Assessing the impact of mRNA vaccination in chronic inflammatory murine model.

The implications of administration of mRNA vaccines to individuals with chronic inflammatory diseases, including myocarditis, rheumatoid arthritis (RA), and inflammatory bowel disease (IBD), are unclear. We investigated mRNA vaccine effects in a chronic inflammation mouse model implanted with an LPS pump, focusing on toxicity and immunogenicity. Under chronic inflammation, mRNA vaccines exacerbated cardiac damage and myocarditis, inducing mild heart inflammation with heightened pro-inflammatory cytokine production and inflammatory cell infiltration in the heart. Concurrently, significant muscle damage occurred, with disturbances in mitochondrial fusion and fission factors signaling impaired muscle repair. However, chronic inflammation did not adversely affect muscles at the vaccination site or humoral immune responses; nevertheless, it partially reduced the cell-mediated immune response, particularly T-cell activation. These findings underscore the importance of addressing mRNA vaccine toxicity and immunogenicity in the context of chronic inflammation, ensuring their safe and effective utilization, particularly among vulnerable populations with immune-mediated inflammatory diseases.

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.

Analysis of the immunostimulatory effects of cytokine-expressing internal ribosome entry site-based RNA adjuvants and their applications.

Developing new adjuvants that can effectively induce both humoral and cellular immune responses while broadening the immune response is of great value. In this study, we aimed to develop GM-CSF- or IL-18-expressing single-stranded RNA (ssRNA) adjuvants based on the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) and tested their efficacy in combination with ovalbumin (OVA) or inactivated influenza vaccines. Notably, cytokine-expressing RNA adjuvants increased the expression of antigen-presenting cell activation markers. Specifically, GM-CSF-expressing RNA adjuvants increased CD4+T cell responses, while IL-18-expressing RNA adjuvants increased CD8+T cell responses in mice when combined with OVA. In addition, cytokine-expressing RNA adjuvants increased the frequency of polyclonal T cells in combination with the influenza vaccine and reduced the clinical illness scores and weight loss of mice after viral challenge. Collectively, our results suggest that cytokine-expressing RNA adjuvants can be applied to protein-based or inactivated vaccines to increase their efficacy.

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.

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.

Effective inactivated influenza vaccine for the elderly using a single-stranded RNA-based adjuvant.

There is an unmet need for new influenza vaccine strategies that compensate for impaired vaccine responses in elderly individuals. Here, we evaluated the effectiveness of a single-stranded RNA (ssRNA) as an adjuvant to enhance the efficacy of inactivated influenza vaccine (IIV) in mouse models. Immunization with the ssRNA along with IIV reduced viral titers as well as pathological and inflammatory scores in the lungs after influenza challenge in aged mice. ssRNA induced balanced Th1/Th2 responses with an increase in IgA titers. Moreover, the ssRNA adjuvant markedly increased the frequency of influenza HA-specific T cells and IFN-γ production along with the expression of genes related to innate and adaptive immune systems that could overcome immunosenescence in aged mice. Our findings indicate that ssRNA is an efficient vaccine adjuvant that boosts cellular and humoral immunity in aged mice, demonstrating its potential as a novel adjuvant for currently available influenza virus vaccines for elderly individuals.

Immunization with RBD-P2 and N protects against SARS-CoV-2 in nonhuman primates.

Since the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), various vaccines are being developed, with most vaccine candidates focusing on the viral spike protein. Here, we developed a previously unknown subunit vaccine comprising the receptor binding domain (RBD) of the spike protein fused with the tetanus toxoid epitope P2 (RBD-P2) and tested its efficacy in rodents and nonhuman primates (NHPs). We also investigated whether the SARS-CoV-2 nucleocapsid protein (N) could increase vaccine efficacy. Immunization with N and RBD-P2 (RBDP2/N) + alum increased T cell responses in mice and neutralizing antibody levels in rats compared with those obtained using RBD-P2 + alum. Furthermore, in NHPs, RBD-P2/N + alum induced slightly faster SARS-CoV-2 clearance than that induced by RBD-P2 + alum, albeit without statistical significance. Our study supports further development of RBD-P2 as a vaccine candidate against SARS-CoV-2. Also, it provides insights regarding the use of N in protein-based vaccines against SARS-CoV-2.

