213 cells pretreated with gemcitabine were killed by CTLs activated by either SD-DC + Lys or SD-DC-PR more efficiently than those activated by Mo-DC + Lys. Furthermore, KKU-213 cells pretreated with a low dose (2 µM) of gemcitabine significantly enhanced the cytotoxic activity of CTLs activated by either SD-DC + Lys or SD-DC-PR at all evaluated effector (E) to target cell (T) ratios. At an E:T ratio of 5:1, KKU-213 cells pretreated with gemcitabine enhanced the cytotoxic activity of CLTs by approximately 2.5-fold (greater than 50% cell death) compared to untreated condition. The upregulation of HLA class I upon pretreatment of KKU-213 cells with gemcitabine may suggest a mechanism that leads to alteration of the antigen presentation process to promote CTL functions. These findings support the concept of combination therapy for overcoming chemo-resistant CCA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cellular immunotherapy is a promising new therapeutic approach for hepatocellular carcinoma (HCC), which has a high recurrence rate, irrespective of the treatment administered. In this study, we attempted to improve the cytolytic activity of effector T-lymphocytes against HCC. T-lymphocytes were activated by monocyte-derived dendritic cells (DCs) pulsed with cell lysate or RNA prepared from HCC cell lines. Monocytes were activated for differentiation into DCs by treatment with the IL4 and GM-CSF. DCs were pulsed with cell lysate or RNA prepared from a single cell line or combinations of two or three HCC cell lines, and then co-cultured with autologous T-lymphocytes with the intent of creating specific cytotoxicity. We discovered that DCs pulsed with total RNA effectuated greater T-lymphocyte function than DCs pulsed with total cell lysate, as evidenced by greater cytolytic activities against HCC target cells. The percentage of Huh7, HepG2, and SNU449 cell apoptosis at effector:target ratio of 10:1 was 42.6 ± 4.5% (p = 0.01), 33.6 ± 3.1% (p = 0.007), and 21.4 ± 1.4% (p < 0.001), respectively. DCs pulsed with pools of antigens prepared from three cell lines improved the cytolytic function of effector T-lymphocytes by approximately two-fold (p < 0.001), which suggests that this approach be further developed and applied for adoptive transfer treatment of HCC.

Skin dendritic cells (DCs) are primary target cells of dengue virus (DENV) infection and they play an important role in its immunopathogenesis. Monocyte-derived dendritic cells (MDDCs) represent dermal and bloodstream DCs that serve as human primary cells for ex vivo studies of DENV infection. Improved understanding of the mechanisms that effectuate the inhibition of DENV replication in MDDCs will accelerate the development of antiviral drugs to treat DENV infection. In this study, we investigated whether or not vivo-morpholino oligomer (vivo-MO), which was designed to target the top of the 3' stem-loop (3' SL) at the 3' UTR of the DENV genome, could inhibit DENV infection and replication in MDDCs. The findings of this study revealed that vivo-MO-1 could inhibit DENV-2 infection in MDDCs, and that it could significantly reduce DENV RNA, protein, and viral production in a dose-dependent manner. Treatment of MDDCs with 4 μM of vivo-MO-1 decreased DENV production by more than 1,000-fold, when compared to that of the vivo-MO-NC control. Thus, vivo-MO-1 targeting of DENV RNA demonstrates potential for further development into an anti-DENV agent., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cholangiocarcinoma (CCA) is a cancer of the bile ducts that is associated with poor prognosis and poor treatment outcome. Approximately one-third of CCA patients can undergo surgery, but the recurrence rate is high and chemotherapy often cannot satisfactorily prolong survival. Cellular immunotherapy based on adoptive T-cell transfer is a potential treatment for CCA; however, the development of this technology and the search for an appropriate tumor-associated antigen are still ongoing. To enhance the cytotoxic activity of effector T cells against CCA, we developed self-differentiated monocyte-derived dendritic cells (SD-DC) presenting cAMP-dependent protein kinase type I-alpha regulatory subunit (PRKAR1A), which is an overexpressed protein that plays a role in the regulation of tumor growth to activate T cells for CCA cell killing. Dendritic cells (DCs) transduced with lentivirus harboring tri-cistronic cDNA sequences (SD-DC-PR) could produce granulocyte-macrophage colony-stimulating factor, interleukin-4, and PRKAR1A. SD-DC showed similar phenotypes to those of DCs derived by conventional method. Autologous effector T cells (CD3+, CD8+) activated by SD-DC-PR exhibited greater cytotoxic activity against CCA than those activated by conventionally-derived DCs. Effector T cells activated by SD-DC-PR killed 60% of CCA cells at an effector-to-target ratio of 15:1, which is approximately twofold greater than the cell killing performance of those stimulated with control DC. The cytotoxic activities of effector T cells activated by SD-DC-PR against CCA cells were significantly associated with the expression levels of PRKR1A in CCA cells. This finding that SD-DC-PR effectively stimulated autologous effector T cells to kill CCA cells may help to accelerate the development of novel therapies for treating CCA.

