Precision immunotherapy for cholangiocarcinoma: Pioneering the use of human-derived anti-cMET single chain variable fragment in anti-cMET chimeric antigen receptor (CAR) NK cells.

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.

Enhancing cholangiocarcinoma immunotherapy with adoptive T cells targeting HLA-restricted neoantigen peptides derived from driver gene mutations.

Precision immunotherapy, driven by genomic and bioinformatic advancements, has emerged as a promising and viable approach to combat cancer. Targeting neoantigens offers the advantage of specific immune responses with minimal off-tumor toxicity. In this study, we investigated the potential of adoptive T cells activated by HLA-restricted neoantigen peptides from driver gene mutations for treating cholangiocarcinoma (CCA), a highly aggressive cancer with poor prognosis and high mortality rates. Through whole exome sequencing of CCA cell lines, KKU-213A and KKU-100, we identified mutations in common driver genes and predicted corresponding HLA-restricted peptides. Peptides from KRAS, RNF43, and TP53 mutations exhibited strong binding affinity to HLA-A11, as validated through molecular docking and T2-cell binding assays. Dendritic cells (DCs) from healthy donors expressing HLA-A* 11:01, pulsed with individual or pooled peptides, showed comparable levels of costimulatory molecules (CD11c, CD40, CD86, and HLA-DR) to conventional DCs but higher expression of maturation markers, CD80 and CD86. Autologous HLA-A* 11:01-restricted T cells, activated by peptide-pulsed DCs, effectively lysed KKU-213A (HLA-A*11:01) cells, outperforming conventional tumor lysate-pulsed DCs. This effect was specific to HLA-A* 11:01-restricted T cells and not observed in KKU-100 (HLA-A*33:03) cells. Moreover, HLA-A* 11:01-restricted T cells exhibited elevated levels of IFN-gamma, granulysin, and granzyme B, indicating their potent anti-tumor capabilities. These findings underscore the specificity and efficiency of HLA-A* 11:01-restricted T cells targeting KRAS, RNF43, TP53 mutated CCA cells, and offer valuable insights for developing immunotherapeutic strategies and therapeutic peptide-vaccines for CCA treatment.

Enhanced antitumor efficacy, proliferative capacity, and alleviation of T cell exhaustion by fifth-generation chimeric antigen receptor T cells targeting B cell maturation antigen in multiple myeloma.

Chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) has been approved for treating multiple myeloma (MM). Some clinical studies reported suboptimal outcomes, including reduced cytotoxicity of CAR-T cells and tumor evasion through increased expression of programmed death-ligand 1 (PD-L1). To enhance CAR-T cell efficiency and overcome PD-L1-mediated T cell suppression, we developed anti-BCMA-CAR5-T cells equipped with three costimulatory domains and the ability to secrete anti-PD-L1 single-chain variable fragment (scFv) blockade molecules. Anti-BCMA-CAR4-T cells contained a fully human anti-BCMA scFv and three intracellular domains (CD28, 4-1BB, and CD27) joined with CD3ζ. Anti-BCMA-CAR5-T cells were generated by fusing anti-BCMA-CAR4 with anti-PD-L1 scFv. Both anti-BCMA-CAR4-T and anti-BCMA-CAR5-T cells demonstrated comparable antitumor activity against parental MM cells. However, at an effector-to-target ratio of 1:2, only anti-BCMA-CAR5-T cells maintained cytolytic activity against PD-L1 high MM cells, unlike anti-BCMA-CAR4 T cells. Anti-BCMA-CAR5-T cells were specifically activated by BCMA-expressing target cells, resulting in increased CAR-T cell proliferation, release of cytolytic mediators, and pro-inflammatory cytokines. Anti-BCMA-CAR5-T cells demonstrated specific cytotoxicity against BCMA-expressing target cells, leading to decreased target cell numbers, increased CAR-T cell numbers, and preserved CAR expression during antigenic re-stimulation. Interestingly, only anti-BCMA-CAR5-T cells showed reduced PD-1 receptor levels, which correlated with decreased PD-L1 expression on target cells. We successfully generated anti-BCMA-CAR5-T cells capable of secreting anti-PD-L1 scFv. These cells exhibited superior antitumor efficiency, proliferative capacity, and alleviated T-cell exhaustion against MM cells. Further investigation into the antitumor efficacy of anti-BCMA-CAR5-T cells is warranted in ex vivo and clinical research settings.

