The significance of T-BET-positive CD8 T-cells with diminished CD5 expression in Kikuchi-Fujimoto disease.

Kikuchi-Fujimoto disease (KFD), also known as histiocytic necrotizing lymphadenitis, is a rare condition characterized by benign localized lymphadenopathy and clinical symptoms such as fever, sore throat, odynophagia, and leukopenia. Though the etiology of KFD is unknown, this condition is similar to viral infection, including increased infiltration of activated plasmacytoid dendritic cells. KFD exhibits three histological phases that reflect its progression status: proliferative, necrotic, and xanthomatous lesions. The expression loss of pan T-cell markers, such as CD2, CD5, and CD7, of infiltrating T-cells is observed in KFD cases, complicating the distinction from T-cell lymphoma. However, reports on the loss of their expression in KFD have been limited. Furthermore, the precise population of the T-cell subset in KFD is still unclear. Here, we focused on surface markers and transcription factors for T-cell differentiation and analyzed them immunohistochemically in 46 KFD cases. We observed diminished CD5 expression of CD8-positive (CD5dim CD8+) T-cells in the proliferative lesion of KFD cases. Furthermore, these CD5dim CD8+ T-cells expressed T-BET, a master regulator of type 1 helper T-cells. The upregulation of T-BET and downregulation of CD5 in CD8+ T-cells causes dysregulated activation and proliferation of CD8+ T-cells, potentially contributing to the unique histopathological features of KFD. Recognizing the frequent infiltration of T-BET-positive CD5dim CD8+ T-cells in KFD is important for distinguishing it from mature T-cell lymphoma. Our findings suggest that the immune response in KFD shares similarities with viral infections and highlight the importance of characterizing T-BET-positive CD5dim CD8+ T-cell populations for understanding KFD pathogenesis.

Anti-CX3CL1 (fractalkine) monoclonal antibody attenuates lung and skin fibrosis in sclerodermatous graft-versus-host disease mouse model.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular injury and inflammation, followed by excessive fibrosis of the skin and other internal organs, including the lungs. CX3CL1 (fractalkine), a chemokine expressed on endothelial cells, supports the migration of macrophages and T cells that express its specific receptor CX3CR1 into targeted tissues. We previously reported that anti-CX3CL1 monoclonal antibody (mAb) treatment significantly inhibited transforming growth factor (TGF)-ß1-induced expression of type I collagen and fibronectin 1 in human dermal fibroblasts. Additionally, anti-mouse CX3CL1 mAb efficiently suppressed skin inflammation and fibrosis in bleomycin- and growth factor-induced SSc mouse models. However, further studies using different mouse models of the complex immunopathology of SSc are required before the initiation of a clinical trial of CX3CL1 inhibitors for human SSc. METHODS: To assess the preclinical utility and functional mechanism of anti-CX3CL1 mAb therapy in skin and lung fibrosis, a sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) mouse model was analyzed with immunohistochemical staining for characteristic infiltrating cells and RNA sequencing assays. RESULTS: On day 42 after bone marrow transplantation, Scl-cGVHD mice showed increased serum CX3CL1 level. Intraperitoneal administration of anti-CX3CL1 mAb inhibited the development of fibrosis in the skin and lungs of Scl-cGVHD model, and did not result in any apparent adverse events. The therapeutic effects were correlated with the number of tissue-infiltrating inflammatory cells and α-smooth muscle actin (α-SMA)-positive myofibroblasts. RNA sequencing analysis of the fibrotic skin demonstrated that cGVHD-dependent induction of gene sets associated with macrophage-related inflammation and fibrosis was significantly downregulated by mAb treatment. In the process of fibrosis, mAb treatment reduced cGVHD-induced infiltration of macrophages and T cells in the skin and lungs, especially those expressing CX3CR1. CONCLUSIONS: Together with our previous findings in other SSc mouse models, the current results indicated that anti-CX3CL1 mAb therapy could be a rational therapeutic approach for fibrotic disorders, such as human SSc and Scl-cGVHD.

Analysis of MicroRNA in Bile Cytologic Samples Is Useful for Detection and Diagnosis of Extrahepatic Cholangiocarcinoma.