Hyperglycemia stimulates several pathways to induce pancreatic β-cell apoptosis. In our previous study by mRNA analysis, we demonstrated that B-cell translocation gene 2 (BTG 2 ) expression was up-regulated in INS-1 cells cultured under high glucose conditions, but this effect was reversed by estrogen. In the present study, we demonstrated that BTG 2 mRNA and protein expressions in both INS-1 cells and mouse pancreatic islets increased under high glucose conditions compared to those cultured under basal glucose conditions, while in the presence of estrogen, the BTG 2 mRNA and protein expressions decreased. SiRNA-BTG 2 significantly reduced cell apoptosis, cleaved-caspase 3, and Bax, compared to the siRNA-control in INS-1 cultured under high glucose conditions. We further demonstrated that BTG 2 promoter activity was activated under high glucose conditions whereas estrogen significantly reduced it. The effects of estrogen on BTG 2 expression were inhibited by estrogen receptor inhibitors. Also, under high glucose conditions, p53 and Bax mRNA and protein expressions increased, but they decreased in the presence of estrogen. Again, the effect of estrogen on p53 and Bax expression was inhibited by estrogen receptor inhibitors. Taken together, this study demonstrates that estrogen reduces pancreatic β-cell apoptosis under high glucose conditions via suppression of BTG2, p53, and Bax expressions.

Human kidney stone disease (KSD) causes significant morbidity and public health burden worldwide. The etiology of KSD is heterogeneous, ranging from monogenic defects to complex interaction between genetic and environmental factors. However, the genetic defects causing KSD in the majority of affected families are still unknown. Here, we report the discovery of mutations of SCN10A, encoding Na V 1.8 α subunit of voltage-gated sodium channel, in families with KSD. The region on chromosome 3 where SCN10A locates was initially identified in a large family with KSD by genome-wide linkage analysis and exome sequencing. Two mutations (p.N909K and p.K1809R) in the same allele of SCN10A co-segregated with KSD in the affected family. Additional mutation (p.V1149M) of SCN10A was identified in another affected family, strongly supporting the causal role of SCN10A for KSD. The amino acids at these three positions, N909, K1809, and V1149, are highly conserved in vertebrate evolution, indicating their structural and functional significances. Na V 1.8 α subunit mRNA and protein were found to express in human kidney tissues. The mutant proteins expressed in cultured cells were unstable and causing reduced current density as analyzed by whole-cell patch-clamp technique. Thus, loss-of-function mutations of SCN10A were associated with KSD in the families studied.