Anti-cancer effect of a phytochemical compound - 7R-acetylmelodorinol - against triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a highly aggressive subtype currently lacking effective treatment options. Consequently, novel and effective drugs or compounds are urgently needed to treat TNBC. Therefore, this study aimed to evaluate the potential of 7R-acetylmelodorinol (7R-AMDL), a phytochemical compound isolated from Xylopia pierrei Hance, a plant found in Thailand, as a novel therapeutic agent for TNBC. MTT and clonogenic assays showed that 7R-AMDL significantly reduced the survival of breast cancer cell lines, with a markedly potent effect on MDA-MB-231 cells. Flow cytometry showed that treating MDA-MB-231 cells with 7R-AMDL at the concentration of dose 8 µM significantly increased early and late apoptosis after 24 and 48 h compared to the control group (p < 0.0001). The highest tested 7R-AMDL dose upregulated the death receptors and their ligands, with extrinsic and intrinsic apoptosis pathways significantly activated via the caspase cascade, compared to the untreated group (p < 0.05). In addition, immunoblots showed decreased BCL2-like 1 (BCL2L1/Bcl-xL) expression (p < 0.0001). Furthermore, wound healing and Transwell assays showed that at a non-cytotoxic dose (≤4 µM), 7R-AMDL significantly inhibited the MDA-MB-231 cell migration and invasion. This reduction in cell migration was associated with decreased matrix metallopeptidase 9 (MMP-9) expression (p < 0.01) and nuclear factor kappa B (NF-κB) activation (p < 0.05). Altogether, 7R-AMDL has anti-cancer effects against TNBC and the potential to be further developed and evaluated for treating this disease.

Microalga Chlorella sp. extract induced apoptotic cell death of cholangiocarcinoma via AKT/mTOR signaling pathway.

Cancer is the leading cause of death worldwide. Drug resistance and relapse after current standard treatments frequently occur; thus, alternative and effective treatments are required. Algae and cyanobacteria are abundant organisms that serve as bioresources of nutrients/metabolites, which are attractive sources of numerous bioactive compounds for drug discovery. In the present study, we, therefore, investigated anti-cancer activities of crude polysaccharide and ethanolic extracts from Chlorella sp., Sargassum spp., and Spirulina sp. against cell lines of five top-leading cancers including lung cancer (A549), cervical cancer (Hela), breast cancer (MCF7), hepatocellular carcinoma (Huh7), and cholangiocarcinoma (CCA; KKU213A). Only ethanolic extracts of Chlorella sp. showed consistent inhibition of growth of all cancer cell types. CCA was the most sensitive to Chlorella sp. ethanolic extract with CC50 of 277.4, 400.5, and 313.4 µg/mL for KKU055, KKU100, and KKU213A cells, respectively. Flow cytometric analysis demonstrated that CCA cell death was triggered via apoptosis pathway in accompany with lowering procaspase-3, -8, and -9 and increasing caspase enzymatic activity in addition to reducing anti-apoptosis Bcl-2 protein. Interestingly, the treatment of the extract at 400 µg/mL greatly inhibited the AKT/mTOR survival signaling as evidenced by significant reduction of phosphorylated-AKT and phosphorylated-mTOR proteins. The presence of reported bioactive compounds, gallic acid, and lutein, were confirmed in Chlorella sp. extract by high-performance liquid chromatography. Gallic acid and lutein treatment caused a significant reduction of KKU055, KKU100, and KKU213A cell viability. This study demonstrated the anti-cancer effect of Chlorella sp. ethanolic extract to promote cancer cell death via inhibition of AKT/mTOR pathway.

An induced pluripotent stem cell line (MUSIi019-A) generated from a patient with distal renal tubular acidosis carrying a compound heterozygous mutation in solute carrier family 4 member 1 (SLC4A1) gene.

