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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.

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.

Steroid-induced diabetes is a well-known metabolic side effect of long-term use of glucocorticoid (GC). Our group recently demonstrated dexamethasone-induced pancreatic β-cell apoptosis via upregulation of TRAIL and TRAIL death receptor (DR5). Genistein protects against pancreatic β-cell apoptosis induced by toxic agents. This study aimed to investigate the cytoprotective effect of genistein against dexamethasone-induced pancreatic β-cell apoptosis in cultured rat insulinoma (INS-1) cell line and in isolated mouse islets. In the absence of genistein, dexamethasone-induced pancreatic β-cell apoptosis was associated with upregulation of TRAIL, DR5, and superoxide production, but downregulation of TRAIL decoy receptor (DcR1). Dexamethasone also activated the expression of extrinsic and intrinsic apoptotic proteins, including Bax, NF-κB, caspase-8, and caspase-3, but suppressed the expression of the anti-apoptotic Bcl-2 protein. Combination treatment with dexamethasone and genistein protected against pancreatic β-cell apoptosis, and reduced the effects of dexamethasone on the expressions of TRAIL, DR5, DcR1, superoxide production, Bax, Bcl-2, NF-κB, caspase-8, and caspase-3. Moreover, combination treatment with dexamethasone and genistein reduced the expressions of TRAIL and DR5 in isolated mouse islets. The results of this study demonstrate the cytoprotective effect of genistein against dexamethasone-induced pancreatic β-cell apoptosis in both cell line and islets via reduced TRAIL and DR5 protein expression.

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 p

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. A newly potential therapy – chimeric antigen receptor (CAR) T cell therapy utilizes genetically modified T cells that specifically recognize surface antigen on cancer cells without restriction by major histocompatibility complex (MHC). Mucin 1 (MUC1) is an attractive candidate antigen due to its high expression in CCA cells, and its association with poor prognosis and survival. Since anti-MUC1-CAR T cells have not previously been tested for activity in models of CCA, we set forth to test their utility in this setting. A fourth generation anti-MUC1-CAR construct was engineered to contain anti-MUC1-single-chain variable fragment (scFv) and three co-stimulatory domains (CD28, CD137, and CD27) linked to CD3ζ. Cultures of anti-MUC1-CAR T cells consisted primarily of cytotoxic (CD8+) T cells (75.13±11.65%, p

Viruses manipulate the life cycle in host cells via the use of viral properties and host machineries. Development of antiviral peptides against dengue virus (DENV) infection has previously been concentrated on blocking the actions of viral structural proteins and enzymes in virus entry and viral RNA processing in host cells. In this study, we proposed DENV NS1, which is a multifunctional non-structural protein indispensable for virus production, as a new target for inhibition of DENV infection by specific peptides. We performed biopanning assays using a phage-displayed peptide library and identified 11 different sequences of 12-mer peptides binding to DENV NS1. In silico analyses of peptide-protein interactions revealed 4 peptides most likely to bind to DENV NS1 at specific positions and their association was analysed by surface plasmon resonance. Treatment of Huh7 cells with these 4 peptides conjugated with N-terminal fluorescent tag and C-terminal cell penetrating tag at varying time-of-addition post-DENV infection could inhibit the production of DENV-2 in a time- and dose-dependent manner. The inhibitory effects of the peptides were also observed in other virus serotypes (DENV-1 and DENV-4), but not in DENV-3. These findings indicate the potential application of peptides targeting DENV NS1 as antiviral agents against DENV infection.

Chronic exposure of pancreatic β-cells to high glucose levels results in β-cell dysfunction and death. These effects can be protected by estrogen. The local pancreatic renin-angiotensin system (RAS) has been shown as a novel pathological pathway of high-glucose-induced cell death. The effect of estrogen on pancreatic RAS is still unknown. This study examines whether estrogen protects against pancreatic β-cell death caused by glucotoxicity via a decrease in the pancreatic β-cell RAS pathway. When INS-1 cells were cultured in a high glucose medium, cell death was significantly higher than when the cells were cultured in a basal glucose medium; similarly, there were also higher levels of AGTR1 and p47 ph ° x mRNA, and protein expression. Moreover, the addition of 10−8 M 17β-estradiol to INS-1 cells cultured in a high glucose medium markedly reduced cell death, AGTR1 and p47 ph ° x mRNA levels, and protein expression. Similar results were demonstrated in the pancreatic islets. The presence of 10−8 M 17β-estradiol, losartan, or a combination of both, in a high glucose medium had similar levels of reduction of p47 ph ° x mRNA and protein expression, compared with those cultured in high glucose. Taken together, estrogen protected pancreatic β-cells from high-glucose-induced cell death by reducing the AGTR1 pathway.

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 NaV1.8 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. NaV1.8 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.