Tumor escapes host immune responses by producing immunosuppressive cytokines, such as IL-10 and TGF-β, secreted into the tumor microenvironment. These cytokines play important roles in the suppression of dendritic cell (DC) function, leading to decreased immune responses of the effector CD4 + and CD8 + T cells. To improve DC functions and enhance cytolytic activity of activated effector T-cells, we suppressed the effect of these cytokines on DCs by using specific neutralizing antibodies that inhibit IL-10 and TGF-β receptors. Monocyte-derived DCs generated in vitro showed up-regulation of MHC (HLA-DR) and co-stimulatory molecules (CD40 and CD86). The IL-10 and TGF-β receptors were expressed and localized on cell membrane of DCs, as shown by Western blot analysis and immunofluorescence staining, whereas the IL-10 and TGF-β ligands were detected in the culture supernatants of DCs and cholangiocarcinoma (CCA) cell line, respectively. Inhibition of the IL-10 and TGF-β receptors on DCs by specific neutralizing antibodies significantly increased level of IFN-γ and enhanced cytolytic activity of the DC-activated effector T-cells against CCA cell line. These results indicate that the IL-10 and TGF-β receptors are the targets for inhibition to increase DC functions and enhance cytolytic activity of the DC-activated effector T-cells against CCA cells. Thus, inhibition of the IL-10 and TGF-β receptors on DCs is crucial in the preparation of DC-activated effector T cells for adoptive T-cell therapy.

Dengue virus (DENV) infection is a worldwide public health problem, which can cause severe dengue hemorrhagic fever (DHF) and life-threatening dengue shock syndrome (DSS). There are currently no anti-DENV drugs available, and there has been an intensive search for effective anti-DENV agents that can inhibit all four DENV serotypes. In this study, we tested whether vivo-morpholino oligomers (vivo-MOs), whose effect on DENV infection has not previously been studied, can inhibit DENV infection. Vivo-MOs were designed to target the top of 3' stem-loop (3' SL) in the 3' UTR of the DENV genome and tested for inhibition of DENV infection in monkey kidney epithelial (Vero) cells and human lung epithelial carcinoma (A549) cells. The results showed that vivo-MOs could bind to a DENV RNA sequence and markedly reduce DENV-RNA, protein, and virus production in infected Vero and A549 cells. Vivo-MOs at a concentration of 4 µM could inhibit DENV production by more than 10 4 -fold when compared to that of an untreated control. In addition, vivo-MOs also inhibited DENV production in U937 cells and primary human monocytes. Therefore, vivo-MOs targeting to the 3' SL in the 3' UTR of DENV genomes are effective and have the potential to be developed as anti-DENV agents.

Cholangiocarcinoma is a malignancy of bile duct epithelia with an increasing in incidence rate worldwide. Surgery is the only curative treatment, while adjuvant chemotherapy and radiotherapy render poor responses. Cell-based immunotherapy is a potential strategy for cholangiocarcinoma treatment. However, variation of tumor antigens in cholangiocarcinoma leads to the ineffectiveness of cell-based immunotherapy. In this study, we examined the activation of effector T-cells by dendritic cells pulsed with protein lysate or total RNA from cholangiocarcinoma cell lines for their cytolytic activity against cholangiocarcinoma. Broad-spectrum antigen types with respect to RNA antigen sources were obtained from combination of three cholangiocarcinoma cell lines (KKU-213, KKU-100, and KKU-055). Compared with protein lysate-pulsed dendritic cells, total RNA-pulsed dendritic cells induced anti-tumor effector T-cell response with higher killing ability to KKU-100 and KKU-213 cells compared with protein lysate-pulsed dendritic cells. Moreover, pooled messenger RNA from three cholangiocarcinoma cell lines significantly increased the specific killing capacity of activated lymphocytes against KKU-213 cells. These results suggest that activation of anti-tumor effector T-cells against cholangiocarcinoma by RNA-pulsed dendritic cells is more effective than that by protein lysate-pulsed dendritic cells. In addition, pulsing dendritic cells with pooled messenger RNA from multiple cell lines enhanced the efficacy of a cellular immune response against cholangiocarcinoma.