OBJECTIVES: This study aimed to develop reliable biomarkers that improve the ability of bile cytology to diagnose cholangiocarcinoma vs benign biliary lesions. METHODS: Many studies indicate that microRNAs (miRNAs) are potential candidates for the early diagnosis of cancer. We analyzed the expression of five tumor-associated miRNAs (miR-31-5p, miR-122-5p, miR-378d, miR-182-5p, and miR-92a-3p) in cytology samples using quantitative reverse transcription polymerase chain reaction. We collected 52 surgically resected tissue samples, 84 cytologic specimens from smears (53 cases of cancer and 31 cases of noncancer), and 40 residual sediments after smearing for routine cytology at Hiroshima University Hospital. RESULTS: The expression of miR-31-5p, miR-378d, and miR-122-5p was significantly higher in cancer tissues than those in normal tissues, while miR-182-5p expression was lower. The expression of miR-31-5p, miR-378d, miR-182-5p, and miR-92a-3p was significantly higher in detached cell samples from smears of cholangiocarcinoma cases than in those from noncancer cases. CONCLUSIONS: These results suggest that the analysis of miRNAs in bile cytologic specimens is a promising auxiliary tool for distinguishing cholangiocarcinoma from benign biliary lesions.

Intramolecular Activation of C-O Bond by an o-Boryl Group in o-(Alkoxysilyl)(diarylboryl)benzenes.

Halogen-lithium exchange reaction of o-(silyl)bromobenzene 5 with tert-BuLi afforded o-(silyl)lithiobenzene 6, which was reacted with (alkoxy)diarylboranes 7 to form borate intermediates 8. Treatment of 8 with chlorotrimethylsilane formed o-(alkoxysilyl)(diarylboryl)benzenes 4. The C-O bond in 4 was activated by intramolecular interaction between the oxygen atom and the boron atom. 4a readily reacted with MeOH and EtOH to afford the corresponding alkoxysilanes 10 and 11, respectively. Treatment of 10 with 1,4-diazabicyclo[2.2.2]octane (DABCO) afforded the silyloxyborate complex 13.

Five major sequence variants and copy number variants in the EYS gene account for one-third of Japanese patients with autosomal recessive and simplex retinitis pigmentosa.

Purpose: To elucidate the variant spectrum of the EYS gene in a large cohort of Japanese patients with autosomal recessive and simplex retinitis pigmentosa (arRP and sRP). Methods: We performed a direct sequencing analysis of 44 exons of the EYS gene in 469 patients with RP (including 144 arRP, 288 sRP, and 17 autosomal dominant RP (adRP) cases) in eastern and western regions of Japan and a multiplex ligation-dependent probe amplification (MLPA) of patients who had a single heterozygous pathogenic variant. Results: We identified six pathogenic and 16 likely pathogenic variants from a total of 186 nucleotide sequence variants, of which five variants, c.2528G>A (p.(Gly843Glu)), c.4957dupA (p.(Ser1653Lysfs*2)), c.6557G>A (p.(Gly2186Glu)), c.6563T>C (p.(Ile2188Thr)), and c.8868C>A (p.(Tyr2956*)), were prevalent in patients with arRP and sRP. The homozygous and heterozygous combinations of these five variants accounted for 32.4% (140/432) of Japanese patients with arRP and sRP. Five patients with adRP also had these variants. These five variants segregated with the phenotype in 15 families with RP. MLPA revealed seven copy number variations (CNVs) of the EYS exon(s). Conclusions: This study showed that five major sequence variants and CNVs in the EYS gene account for one-third of Japanese patients with arRP and sRP, and these variants are also responsible for RP showing an autosomal dominant inheritance pattern. This is the first report showing the pathogenicity of three missense variants (p.(Gly843Glu), p.(Gly2186Glu), and p.(Ile2188Thr)) and the presence of CNVs in the EYS gene of Japanese patients with arRP and sRP.

[Analysis of the Risk Factors Associated with Minor Bleeding in Patients with Venous Thromboembolism during Treatment with Direct Oral Anticoagulants].

Direct oral anticoagulants (DOACs) are safe and efficacious when compared to warfarin for patients with venous thromboembolism (VTE). However, bleeding is a major side effect of anticoagulant therapy in VTE patients. Discontinuation of the DOACs associated to adverse events such as bleeding. The HAS-BLED score predicts warfarin-associated hemorrhage. However, little is known about risk factors for DOAC-associated minor bleeding in VTE patients. We aimed to identify risk factors for minor bleeding in VTE patients that were treated with edoxaban, rivaroxaban, or apixaban. We retrospectively evaluated the data of 212 VTE patients who received treatment with a DOAC. The study endpoint was defined as the occurrence of minor bleeding. Logistic regression analysis was used to determine risk factors that were significantly associated with minor bleeding. A total of 36 (17.0%) patients experienced minor bleeding, with rates of 15.7%, 0%, and 21.3% for edoxaban, rivaroxaban, and apixaban, respectively. In the multivariate analysis, bleeding history or predisposition [odds ratio (OR) 6.083, 95% confidence interval (CI) 2.131-17.364, p=0.001] and cancer (OR 6.397, 95% CI 2.858-14.317, p<0.001) were significantly associated with minor bleeding. Bleeding history or predisposition and cancer were the most important risk factors for DOAC-induced minor bleeding in VTE patients in this study. To continue anticoagulant therapy of the DOACs, further management systems by minor bleeding risk factors for patients with VTE will be required.

