New Function Annotation of PROSER2 in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to the absence of diagnostic markers and molecular targets. Here, we took an unconventional approach to identify new molecular targets for pancreatic cancer. We chose uncharacterized protein evidence level 1 without function annotation from extensive proteomic research on pancreatic cancer and focused on proline and serine-rich 2 (PROSER2), which ranked high in the cell membrane and cytoplasm. In our study using cell lines and patient-derived orthotopic xenograft cells, PROSER2 exhibited a higher expression in cells derived from primary tumors than in those from metastatic tissues. PROSER2 was localized in the cell membrane and cytosol by immunocytochemistry. PROSER2 overexpression significantly reduced the metastatic ability of cancer cells, whereas its suppression had the opposite effect. Proteomic analysis revealed that PROSER2 interacts with STK25 and PDCD10, and their binding was confirmed by immunoprecipitation and immunocytochemistry. STK25 knockdown enhanced metastasis by decreasing p-AMPK levels, whereas PROSER2-overexpressing cells increased the level of p-AMPK, indicating that PROSER2 suppresses invasion via the AMPK pathway by interacting with STK25. This is the first demonstration of the novel role of PROSER2 in antagonizing tumor progression via the STK25-AMPK pathway in PDAC. LC-MS/MS data are available at MassIVE (MSV000092953) and ProteomeXchange (PXD045646).

mRNA-HPV vaccine encoding E6 and E7 improves therapeutic potential for HPV-mediated cancers via subcutaneous immunization.

The E6 and E7 proteins of specific subtypes of human papillomavirus (HPV), including HPV 16 and 18, are highly associated with cervical cancer as they modulate cell cycle regulation. The aim of this study was to investigate the potential antitumor effects of a messenger RNA-HPV therapeutic vaccine (mHTV) containing nononcogenic E6 and E7 proteins. To achieve this, C57BL/6j mice were injected with the vaccine via both intramuscular and subcutaneous routes, and the resulting effects were evaluated. mHTV immunization markedly induced robust T cell-mediated immune responses and significantly suppressed tumor growth in both subcutaneous and orthotopic tumor-implanted mouse model, with a significant infiltration of immune cells into tumor tissues. Tumor retransplantation at day 62 postprimary vaccination completely halted progression in all mHTV-treated mice. Furthermore, tumor expansion was significantly reduced upon TC-1 transplantation 160 days after the last immunization. Immunization of rhesus monkeys with mHTV elicited promising immune responses. The immunogenicity of mHTV in nonhuman primates provides strong evidence for clinical application against HPV-related cancers in humans. All data suggest that mHTV can be used as both a therapeutic and prophylactic vaccine.

Heterologous vaccination utilizing viral vector and protein platforms confers complete protection against SFTSV.

Severe fever with thrombocytopenia syndrome virus was first discovered in 2009 as the causative agent of severe fever with thrombocytopenia syndrome. Despite its potential threat to public health, no prophylactic vaccine is yet available. This study developed a heterologous prime-boost strategy comprising priming with recombinant replication-deficient human adenovirus type 5 (rAd5) expressing the surface glycoprotein, Gn, and boosting with Gn protein. This vaccination regimen induced balanced Th1/Th2 immune responses and resulted in potent humoral and T cell-mediated responses in mice. It elicited high neutralizing antibody titers in both mice and non-human primates. Transcriptome analysis revealed that rAd5 and Gn proteins induced adaptive and innate immune pathways, respectively. This study provides immunological and mechanistic insight into this heterologous regimen and paves the way for future strategies against emerging infectious diseases.

Complexation of drug and hapten-conjugated aptamer with universal hapten antibody for pancreatic cancer treatment.

Owing to a lack of reliable markers and therapeutic targets, pancreatic ductal adenocarcinoma (PDAC) remains the most lethal malignant tumor despite numerous therapeutic advances. In this study, we utilized cell-SELEX to isolate a DNA aptamer recognizing the natural conformation of the target on the cell surface. PAp7T8, an aptamer optimized by size and chemical modification, exhibited specific targeting to pancreatic cancer cells and orthotopic xenograft pancreatic tumors. To confer therapeutic functions to the aptamer, we adopted a drug-conjugated oligobody (DOligobody) strategy. Monomethyl auristatin E was used as a cytotoxic drug, digoxigenin acted as a hapten, and the humanized anti-digoxigenin antibody served as a universal carrier of the aptamer. The resulting PAp7T8-DOligobody showed extended in vivo half-life and markedly inhibited tumor growth in an orthotopic pancreatic cancer xenograft model without causing significant toxicity. Therefore, PAp7T8-DOligobody represents a promising novel therapeutic delivery platform for PDAC.