Mutations of the solute carrier family 4 member 1 ( SLC4A1 ) gene encoding kidney anion (chloride/bicarbonate ion) exchanger 1 (kAE1) can cause genetic distal renal tubular acidosis (dRTA). Different SLC4A1 mutations give rise to mutant kAE1 proteins with distinct defects in protein trafficking. The mutant kAE1 protein may be retained in endoplasmic reticulum (ER) or Golgi apparatus, or mis-targeted to the apical membrane, failing to display its function at the baso-lateral membrane. The ER-retained mutant kAE1 interacts with calnexin chaperone protein; disruption of this interaction permits the mutant kAE1 to reach the cell surface and display anion exchange activity. However, the mechanism of Golgi retention of mutant kAE1 G701D protein, which is otherwise functional, is still unclear. In the present study, we show that Golgi retention of kAE1 G701D is due to a stable interaction with the Golgi-resident protein, coat protein complex I (COPI), that plays a role in retrograde vesicular trafficking and Golgi-based quality control. The interaction and co-localization of kAE1 G701D with the γ-COPI subunit were demonstrated in human embryonic kidney (HEK-293T) cells by co-immunoprecipitation and immunofluorescence staining. Small interference RNA (siRNA) silencing of COPI expression in the transfected HEK-293T cells increased the cell surface expression of transgenic kAE1 G701D, as shown by immunofluorescence staining. Our data unveil the molecular mechanism of Golgi retention of kAE1 G701D and suggest that disruption of the COPI-kAE1 G701D interaction could be a therapeutic strategy to treat dRTA caused by this mutant., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Dengue virus (DENV) infection is a worldwide public health problem with an increasing magnitude. The severity of disease in the patients with DENV infection correlates with high viral load and massive cytokine production - the condition referred to as "cytokine storm". Thus, concurrent inhibition of DENV and cytokine production should be more effective for treatment of DENV infection. In this study, we investigated the effects of the antiviral agent - ribavirin (RV), and the anti-inflammatory compound - compound A (CpdA), individually or in combination, on DENV production and cytokine/chemokine transcription in human lung epithelial carcinoma (A549) cells infected with DENV. Initially, the cells infected with DENV serotype 2 (DENV2) was studied. The results showed that treatment of DENV-infected cells with RV could significantly reduce both DENV production and cytokine (IL-6 and TNF-α) and chemokine (IP-10 and RANTES) transcription while treatment of DENV-infected cells with CpdA could significantly reduce cytokine (IL-6 and TNF-α) and chemokine (RANTES) transcription. Combined RV and CpdA treatment of the infected cells showed greater reduction of DENV production and cytokine/chemokine transcription. Similar results of this combined treatment were observed for infection with any one of the four DENV (DENV1, 2, 3, and 4) serotypes. These results indicate that combination of the antiviral agent and the anti-inflammatory compound offers a greater efficiency in reduction of DENV and cytokine/chemokine production, providing a new therapeutic approach for DENV infection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dengue virus (DENV) infection is a public health problem worldwide; thus, the development of a vaccine and anti-DENV drugs is urgently needed. It has been observed that low levels of viremia in DENV-infected individuals are associated with mild disease outcomes; therefore, reduction of DENV load should offer therapeutic benefits. Disruption of protein-protein interactions on the surface of DENV by a peptide that mimics part of its structural protein may affect stability of the virion structure and inhibit viral entry into host cells. To test this hypothesis, we generated a novel peptide inhibitor that mimics the conserved ectodomain region of DENV membrane (M) protein, MLH40 peptide, for DENV inhibition assays. MLH40 inhibited all four serotypes of the virus (DENV1-4) at half maximal inhibition concentration of 24-31 μm. MLH40 at 100 μm blocked DENV2 attachment to cells by 80%. The inhibitory activity of MLH40 against DENV was consistently observed with different cell types, including Vero, A549, and Huh7 cells. Prediction of MLH40 binding by a molecular docking program indicated that its N-terminal loop may interact with DENV envelope (E) proteins and alter their dimer conformation. Thus, MLH40 may serve as a lead-peptide inhibitor for the development of an anti-DENV drug., (© 2015 John Wiley & Sons A/S.)