Genetic Analysis of Human Norovirus Strains in Japan in 2016-2017.

In the 2016/2017 winter season in Japan, HuNoV GII.P16-GII.2 strains (2016 strains) emerged and caused large outbreaks of acute gastroenteritis. To better understand the outbreaks, we examined the molecular evolution of the VP1 gene and RdRp region in 2016 strains from patients by studying their time-scale evolutionary phylogeny, positive/negative selection, conformational epitopes, and phylodynamics. The time-scale phylogeny suggested that the common ancestors of the 2016 strains VP1 gene and RdRp region diverged in 2006 and 1999, respectively, and that the 2016 strain was the progeny of a pre-2016 GII.2. The evolutionary rates of the VP1 gene and RdRp region were around 10-3 substitutions/site/year. Amino acid substitutions (position 341) in an epitope in the P2 domain of 2016 strains were not found in pre-2016 GII.2 strains. Bayesian skyline plot analyses showed that the effective population size of the VP1 gene in GII.2 strains was almost constant for those 50 years, although the number of patients with NoV GII.2 increased in 2016. The 2016 strain may be involved in future outbreaks in Japan and elsewhere.

Phylogeny and Immunoreactivity of Norovirus GII.P16-GII.2, Japan, 2016-17.

During the 2016-17 winter season in Japan, human norovirus GII.P16-GII.2 strains (2016 strains) caused large outbreaks of acute gastroenteritis. Phylogenetic analyses suggested that the 2016 strains derived from the GII.2 strains detected during 2010-12. Immunochromatography between 2016 strains and the pre-2016 GII.2 strains showed similar reactivity.

A method for detecting rash and fever illness-associated viruses using multiplex reverse transcription polymerase chain reaction.

In this study, a new multiplex RT-PCR method for detecting various viral genes in patients with rash and fever illnesses (RFIs) was constructed. New primer sets were designed for detection of herpes simplex viruses 1 and 2 (HSV1 and 2), and Epstein-Barr virus (EBV). The newly designed and previously reported primer sets were used to detect 13 types of RFI-associated viruses by multiplex RT-PCR assay systems. Moreover, to eliminate non-specific PCR products, a double-stranded specific DNase was used to digest double-stranded DNA derived from the templates in clinical specimens. RFI-associated viruses were detected in 77.0% of the patients (97/126 cases) by the presented method, multiple viruses being identified in 27.8% of the described cases (35/126 cases). Detected viruses and clinical diagnoses were compatible in 32.5% of the patients (41/126 cases). Sensitivity limits for these viruses were estimated to be 101 -103 copies/assay. Furthermore, non-specific PCR products were eliminated by a double-stranded specific DNase with no influence on sensitivity. These results suggest that this method can detect various RFI-associated viruses in clinical specimens with high sensitivity and specificity.

Inhibition of phosphatidylinositide 3-kinase ameliorates antiproliferation by benzyl isothiocyanate in human colon cancer cells.

In the present study, we clarified the role of phosphatidylinositide 3-kinase (PI3K) in antiproliferation induced by benzyl isothiocyanate (BITC) in human colorectal cancer cells. BITC simultaneously activated the PI3K/Akt/forkhead box O (FoxO) pathway, whereas it significantly inhibited the proliferation in human colorectal cancer cells. Inhibitory experiments using a PI3K selective inhibitor, LY294002 or NVP-BEZ235, significantly enhanced the BITC-induced antiproliferation and apoptotic cell population with the attenuation of the BITC-induced activation of the PI3K/Akt/FoxO survival pathway. Furthermore, BITC enhanced the insulin-activated PI3K/Akt/FoxO pathway, possibly through its inhibition of the protein tyrosine phosphatase 1B enzymatic activity. Taken together, these results suggested that the PI3K/Akt/FoxO pathway negatively regulates the BITC-induced antiproliferation in human colorectal cancer cells.

Identification of Datura Species Involved in a Food-Poisoning Case Using LC-MS/MS and DNA Barcording.