Adjuvant effect of IRES-based single-stranded RNA on melanoma immunotherapy.

BACKGROUND: Adjuvant therapies such as radiation therapy, chemotherapy, and immunotherapy are usually given after cancer surgery to improve the survival of cancer patients. However, despite advances in several adjuvant therapies, they are still limited in the prevention of recurrences. METHODS: We evaluated the immunological effects of RNA-based adjuvants in a murine melanoma model. Single-stranded RNA (ssRNA) were constructed based on the cricket paralysis virus (CrPV) internal ribosome entry site (IRES). Populations of immune cells in bone marrow cells and lymph node cells following immunization with CrPVIRES-ssRNA were determined using flow cytometry. Activated cytokine levels were measured using ELISA and ELISpot. The tumor protection efficacy of CrPVIRES-ssRNA was analyzed based on any reduction in tumor size or weight, and overall survival. RESULTS: CrPVIRES-ssRNA treatment stimulated antigen-presenting cells in the drain lymph nodes associated with activated antigen-specific dendritic cells. Next, we evaluated the expression of CD40, CD86, and XCR1, showing that immunization with CrPVIRES-ssRNA enhanced antigen presentation by CD8a+ conventional dendritic cell 1 (cDC1), as well as activated antigen-specific CD8 T cells. In addition, CrPVIRES-ssRNA treatment markedly increased the frequency of antigen-specific CD8 T cells and interferon-gamma (IFN-γ) producing cells, which promoted immune responses and reduced tumor burden in melanoma-bearing mice. CONCLUSIONS: This study provides evidence that the CrPVIRES-ssRNA adjuvant has potential for use in therapeutic cancer vaccines. Moreover, CrPVIRES-ssRNA possesses protective effects on various cancer cell models.

Single-stranded RNA adjuvant enhances the efficacy of 10-valent human papilloma virus-like particle vaccine.

Following the development of various types of vaccines, the use of adjuvants to boost vaccine efficacy has become a focus of research. Aluminum hydroxide (alum), the most commonly used adjuvant, induces a certain immune response and ensures safety in human trials. However, alum mainly induces only a Th2 response; its Th1 response is weak. Thus, we previously developed a single-stranded ribose nucleic acid (ssRNA) adjuvant that induces a Th1 response through toll-like receptors. Here, we explored whether 10-valent human papilloma virus (HPV)-like particle (VLP) vaccine formulated with ssRNA adjuvant and alum helped to enhance immune response and maintained memory response. The mice were immunized intramuscularly twice at 2 week intervals and were inoculated 4 days after the second boost (after about 1 year). The antibody response and T cell activation were measured by Elispot, ELISA using harvested serum and splenocytes. The 10-valent HPV VLP vaccine formulated with ssRNA adjuvant and alum increased the antigen-specific immune response more than alum used alone. It increased each type-specific IgG1/IgG2a titer, and antigen-specific IFN-γ cells. Furthermore, the ssRNA adjuvant with alum induced memory response. In memory response, each type-specific IgG1/IgG2c, IFN-γ, and IL-6 cytokine, and neutralizing antibodies were increased by the ssRNA adjuvant with alum. Overall, the ssRNA adjuvant with alum induced memory responses and balanced Th1/Th2 responses. The ssRNA adjuvant and alum may help to enhance prophylactic vaccine efficacy.

Comparison of remission criteria in patients with rheumatoid arthritis treated with biologic or targeted synthetic disease-modifying anti-rheumatic drugs: results from a nationwide registry.

Background: Biologic or targeted synthetic disease-modifying anti-rheumatic drugs (b/tsDMARD) are widely used for treatment of rheumatoid arthritis (RA), enabling patients to better achieve remission. Objective: The objective of the study was to investigate and compare remission rates in RA patients treated with different b/tsDMARDs during the period 2013-2019. Design: A longitudinal observational analysis was performed on data from a nationwide RA registry. Methods: Remission rates in the KOBIO-RA registry were defined by a disease activity score in 28 joints (DAS28), clinical disease activity index (CDAI), simplified disease activity index (SDAI), and Boolean-based assessment. After initiating treatment with b/tsDMARDs, yearly remission rates in response to b/tsDMARDs, either all or as subgroups (tumor necrosis factor-α inhibitors, tocilizumab, abatacept, and Janus kinase inhibitors), were investigated for 5 years. Sustained remission was defined as remission maintained for two consecutive years. Results: Patients (N = 1805) who completed at least one follow-up visit were analyzed (mean age = 55 years; 83.2% female). At month 12, 56.0% of patients achieved remission based on DAS28-C-reactive protein (CRP), 36.2% on DAS28-erythrocyte sedimentation rate (ESR), 10.4% on CDAI, 12.7% on SDAI, and 12.9% on Boolean criteria. Sustained remission rates were 62%, 40%, 13%, 11%, and 8% for the DAS28-CRP, DAS28-ESR, Boolean, SDAI, and CDAI remission criteria, respectively. Remission rates using the DAS28 definition varied most among the b/tsDMARD subgroups. Conclusion: Assessment of sustained remission using the CDAI, SDAI, or Boolean criteria is more stringent, yet congruous with the DAS28-based criteria in RA patients treated with b/tsDMARDs.