Distal renal tubular acidosis (dRTA), a disease characterized by the failure of the distal nephron to secrete acid into the urine, can be caused by mutations in SLC4A1 gene encoding erythroid and kidney anion exchanger 1 (AE1). Here, an induced pluripotent stem cell (iPSC) line was generated from a patient with dRTA and hemolytic anemia carrying compound heterozygous SLC4A1 mutations containing c.1199_1225del (p.Ala400_Ala408del), resulting in Southeast Asian ovalocytosis (SAO), and c.1331C>A (p.Thr444Asn). Peripheral blood mononuclear cells (PBMCs) were reprogrammed using Sendai viral reprogramming. The established iPSC line, MUSIi019-A, exhibited pluripotent property and retained the same mutations observed in the patients.

Impaired trafficking and instability of mutant kidney anion exchanger 1 proteins associated with autosomal recessive distal renal tubular acidosis.

BACKGROUND: Mutations in solute carrier family 4 member 1 (SLC4A1) encoding anion exchanger 1 (AE1) are the most common cause of autosomal recessive distal renal tubular acidosis (AR dRTA) in Southeast Asians. To explain the molecular mechanism of this disease with hematological abnormalities in an affected family, we conducted a genetic analysis of SLC4A1 and studied wild-type and mutant AE1 proteins expressed in human embryonic kidney 293T (HEK293T) cells. METHODS: SLC4A1 mutations in the patient and family members were analyzed by molecular genetic techniques. Protein structure modeling was initially conducted to evaluate the effects of mutations on the three-dimensional structure of the AE1 protein. The mutant kidney anion exchanger 1 (kAE1) plasmid construct was created to study protein expression, localization, and stability in HEK293T cells. RESULTS: We discovered that the patient who had AR dRTA coexisting with mild hemolytic anemia carried a novel compound heterozygous SLC4A1 mutations containing c.1199_1225del (p.Ala400_Ala408del), resulting in Southeast Asian ovalocytosis (SAO), and c.1331C > A (p.Thr444Asn). Homologous modeling and in silico mutagenesis indicated that these two mutations affected the protein structure in the transmembrane regions of kAE1. We found the wild-type and mutant kAE1 T444N to be localized at the cell surface, whereas the mutants kAE1 SAO and SAO/T444N were intracellularly retained. The half-life of the kAE1 SAO, T444N, and SAO/T444N mutants was shorter than that of the wild-type protein. CONCLUSION: These results suggest impaired trafficking and instability of kAE1 SAO/T444N as the likely underlying molecular mechanism explaining the pathogenesis of the novel SLC4A1 compound heterozygous mutation identified in this patient.

Anti-CD19 chimeric antigen receptor T cells secreting anti-PD-L1 single-chain variable fragment attenuate PD-L1 mediated T cell inhibition.

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-L1high 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-L1high cells compared to that of anti-CD19-CAR4-T cells. The cytotoxicity of anti-CD19-CAR4-T cells against CD19+/PD-L1high 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-L1high 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.

Genistein Sensitizes Human Cholangiocarcinoma Cell Lines to Be Susceptible to Natural Killer Cells.

Cholangiocarcinoma (CCA) is a lethal bile duct cancer, which has poor treatment outcomes due to its high resistance to chemotherapy and cancer recurrence. Activation of aberrant anti-apoptotic signaling pathway has been reported to be a mechanism of chemoresistance and immune escape of CCA. Therefore, reversal of anti-apoptotic signaling pathway represents a feasible approach to potentiate effective treatments, especially for CCA with high chemoresistance. In this study, we demonstrated the effects of genistein on reactivation of apoptosis cascade and increase the susceptibility of CCA cells to natural killer (NK-92) cells. Genistein at 50 and 100 µM significantly activated extrinsic apoptotic pathway in CCA cells (KKU055, KKU100, and KKU213A), which was evident by reduction of procaspase-8 and -3 expression. Pretreatment of CCA cells with genistein at 50 µM, but not NK-92 cells, significantly increased NK-92 cell killing ability over the untreated control, suggesting the ability of genistein to sensitize CCA cells. Interestingly, genistein treatment could greatly lower the expression of cFLIP, an anti-apoptotic protein involved in the immune escape pathway, in addition to upregulation of death receptors, Fas- and TRAIL-receptors, in CCA cells, which might be the underlying molecular mechanism of genistein to sensitize CCA to be susceptible to NK-92 cells. Taken together, this finding revealed the benefit of genistein as a sensitizer to enhance the efficiency of NK cell immunotherapy for CCA.