Human kidney anion exchanger 1 (kAE1) mediates Cl(-)/HCO3(-) exchanges at the basolateral membrane of the acid-secreting α-intercalated cells. Mutations in SLC4A1 gene encoding kAE1 are associated with distal renal tubular acidosis (dRTA). Several studies have shown that impaired trafficking of the mutant kAE1 is an important molecular mechanism underlying the pathogenesis of dRTA. Proteins involved in kAE1 trafficking were identified but the mechanism resulting in dRTA remained unclear. Thus, this study attempted to search for additional proteins interacting with C-terminal of kAE1 (Ct-kAE1) and involved in kAE1 trafficking to cell membrane. Transmembrane protein 139 (TMEM139) was identified as a protein interacting with Ct-kAE1 by yeast two-hybrid screening. The interaction between kAE1 and TMEM139 was confirmed by affinity co-purification, co-immunoprecipitation (co-IP) and yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA). In addition, flow cytometry results showed that suppression of endogenous TMEM139 by small interfering RNA (siRNA) and over-expression of TMEM139 in HEK293T cells could reduce and increase membrane localization of kAE1, respectively. The presented data demonstrate that TMEM139 interacts with kAE1 and promotes its intracellular trafficking., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Rearrangement of membrane structure induced by dengue virus (DENV) is essential for replication, and requires host cellular machinery. Adaptor protein complex (AP)-1 is a host component, which can be recruited to components required for membrane rearrangement. Therefore, dysfunction of AP-1 may affect membrane organization, thereby decreasing replication of virus in infected cells. In the present study, AP-1-dependent traffic inhibitor inhibited DENV protein expression and virion production. We further clarified the role of AP-1A in the life cycle of DENV by RNA interference. AP-1A was not involved in DENV entry into cells. However, it facilitated DENV RNA replication. Viral RNA level was reduced significantly in Huh7 cells transfected with AP-1A small interfering RNA (siRNA) compared with control siRNA. Transfection of naked DENV viral RNA into Huh7 cells transfected with AP-1A siRNA resulted in less viral RNA and virion production than transfection into Huh7 cells transfected with control siRNA. Huh7 cells transfected with AP-1A siRNA showed greater modification of membrane structures and fewer vesicular packets compared with cells transfected with control siRNA. Therefore, AP-1A may partly control DENV-induced rearrangement of membrane structures required for viral replication.

Hypogonadism in men is associated with an increased incidence of type 2 diabetes. Supplementation with testosterone has been shown to protect pancreatic β-cell against apoptosis due to toxic substances including streptozotocin and high glucose. One of the pathological mechanisms of glucose-induced pancreatic β-cell apoptosis is the induction of the local rennin-angiotensin-aldosterone system (RAAS). The role of testosterone in regulation of the pancreatic RAAS is still unknown. This study aims to investigate the protective action of testosterone against glucotoxicity-induced pancreatic β-cell apoptosis via alteration of the pancreatic RAAS pathway. Rat insulinoma cell line (INS-1) cells or isolated male mouse islets were cultured in basal and high-glucose media in the presence or absence of testosterone, losartan, and angiotensin II (Ang II), then cell apoptosis, cleaved caspase 3 expression, oxidative stress, and expression of angiotensin II type 1 receptor (AGTR1) and p47(phox) mRNA and protein were measured. Testosterone and losartan showed similar effects in reducing pancreatic β-cell apoptosis. Testosterone significantly reduced expression of AGTR1 protein in INS-1 cells cultured in high-glucose medium or high-glucose medium with Ang II. Testosterone decreased the expression of AGTR1 and p47(phox) mRNA and protein in comparison with levels in cells cultured in high-glucose medium alone. Furthermore, testosterone attenuated superoxide production when co-cultured with high-glucose medium. In contrast, when cultured in basal glucose, supplementation of testosterone did not have any effect on cell apoptosis, oxidative stress, and expression of AGT1R and p47(phox). In addition, high-glucose medium did not increase cleaved caspase 3 in AGTR1 knockdown experiments. Thus, our results indicated that testosterone prevents pancreatic β-cell apoptosis due to glucotoxicity through reduction of the expression of ATGR1 and its signaling pathway., (© 2015 Society for Endocrinology.)