Risk of Acute Anterior Uveitis in Ankylosing Spondylitis According to the Type of Tumor Necrosis Factor-Alpha Inhibitor and History of Uveitis: A Nationwide Population-Based Study.

BACKGROUND: We evaluated the risk of acute anterior uveitis (AAU) in patients with ankylosing spondylitis (AS) during treatment with tumor necrosis factor-alpha inhibitors (TNFis). METHODS: This study was performed on AS patients using the Korean National Health Insurance claims database. We analyzed the first and total occurrence of AAU during the first 2 years of TNFis use according to the type of TNFis. Additionally, the occurrence of AAU was assessed in subgroups with or without prior AAU before TNFis initiation. RESULTS: In total, 5938 AS patients initiated TNFis use between 2009 and 2017 and used them for more than 2 years. Among them, 1488 (25.1%) patients had a history of AAU before starting TNFis treatment. Compared to adalimumab, the use of etanercept (hazard ratio [HR] 1.77) increased the risk of AAU. The incidence rate ratio (IRR) of AAU with etanercept was significantly higher than that of adalimumab (IRR 1.78). The IRR of AAU was also higher for etanercept than adalimumab use in patients with (IRR 1.86) and without (IRR 2.92) a history of AAU. CONCLUSION: These data suggest that compared to anti-TNF-alpha monoclonal antibodies, etanercept has a higher incidence of AAU regardless of a history of AAU.

Establishment of Patient-derived Preclinical Models for Invasive Papillary Cholangiocarcinoma.

BACKGROUND/AIM: Invasive papillary cholangio-carcinoma (IPC) is a minor subtype of extrahepatic cholangiocarcinoma. However, its etiology and characteristics remain unknown because of the unavailability of in vitro and in vivo models. We aimed to establish a novel preclinical model for translational research of IPC. MATERIALS AND METHODS: A patient-derived xenograft (PDX) was engrafted in NOG mice and the cell line National Cancer Center human IPC (NCChIPC) was subsequently established from the PDX tumors. Immunohistochemistry and RNA-sequencing were used to determine the retention of original characteristics of patient tissues. RESULTS: PDX tumors showed successful amplification, and the NCChIPC-derived xenograft largely retained the histopathological features of the original tumor with CK19, MUC1 and MUC5AC expression. Transcriptome analysis showed a high correlation between patient and preclinical models. Additionally, anticancer drugs response was analyzed in the NCChIPC PDX. CONCLUSION: These novel preclinical models here will help elucidate IPC etiology and facilitate translational research.

Peptides Derived From S and N Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 Induce T Cell Responses: A Proof of Concept for T Cell Vaccines.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape vaccine-induced neutralizing antibodies has indicated the importance of T cell responses against this virus. In this study, we highlight the SARS-CoV-2 epitopes that induce potent T cell responses and discuss whether T cell responses alone are adequate to confer protection against SARS-CoV-2 and describe the administration of 20 peptides with an RNA adjuvant in mice. The peptides have been synthesized based on SARS-CoV-2 spike and nucleocapsid protein sequences. Our study demonstrates that immunization with these peptides significantly increases the proportion of effector memory T cell population and interferon-γ (IFN-γ)-, interleukin-4 (IL-4)-, tumor necrosis factor-α (TNF-α)-, and granzyme B-producing T cells. Of these 20 peptides, four induce the generation of IFN-γ-producing T cells, elicit CD8+ T cell (CTL) responses in a dose-dependent manner, and induce cytotoxic T lymphocytes that eliminate peptide-pulsed target cells in vivo. Although it is not statistically significant, these peptide vaccines reduce viral titers in infected hamsters and alleviate pulmonary pathology in SARS-CoV-2-infected human ACE2 transgenic mice. These findings may aid the design of effective SARS-CoV-2 peptide vaccines, while providing insights into the role of T cells in SARS-CoV-2 infection.