A food-poisoning case due to eating the roots of Datura occurred in Kawasaki City, Japan in 2014. The Datura plant was mistakenly collected instead of burdock in a domestic garden. The roots of these plants are quite similar to each other. We presumed that the specimen was the root of Datura, but it was difficult to classify it only from the morphology. Using LC-MS/MS, we detected atropine and scopolamine from the remaining plant specimen. Therefore, we applied the DNA barcoding method. The results showed that the specimen was classified into Solanaceae family, but not Asteraceae family. Thus, the specimen was confirmed to be Datura species based on both chemical and genetic analyses.

Complete Genome Sequence of a Recombinant GII.P16-GII.4 Norovirus Detected in Kawasaki City, Japan, in 2016.

A recombinant norovirus, GII.P16-GII.4_Sydney2012, was first detected from nine patients with gastroenteritis in Kawasaki City, Japan, in 2016. The viral genome showed nucleotide sequence identities of 95.1% and 97.2% to the closest strains in the regions of 5' terminus to ORF1 and ORF2 to 3' terminus, respectively.

Utility of cytopathological specimens and an automated image analysis for the evaluation of HER2 status and intratumor heterogeneity in breast carcinoma.

OBJECTIVES: Although updated HER2 testing guidelines have been improved by a collaboration between the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) in 2013, HER2 evaluation is still problematic because of issues involving CEP17 polysomy, heterogeneity, and HER2 score 2+ cases. The aim of this retrospective study was to evaluate the relationship between HER2 gene heterogeneity, or so called CEP17 polysomy, using breast carcinoma cells sampled by scraping and the IHC score graded by automated image analysis using whole slide image. MATERIAL AND METHODS: We randomly selected 23 breast carcinoma cases with a HER2 score 0, 24 cases with a HER2 score 1+, 24 cases with HER2 score 2+, and 23 cases with HER2 score 3+ from the records of patients with breast cancer at Hiroshima University Hospital. We compared the results of fluorescent in situ hybridization (FISH) using formalin-fixed, paraffin-embedded (FFPE) tissues and cytological samples and compared the HER2 score calculated using an automated image analysis using wholly scanned slide images and visual counting. RESULTS: We successfully performed the FISH assay in 78 of 94 cases (83%) using FFPE tissues and in all 94 (100%) cases using cytological samples. Frequency of both HER2 amplification and CEP17 polysomy was higher when cytological samples were used than when FFPE tissue was used. Frequency of HER2 heterogeneity using cytological samples was higher that than using FFPE tissue, except for the IHC score 3+ cases. CONCLUSIONS: When assessment of HER2 status based on FISH using FFPE tissue cannot be accomplished, FISH using cytological samples should be considered. When intensity of HER2 is heterogeneous in the tumor tissue, particularly in cases regarded as score 2+, they should be evaluated by automated image analysis using the whole slide image.

Different training status may alter the continuous blood glucose kinetics in self-paced endurance running.

The main purpose of the systemic energy metabolism is to provide a source of energy, mainly glucose, for the brain; therefore, blood glucose levels would be expected to correlate with exercise performance. The individual training status may also affect the blood glucose levels. The aim of the present study was to assess the association between blood glucose levels and running velocity during prolonged running in athletes with different training statuses. Two female college athletes, a triathlete and a tennis player, ran a course that was 247.4 m in circumference for 5 h while wearing a continuous glucose monitoring system. Blood was obtained at time-points of -1, 1, 3 and 5 h. The athletes had free access to food and fluids throughout the run. The athletes ran at almost the same pace without a sudden decrease in pace. The blood glucose levels increased and remained high in the triathlete, whereas the tennis player remained hypoglycemic throughout the run. Carbohydrate ingestion did not affect the blood glucose levels. The magnitude of hormonal changes, e.g. insulin, adrenaline and cortisol, was greater in the tennis player. The blood glucose concentration did not correlate with the running velocity or the carbohydrate ingestion; however, a discrepancy in blood glucose transition was observed between the triathlete and the tennis player, indicating a possible association between the adaptation to endurance exercise and the blood glucose kinetics during prolonged running.

Discordant HER2 status between primary breast carcinoma and recurrent/metastatic tumors using fluorescence in situ hybridization on cytological samples.