Combination gemcitabine and PD-L1xCD3 bispecific T cell engager (BiTE) enhances T lymphocyte cytotoxicity against cholangiocarcinoma cells.

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.

Doxorubicin sensitizes breast cancer cells to natural killer cells in connection with increased Fas receptors.

Breast cancer (BC) is the most common cancer in women. Although standard treatments are successful in patients with BC diagnosed at an early stage, an alternative treatment is required for patients with advanced­stage disease who do not respond to these treatments. The concept of using chemotherapy to sensitize cancer cells to become susceptible to immunotherapy was recently introduced and may be used as an alternative treatment for BC. The chemotherapeutic drug doxorubicin has been reported to sensitize cancer cells; however, the efficacy to sensitize the solid spheroids, in addition to its underlying mechanism regarding how doxorubicin sensitizes BC, has not previously been explored. In the present study, the effectiveness of a combined treatment of doxorubicin and natural killer­92 (NK­92) cells against BC in either 2D or 3D spheroid models, and its association with Fas receptor (FasR) expression, was demonstrated. The BC (MCF7) cell line expressing a higher level of FasR was more sensitive to NK­92 cell killing than the MDA­MB­231 cell line, which expressed a lower level of FasR. A sublethal dose of doxorubicin caused a significant improvement in NK cytotoxicity. Concordantly, a significant reduction in cell viability was observed in the doxorubicin­treated MCF7 spheroids. Notably, flow cytometric analysis revealed significantly increased FasR expression in the MCF7 cells, suggesting the underlying sensitization mechanism of doxorubicin in BC was related to the FasR upregulation. The present findings supported the use of combined doxorubicin and NK immunotherapy in BC treatment.

Establishment and characterization of novel highly aggressive HER2­positive and triple­negative breast cancer cell lines.

Breast cancer cell lines are widely used as an in vitro system with which to study the mechanisms underlying biological and chemotherapeutic resistance. In the present study, two novel breast cancer cell lines designated as PC­B­142CA and PC­B­148CA were successfully established from HER2­positive and triple­negative (TN) breast cancer tissues. The cell lines were characterized by cytokeratin (CK), α­smooth muscle actin (α­SMA), fibroblast­activation protein (FAP) and programmed death­ligand 1 (PD­L1). Cell proliferation was assessed using a colony formation assay, an MTS assay, 3­dimensional (3­D) spheroid and 3­D organoid models. Wound healing and Transwell migration assays were used to explore the cell migration capability. The responses to doxorubicin (DOX) and paclitaxel (PTX) were evaluated by 3­D spheroids. The results showed that the PC­B­142CA and PC­B­148CA cell lines were α­SMA­negative, FAP­negative, CK­positive and PD­L1­positive. Both cell lines were adherent with the ability of 3­D­multicellular spheroid and organoid formations; invadopodia were found in the spheroids/organoids of only PC­B­148CA. PC­B­142CA had a faster proliferative but lower metastatic rate compared to PC­B­148CA. Compared to MDA­MB­231, a commercial TN breast cancer cell line, PC­B­148CA had a similar CD44+/CD24­ stemness property (96.90%), whereas only 8.75% were found in PC­B­142CA. The mutations of BRCA1/2, KIT, PIK3CA, SMAD4, and TP53 were found in PC­B­142CA cells related to the resistance of several drugs, whereas PC­B­148CA had mutated BRCA2, NRAS and TP53. In conclusion, PC­B­142CA can serve as a novel HER2­positive breast cancer cell line for drug resistance studies; while PC­B­148CA is a novel TN breast cancer cell line suitable for metastatic and stemness­related properties.

Cordycepin Sensitizes Cholangiocarcinoma Cells to Be Killed by Natural Killer-92 (NK-92) Cells.