Dengue virus (DENV) infection associates with renal disorders. Patients with dengue hemorrhagic fever and acute kidney injury have a high mortality rate. Increased levels of cytokines may contribute to the pathogenesis of DENV-induced kidney injury. Currently, molecular mechanisms how DENV induces kidney cell injury has not been thoroughly investigated. Excessive cytokine production may be involved in this process. Using human cytokine RT(2) Profiler PCR array, 14 genes including IP-10, RANTES, IL-8, CXCL-9 and MIP-1β were up-regulated more than 2 folds in DENV-infected HEK 293 cells compared to that of mock-infected HEK 293 cells. In the present study, RANTES was suppressed by the NF-κB inhibitor, compound A (CpdA), in DENV-infected HEK 293 cells implying the role of NF-κB in RANTES expression. Chromatin immunoprecipitation (ChIP) assay showed that NF-κB binds more efficiently to its binding sites on the RANTES promoter in NS5-transfected HEK 293 cells than in HEK 293 cells expressing the vector lacking NS5 gene. To further examine whether the NS5-activated RANTES promoter is mediated through NF-κB, the two NF-κB binding sites on the RANTES promoter were mutated and this promoter was coupled to the luciferase cDNA. The result showed that when both binding sites of NF-κB in the RANTES promoter were mutated, the ability of NS5 to induce the luciferase activity was significantly decreased. Therefore, DENV NS5 activates RANTES production by increasing NF-κB binding to its binding sites on the RANTES promoter., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called 'cytokine storm', is observed in the patients with DHF/DSS. A complex interaction between DENV proteins and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of α-intercalated cells of the kidney collecting duct leads to the defect of the Cl(-)/HCO(3)(-) exchange and the failure of proton (H(+)) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney α-intercalated cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Kidney anion exchanger 1 (kAE1) mediates chloride (Cl⁻) and bicarbonate (HCO₃⁻) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl⁻/HCO₃⁻ exchange at the basolateral membrane and failure of proton (H+) secretion at the apical membrane, causing a kidney disease--distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Cholangiocarcinoma (CCA) is a fatal disease with high resistance to anticancer drugs. This is probably in part due to enhanced resistance to apoptosis. We have previously shown that galectin-3 (Gal-3), a beta-galactoside-binding lectin, is highly expressed in CCA tissues. In this study, we demonstrated further that Gal-3 plays a direct role in anti-apoptosis regardless of the apoptotic insults. The anti-apoptotic activity and chemoresistance of CCA cells were related to Gal-3 expression level. Suppression of Gal-3 expression with siRNA stimulated apoptosis. siGal-3-K626 transiently depleted Gal-3 expression to the baseline and dramatically induced apoptosis, while siGal-3-K402 suppressed Gal-3 expression by 50% and provoked cell apoptosis, but only under apoptotic insults (hypoxic conditions or short UV radiation). These actions were reversed in Gal-3 overexpressing CCA cells. The correlation between the degree of anti-apoptotic activity and the level of endogenous Gal-3 was demonstrated. Suppression of Gal-3 expression in CCA cells with siGal-3-K402 significantly enhanced apoptosis induced by cisplatin or 5-fluorouracil by approximately 10 times, whereas overexpression of Gal-3 led to an increased resistance to drugs. In summary, the present study showed that the cellular level of Gal-3 might contribute to the anti-apoptotic activity and chemoresistance of CCA cells. Hence, Gal-3 expression level in cancer cells or tissues may be a marker for predicting chemotherapeutic response, and Gal-3 may be a specific gene-targeting therapy option for treating CCA.

Galectin-3, a beta-galactoside-binding lectin, is a multifunctional protein implicated in a variety of biological functions, including tumour cell adhesion, proliferation, differentiation, cancer progression and metastasis. The present study was performed to clarify the impact of galectin-3 expression on patients with liver fluke-associated cholangiocarcinoma. Galectin-3 expression was examined immunohistochemically in 53 patients with intrahepatic cholangiocarcinoma, who had undergone surgery without pre-operative therapy. All bile duct epithelium expressed galectin-3 with different intensities, according to the different histological subtypes. The poorly-differentiated type expressed galectin-3 less intensely than the papillary, well- to moderately-differentiated types (P=0.012). We observed the association of low galectin-3 expression with lymphatic invasion (P=0.002). Suppression of galectin-3 expression in two human cholangiocarcinoma cell lines using siRNA targeted to galectin-3 significantly increased cell migration and invasion without alterations in cell proliferation. Regulation of galectin-3 expression may therefore be an alternative therapeutic approach to control metastasis of cholangiocarcinoma.