MERS-CoV Spike Protein Vaccine and Inactivated Influenza Vaccine Formulated with Single Strand RNA Adjuvant Induce T-Cell Activation through Intranasal Immunization in Mice.

The effectiveness of vaccines is enhanced by adding adjuvants. Furthermore, the selection of an inoculation route depends on the type of adjuvant used and is important for achieving optimum vaccine efficacy. We investigated the immunological differences between two types of vaccines-spike protein from the Middle East respiratory syndrome virus and inactivated influenza virus vaccine, in combination with a single-stranded RNA adjuvant-administered through various routes (intramuscular, intradermal, and intranasal) to BALB/c mice. Intramuscular immunization with the RNA adjuvant-formulated spike protein elicited the highest humoral immune response, characterized by IgG1 and neutralizing antibody production. Although intranasal immunization did not elicit a humoral response, it showed extensive T-cell activation through large-scale induction of interferon-γ- and interleukin-2-secreting cells, as well as CD4+ T-cell activation in mouse splenocytes. Moreover, only intranasal immunization induced IgA production. When immunized with the inactivated influenza vaccine, administration of the RNA adjuvant via all routes led to protection after viral challenge, regardless of the presence of a vaccine-specific antibody. Therefore, the inoculation route should depend on the type of immune response needed; i.e., the intramuscular route is suitable for eliciting a humoral immune response, whereas the intranasal route is useful for T-cell activation and IgA induction.

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.

Inhibition of BMP signaling overcomes acquired resistance to cetuximab in oral squamous cell carcinomas.

Despite expressing high levels of the epidermal growth factor receptor (EGFR), a majority of oral squamous cell carcinoma (OSCC) patients show limited response to cetuximab and ultimately develop drug resistance. However, mechanism underlying cetuximab resistance in OSCC is not clearly understood. Here, using a mouse orthotopic xenograft model of OSCC, we show that bone morphogenic protein-7-phosphorylated Smad-1, -5, -8 (BMP7-p-Smad1/5/8) signaling contributes to cetuximab resistance. Tumor cells isolated from the recurrent cetuximab-resistant xenograft models exhibited low EGFR expression but extremely high levels of p-Smad1/5/8. Treatment with the bone morphogenic protein receptor type 1 (BMPRI) inhibitor, DMH1 significantly reduced cetuximab-resistant OSCC tumor growth, and combined treatment of DMH1 and cetuximab remarkably reduced relapsed tumor growth in vivo. Importantly, p-Smad1/5/8 level was elevated in cetuximab-resistant patients and this correlated with poor prognosis. Collectively, our results indicate that the BMP7-p-Smad1/5/8 signaling is a key pathway to acquired cetuximab resistance, and demonstrate that combination therapy of cetuximab and a BMP signaling inhibitor as potentially a new therapeutic strategy for overcoming acquired resistance to cetuximab in OSCC.

Erratum: the palatability of cereal based nutritional supplements in cancer patients.

[This corrects the article on p. 48 in vol. 3, PMID: 24527420.].

The palatability of cereal based nutritional supplements in cancer patients.

Recently, it is reported that intervention of oral nutritional supplement improves the nutritional status of cancer patients, and the effectiveness is affected by the sensory preference of cancer patients on the oral nutritional supplement. However, the variety of oral nutritional supplement is extremely limited and the number of patient's benefits from using the products are restricted mostly due to sensory dislikes. The objective of this study was to provide sensory preference score of trial manufactured products with different accessory ingredients to maximize the use of oral nutritional supplements. Cancer patients (n = 30) and age, sex-matched healthy volunteers (n = 30) participated in the sensory assessments (taste, flavor, viscosity, color and overall preference) of three types of oral supplements (cereal base, cereal base+herb and cereal base+fruit) and a control supplement product with scorched cereal flavor, a top seller in current Korean market. Results indicate that the cancer patients' overall preference was significantly higher for the control supplement, and fruit added supplement was preferred over plain cereal and herb added products, although the difference was insignificant. However, there was no significant preference difference for the supplements among the control group for all sensory factors. These results suggest that cancer patients are more sensitive to sensory preferences compared to the control group, and the patients prefer the flavor of cooked cereal which is a staple food in Korea.