Immunotherapy harnessing immune functions is a promising strategy for cancer treatment. Tumor sensitization is one approach to enhance tumor cell susceptibility to immune cell cytotoxicity that can be used in combination with immunotherapy to achieve therapeutic efficiency. Cordycepin, a bioactive compound that can be extracted from some Cordyceps spp. has been reported to effectively inhibit tumor growth, however, the mechanism of its tumor sensitization activity that enhances immune cell cytotoxicity is unknown. In the present study, we investigated the potency of cordycepin to sensitize a lethal cancer, cholangiocarcinoma (CCA), to natural killer (NK) cells. Treatment with cordycepin prior to and during co-culturing with NK-92 cells significantly increased cell death of KKU-213A as compared to solitary cordycepin or NK treatment. Moreover, sensitization activity was also observed in the combination of NK-92 cells and Cordyceps militaris extract that contained cordycepin as a major component. The cordycepin treatment remarkably caused an increase in TRAIL receptor (DR4 and DR5) expression in KKU-213A, suggesting the possible involvement of TRAIL signaling in KKU-213A sensitization to NK-92 cells. In conclusion, this is the first report on the sensitization activity of cordycepin on CCA cells to NK cytotoxicity, which supports that cordycepin can be further developed as an alternate immunomodulating agent.

Cordycepin Inhibits Virus Replication in Dengue Virus-Infected Vero Cells.

Dengue virus (DENV) infection causes mild to severe illness in humans that can lead to fatality in severe cases. Currently, no specific drug is available for the treatment of DENV infection. Thus, the development of an anti-DENV drug is urgently required. Cordycepin (3'-deoxyadenosine), which is a major bioactive compound in Cordyceps (ascomycete) fungus that has been used for centuries in Chinese traditional medicine, was reported to exhibit antiviral activity. However, the anti-DENV activity of cordycepin is unknown. We hypothesized that cordycepin exerts anti-DENV activity and that, as an adenosine derivative, it inhibits DENV replication. To test this hypothesis, we investigated the anti-DENV activity of cordycepin in DENV-infected Vero cells. Cordycepin treatment significantly decreased DENV protein at a half-maximal effective concentration (EC50) of 26.94 µM. Moreover, DENV RNA was dramatically decreased in cordycepin-treated Vero cells, indicating its effectiveness in inhibiting viral RNA replication. Via in silico molecular docking, the binding of cordycepin to DENV non-structural protein 5 (NS5), which is an important enzyme for RNA synthesis, at both the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, was predicted. The results of this study demonstrate that cordycepin is able to inhibit DENV replication, which portends its potential as an anti-dengue therapy.

Association between intelectin-1 variation and human kidney stone disease in northeastern Thai population.

An interplay of multiple genetic and environmental factors implicates an incidence of human kidney stone disease (KSD). However, the genetic factors associated with KSD are not completely known or understood. To identify KSD-associated genetic variations among the northeastern Thai patients, a genome-wide association study (GWAS) was conducted. We initially employed genotyping of single nucleotide polymorphism (SNP) using Genome-Wide Human SNP Array 6.0 in 105 patients and in 105 normal control subjects. To overcome the limitation of small sample size, we set forth to analyze SNPs as clusters based on the concept of linkage disequilibrium (LD) and haplotype. Using this analysis, 29 genes were identified. Three candidate SNPs, including rs2039415, rs2274907, and rs3747515, were selected on the basis of haplotype analysis, potentially functional SNPs, and the functions of associated genes. Further genotyping of these SNPs in a larger sample size (altogether 216 patients and 216 control subjects) showed that the candidate SNP rs2274907 remained significantly different between case and control subjects in both genotype frequencies (OR 2.44, 95% CI 1.38-4.30; p = 0.0015) and allele frequencies (OR 1.54, 95% CI 1.17-2.03; p = 0.0021). The non-synonymous SNP rs2274907 (c.326T > A) located in exon 4 of the ITLN1 gene results in a substitution of valine (V) by aspartate (D) at position 109 (p.V109D). This substitution could affect the predicted hydrogen (H)-bonds between lysine (K) 107 and glutamine (Q) 104, which supports its association with KSD in this population.

Alpha-mangostin inhibits dengue virus production and pro-inflammatory cytokine/chemokine expression in dendritic cells.