Development of Patient-Derived Preclinical Platform for Metastatic Pancreatic Cancer: PDOX and a Subsequent Organoid Model System Using Percutaneous Biopsy Samples.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignant tumor and more than 50% patients are diagnosed at metastatic stage. The preclinical model systems that reflect the genetic heterogeneity of metastatic tumors are urgently needed to guide optimal treatment. This study describes the development of patient-derived preclinical platform using very small sized-percutaneous liver gun biopsy (PLB) of metastatic pancreatic cancer, based on patient-derived xenograft (PDX)-mediated tissue amplification and subsequent organoid generation. To increase the success rate and shorten the tumor growth period, patient-derived orthotopic xenograft (PDOX) model was developed to directly implant threadlike PLB samples into the pancreas. The engraftment success rate of PDOX samples from 35 patients with metastatic PDAC was 47%, with these samples showing the potential to metastasize to distant organs, as in patients. The PDOX models retained the genetic alterations and histopathological features of the primary tumors. Tumor organoids were subsequently generated from first passage cancer cells isolated from F1 tumor tissue of PDOX that preserve the epithelial cancer characteristics and KRAS mutations of primary tumors. The response to gemcitabine of PDOX-derived organoids correlated with clinical outcomes in corresponding patients as well as PDOX models in vivo, suggesting that this PDOX-organoid system reflects clinical conditions. Collectively, these findings indicate that the proposed PDOX-organoid platform using PLB samples assessed both in vitro and in vivo could predict drug response under conditions closer to those found in actual patients, as well as enhancing understanding of the complexity of metastatic PDAC.

Efficient targeting and tumor retardation effect of pancreatic adenocarcinoma up-regulated factor (PAUF)-specific RNA replacement in pancreatic cancer mouse model.

The soluble protein pancreatic adenocarcinoma up-regulated factor (PAUF) plays an important role in pancreatic tumor progression and has begun to attract attention as a therapeutic target for pancreatic cancer. We herein present PAUF RNA-targeting gene therapy strategies with both targeting and therapeutic function using trans-splicing ribozyme (TSR) in pancreatic cancer. We developed adenoviral PAUF-targeting TSR (Rz) containing a PAUF-specific internal guide sequence (IGS) determined by library screening. This Rz harbors suicide gene, herpes simplex virus thymidine kinase (HSV-tk) or firefly luciferase (Luc) as a transgene for 3' exon replacement of PAUF RNAs. Ad-Rz-TK, Rz harboring the HSV-tk, showed significant inhibition of tumor growth in vivo as well as PAUF-dependent cell death in vitro via a successful trans-splicing reaction. Selective induction of Rz-controlled transgene in PAUF-expressing pancreatic cancer was confirmed through noninvasive in vivo imaging; a luminescence signal from Rz harboring Luc (Ad-Rz-Luc) was detectable only in pancreatic tumor sites, not in normal mice. In addition, a [(125)I] FIAU signal reflecting thymidine kinase expression through SPECT and ex vivo biodistribution was co-localized with the tumor sites when we treated with Ad-Rz-TK in orthotopic xenograft model. Taken together, these results imply that PAUF-targeting TSR can contribute to successful targeted gene therapy for pancreatic cancer.

Relationship between acute stroke outcome, aspirin resistance, and humoral factors.

BACKGROUND: The relationship between biochemical aspirin resistance (AR) and functional outcome of acute ischemic stroke is uncertain. METHODS: Prospectively, 269 patients with acute ischemic stroke were recruited. Their responsiveness to aspirin was evaluated by platelet function analyzer (PFA-100). All patients received blood tests for fibrinogen, high-sensitivity C-reactive protein (hs-CRP), CD40-ligand, P-selectin, intercellular adhesion molecule -1, von Willebrand factor (vWF), and D-dimer. The patients' National Institutes of Health Stroke Scale and modified Rankin Scale scores were recorded on admission, at 30 days, and at 90 days after stroke. RESULTS: Closure-time measured by PFA-100 equipped with epinephrine/collagen cartridge (Epi-CT) was <193 seconds (defined as AR) in 83 patients (30.9%). Patients with AR were less likely to have favorable outcome at 30 days (47.0%, p = 0.047; odds ratio: 0.69, 0.48-0.99) and 90 days (57.8%, p = 0.037; odds ratio: 0.69, 0.47-0.97) after stroke compared with those of patients without AR (60.2% and 71.0%, respectively). The Epi-CT correlated with closure-time measured by adenosine diphosphate/collagen cartridge (r = 0.241, p < 0.001), blood white cell count (r = -0.125, p = 0.041), low density lipoprotein cholesterol (r = 0.120, p = 0.050), hs-CRP (r = -0.150, p = 0.015), vWF (r = -0.134, p = 0.028), and body mass index (r = 0.143, p = 0.019). Multivariate logistic regression analysis showed that higher National Institutes of Health Stroke Scale at admission, atrial fibrillation, increased plasma levels of hs-CRP, and D-dimer were independent predictors for unfavorable stroke outcome at 90 days. CONCLUSION: Aspirin resistance evaluated by PFA-100 test was associated with unfavorable 90-day outcome. However, AR determined by PFA-100 dose not predict 90-day functional outcome. The results of PFA-100 testing represented a complex interaction between drug effect, inflammatory reaction, and prothrombotic activity.