Stereotactic body radiation therapy as an alternative treatment for small hepatocellular carcinoma.

BACKGROUND: Even with early stage hepatocellular carcinoma (HCC), patients are often ineligible for surgical resection, transplantation, or local ablation due to advanced cirrhosis, donor shortage, or difficult location. Stereotactic body radiation therapy (SBRT) has been established as a standard treatment option for patients with stage I lung cancer, who are not eligible for surgery, and may be a promising alternative treatment for patients with small HCC who are not eligible for curative treatment. MATERIALS AND METHODS: A registry database of 93 patients who were treated with SBRT for HCC between 2007 and 2009 was analyzed. A dose of 10-20 Gy per fraction was given over 3-4 consecutive days, resulting in a total dose of 30-60 Gy. The tumor response was determined using dynamic computed tomography or magnetic resonance imaging, which was performed 3 months after completion of SBRT. RESULTS: The median follow-up period was 25.6 months. Median size of tumors was 2 cm (range: 1-6 cm). Overall patients' survival rates at 1 and 3 years were 86.0% and 53.8%, respectively. Complete and partial tumor response were achieved in 15.5% and 45.7% of patients, respectively. Local recurrence-free survival rate was 92.1% at 3 years. Most local failures were found in patients with HCCs > 3 cm, and local control rate at 3 years was 76.3% in patients with HCC > 3 cm, 93.3% in patients with tumors between 2.1-3 cm, and 100% in patients with tumors ≤ 2 cm, respectively. Out-of-field intrahepatic recurrence-free survival rates at 1 and 3 years were 51.9% and 32.4%, respectively. Grade ≥ 3 hepatic toxicity was observed in 6 (6.5%). CONCLUSIONS: SBRT was effective in local control of small HCC. SBRT may be a promising alternative treatment for patients with small HCC which is unsuitable for other curative therapy.

The application of PET-CT to post-mastectomy regional radiation therapy using a deformable image registration.

BACKGROUND: To evaluate the utility of the preoperative PET-CT using deformable image registration (DIR) in the treatment of patients with locally advanced breast cancer and to find appropriate radiotherapy technique for further adequate treatment of axillary nodal area. METHODS: Sixty-five breast cancer patients who had level II, III axillary or supraclavicular lymph node metastasis on ¹8F-FDG PET-CT and received postoperative radiotherapy after modified radical mastectomy were enrolled. One radiation oncologist contoured normal organs (axillary vessels, clavicular head, coracoids process and humeral head) and involved lymph nodes on PET-CT and simulation CT slices. After contouring, deformable image registration of PET-CT on simulation CT was carried out. To evaluate the performance of the DIR, Dice similarity coefficient (DSC) and Center of mass (COM) were used. We created two plans, one was the historically designed three field plan and the other was the modified plan based on the location of axillary lymph node, and we compared the doses that irradiated the axillary lymph nodes. RESULTS: The DSCs for axillary artery, axillary vein, clavicular head, coracoids process and humeral head were 0.43 ± 0.15, 0.39 ± 0.20, 0.85 ± 0.10, 0.72 ± 0.20 and 0.77 ± 0.20, respectively. The distances between the COMs of axillary artery, axillary vein, clavicular head, coracoids process and humeral head in simulation CT and from PET-CT were 13.0 ±7.1, 20.2 ± 11.2, 4.4 ± 6.3, 3.7 ± 6.7, and 9.5 ± 25.0 mm, respectively. In the historically designed plan, only 57.7% of level II lymph nodes received more than 95% of prescribed dose and the coverage was improved to 70.0% with the modified plan (p < 0.01). For level III lymph nodes, the volumes received more than 95% of prescribed dose were similar in both plans (96.8 % vs 97.9%, p = 0.35). CONCLUSION: Deformable image registration of PET-CT on simulation CT was helpful in the identification of the location of the preoperatively involved axillary lymph node. Historically designed three-field plan was not adequate to treat the axillary level II lymph node area. Novel treatment technique based on the location of axillary lymph node from PET-CT using DIR can result in more adequate coverage of nodal area.

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.