Dengue virus (DENV) is transmitted to humans via the bite of an Aedes mosquito, causing dengue fever, dengue hemorrhagic fever, or dengue shock syndrome. In the human skin, DENV first infects keratinocytes, dendritic cells, and macrophages. Monocytes that are recruited to the site of infection and differentiate into monocyte-derived dendritic cells (moDCs) are also infected by DENV. DENV-infected DCs secrete pro-inflammatory cytokines and chemokines to modulate the immune response. The viral load and massive pro-inflammatory cytokine/chemokine production, referred to as a 'cytokine storm', are associated with disease severity. We propose that an ideal drug for treatment of DENV infection should inhibit both virus production and the cytokine storm, and previously, we reported that alpha-mangostin (α-MG) inhibits both DENV replication and cytokine production in hepatocytes. However, the effect of α-MG on DENV-infected moDCs remains unknown. In this study, we investigated the effects of α-MG on DENV infection and pro-inflammatory cytokine/chemokine production in primary moDCs generated ex vivo from monocytes of healthy individuals. α-MG at the non-toxic concentrations of 20 and 25 µM reduced DENV production by more than 10-fold and 1,000-fold, respectively. Treatment with α-MG efficiently inhibited the infection of immature moDCs by all four serotypes of DENV. Time-of-addition studies suggested that α-MG (25 µM) inhibits DENV at the early stage of replication. In addition, α-MG markedly reduced cytokine/chemokine (TNF-α, CCL4, CCL5, CXCL10, IL6, IL1ß, IL10, and IFN-α) transcription in DENV-infected immature moDCs. These findings suggest the potential of α-MG to be developed as a novel anti-DENV drug.

Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains.

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.

A Synthetic Bioactive Peptide Derived from the Asian Medicinal Plant Acacia catechu Binds to Dengue Virus and Inhibits Cell Entry.

Dengue virus (DENV) infection has become a critically important globally prevalent infectious disease, especially in tropical and subtropical countries. Since neither currently exists, there is an urgent need for an effective vaccine to prevent, and a specific drug to treat DENV infection. Therapeutic peptides represent an attractive alternative for development into anti-DENV drugs due to their safety and their diverse biological and chemical properties. We recently reported novel bioactive peptides extracted from the Asian medicinal plant Acacia catechu that efficiently inhibited all four DENV serotypes. In this study, we investigated the anti-DENV activity of a synthetic bioactive peptide derived from this plant. The most effective peptide (designated Pep-RTYM) inhibited DENV infection with a half-maximal inhibition concentration value of 7.9 µM. Time-of-addition study demonstrated that Pep-RTYM interacted with DENV particles and inhibited cellular entry. Pep-RTYM at 50 µM significantly reduced DENV production in Vero-kidney epithelial cells about 1000-fold, but it could decrease the virus production in Huh7 hepatocyte cells approximately 40-fold. Binding of Pep-RTYM to DENV particles may prevent virus interaction with cellular receptor and subsequent virus entry. This finding suggests a potential role of Pep-RTYM in the development of a novel anti-DENV drug.

A novel loss-of-function mutation of PBK associated with human kidney stone disease.

Kidney stone disease (KSD) is a prevalent disorder that causes human morbidity worldwide. The etiology of KSD is heterogeneous, ranging from monogenic defect to complex interaction between genetic and environmental factors. Since mutations of genes responsible for KSD in a majority of families are still unknown, our group is identifying mutations of these genes by means of genomic and genetic analyses. In this study, we identified a novel loss-of-function mutation of PBK, encoding the PDZ binding kinase, that was found to be associated with KSD in an affected Thai family. Glycine (Gly) substituted by arginine (Arg) at position 43 (p.Gly43Arg) in PBK cosegregated with the disease in affected members of this family, but was absent in 180 normal control subjects from the same local population. Gly43 is highly evolutionarily conserved in vertebrates, and its substitution affects protein structure by alterations in H-bond forming patterns. This p.Gly43Arg substitution results in instability of the variant PBK protein as examined in HEK293T cells. The variant PBK protein (p.Gly43Arg) demonstrated decreased kinase activity to phosphorylate p38 MAPK as analyzed by immunoblotting and antibody microarray techniques. Taken together, these findings suggest a possible new mechanism of KSD associated with pathogenic PBK variation.

Suppression of TGF-ß and IL-10 receptors on self-differentiated dendritic cells by short-hairpin RNAs enhanced activation of effector T-cells against cholangiocarcinoma cells.

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-ß (TGF-ß) 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-ß 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-ßRII and IL-10RA mRNAs. Immunoblot confirmed the reduced expression levels of TGF-ß 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-ß 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.