Regulation of glycogen synthase kinase-3 by thymosin beta-4 is associated with gastric cancer cell migration.

Thymosin beta-4 (Tß4), actin-sequestering protein, plays important roles in many cellular functions including cancer cell migrations. Glycogen synthase kinase (GSK) in Wnt signaling pathway is a key molecule to control intercellular interaction. Here, we investigated whether GSK-3 activity is regulated by Tß4 and it is associated with Tß4-mediated migration in gastric cancer cells. Various expression level of Tß4 was observed in human gastric tumor tissues. Migration in gastric cancer cells, SNU638 and SNU668, was dependent on a relative expression level of Tß4. Cell migration was higher in SNU668 with a higher expression level of Tß4 than that in SNU638 with a lower Tß4. Although the level of phosphorylated(p)-GSK-3α (inactive), ß-catenin, E-cadherin and E-cadherin:ß-catenin complex was relatively higher, p-GSK-3ß (inactive) was lower in SNU638 compared to those in SNU668 cells. LiCl, GSK-3α/ß inhibitor, reduced lung metastasis of B16F10 mouse melanoma cells and SNU668 cell migration. Small interference (si)RNA of GSK-3α increased SNU638 cell migration in accordance with the reduction of E-cadherin:ß-catenin complex formation through a decrease in ß-catenin and E-cadherin. Expression level of GSK-3α/ß, ß-catenin and E-cadherin in SNU668 and SNU638 was reversed by Tß4-siRNA and by the treatment with acetylated-serine-aspartic acid-lysine-proline (SDKP) tetrapeptide of Tß4, respectively. E-cadherin expression in SNU638 cells was decreased by ß-catenin-siRNA. PD98059, MEK inhibitor, or U0126, ERK inhibitor, reduced SNU668 cell migration accompanying an increase in p-GSK-3α, ß-catenin and E-cadherin. Taken together, data indicated that the expression of GSK-3α, ß-catenin and E-cadherin could be negatively regulated by Tß4-induced ERK phosphorylation. It suggests that Tß4 could be a novel regulator to control Wnt signaling pathways.

Toll-like receptor 4-mediated cAMP production up-regulates B-cell activating factor expression in Raw264.7 macrophages.

B-cell activating factor (BAFF) plays a role in the generation and the maintenance of mature B cells. Lipopolysaccharide (LPS) increased BAFF expression through the activation of toll-like receptor 4 (TLR4)-dependent signal transduction. Here, we investigated the mechanism of action on mouse BAFF (mBAFF) expression by cAMP production in Raw264.7 mouse macrophages. mBAFF expression was increased by the treatment with a cAMP analogue, dibutyryl-cAMP which is the activator of protein kinase A (PKA), cAMP effector protein. PKA activation was measured by the phosphorylation of cAMP-response element binding protein (CREB) on serine 133 (S133). cAMP production and CREB (S133) phosphorylation were augmented by LPS-stimulation. While mBAFF promoter activity was enhanced by the co-transfection with pS6-RSV-CREB, it was reduced by siRNA-CREB. PKA inhibitor, H-89, reduced CREB (S133) phosphorylation and mBAFF expression in control and LPS-stimulated macrophages. Another principal cAMP effector protein is cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. Epac was activated by the treatment with 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT), Epac activator, as judged by the measurement of Rap1 activation. Basal level of mBAFF expression was increased by CPT treatment. LPS-stimulated mBAFF expression was also slightly enhanced by co-treatment with CPT. In addition, dibutyryl-cAMP and CPT enhanced mBAFF expression in bone marrow-derived macrophages (BMDM). With these data, it suggests that the activation of PKA and cAMP/Epac1/Rap1 pathways could be required for basal mBAFF expression, as well as being up-regulated in the TLR4-induced mBAFF expression.