OBJECTIVE: The aim of this study was to show the usefulness of examining HER2 status on fluorescence in situ hybridization using cytological samples taken from recurrent/metastatic tumors. METHODS: One hundred freshly aspirated or scraped cytological samples were obtained from locoregional recurrences or distant metastases. Fluorescence in situ hybridization assay for HER2 amplification was performed on both these samples and the formalin-fixed, paraffin-embedded tissues of the paired primary tumors of breast cancer, and the relationships between various clinico-pathological factors and HER2 amplification of both tumors were examined. RESULTS: A change in HER2 status was observed in nine cases (9%): six cases (6%) underwent a positive-to-negative conversion in HER2 status and three cases (3%) underwent a negative-to-positive conversion in HER2 status. A positive-to-negative conversion of HER2 status was noted in 4 (36%) of 11 'luminal-B' cases. The change in HER2 status in recurrent or metastatic tumor was noted in more cases treated with drug therapy than in those with no drug therapy (P < 0.05; Fisher's exact probability). Although the time to relapse was 3 years or more in three cases showing a negative-to-positive conversion in HER2 status, the time to relapse was less than 3 years in six cases showing a positive-to-negative conversion (P < 0.05; Fisher's exact probability). CONCLUSIONS: HER2 examination on fluorescence in situ hybridization using fine-needle aspiration cytology samples of tumors in recurrent/metastatic sites or disseminated tumor cells in effusion is beneficial, particularly when the primary tumor is not suitable for the testing of HER2 status or negative for HER2 amplification, because aspiration using a needle is technically feasible and not as traumatic as biopsy.

α-Tocopherol sensitizes human leukemia HL-60 cells to apoptosis induced by benzyl isothiocyanate.

Pre-treatment with α-tocopherol (α-Toc) potentiated cytotoxicity induction by benzyl isothiocyanate (BITC). Biochemical events related to apoptosis, such as DNA ladder formation and caspase-3 activation, were also enhanced by α-Toc. These results suggest a significant role of the caspase-3 pathway in apoptosis induction regulated by α-Toc in combination.

[Sequential changes in inflammatory and stress responses during 24-hour running].

Running for an extended period of time can cause severe stress on the body, subsequently damaging skeletal muscle and resulting in changes in blood components. However, few reports have examined vital responses during and after running. This study analyzed inflammatory responses during and after running and changes in stress responses as determined by serial changes in blood components. Venous blood was obtained before starting, 6 h after starting, 12 h after starting, and immediately after finishing 24 h of continuous running. Samples were analyzed for high-sensitivity C-reactive protein (hsCRP), pentraxin 3 (ptx3), white blood cells (WBC), myoglobin, creatine kinase (CK), and hormones. Diet and physical activity were standardized 24 h before and after running. Subjects comprised 16 men who agreed to participate in experimental running on November 8 and 9, 2008, at Tokyo Gakugei University. Mean running distance was 151.32 +/- 32.1 km (range, 83.6-210.0 km) in 24 h. A significant increase in hsCRP was seen from 12 h after starting to completion. Compared to hsCRP, ptx3 gradually increased from before starting to after completion, showing a significant difference between pre and post-run ptx3 levels. WBC count increased significantly until 6 h after starting. Neutrophils in leukocytosis increased significantly during the first 6 h. Eosinophils decreased significantly over the course of the 24 h. Cortisol increased, and testosterone decreased significantly from 6 h after starting. Dehydroepiandrosterone sulfate (DHEA-S), myoglobin, and CK increased over the course of the 24 h. Reactive oxygen metabolites (d-ROMs) changed within the normal range though there was a significant decrease, and biological anti-oxidant potential (BAP) stabilized. Active natural killer cells decreased significantly after 24 h running. Biopyrrin (BPn) increased significantly. Changes in stress oxide were small both during and after running, and adaptation for antioxidation was good. DHEAS, a biomarker of aging, was found to increase over the course of the 24 h, suggesting that controlling decreases in DHEA-S may be possible using exercise, particularly in males. The key finding was that DHEA S levels tended to increase with continuous aerobic exercise.

Efficacy of Brazilian Propolis against Herpes Simplex Virus Type 1 Infection in Mice and Their Modes of Antiherpetic Efficacies.

Ethanol extracts (AF-06, 07, and 08, 10 mg/kg) of Brazilian propolis were administered orally to cutaneously herpes simplex virus type 1 (HSV-1)-infected mice three times daily on days 0 to 6 after infection to evaluate their efficacies against HSV-1 infection and significantly limited development of herpetic skin lesions. AF-07 and 08 significantly reduced virus titers in brain and/or skin on day 4 without toxicity, but AF-08 had no anti-HSV-1 activity in vitro. AF-06 and 08 significantly enhanced delayed-type hypersensitivity (DTH) to inactivated HSV-1 antigen in infected mice. Oral AF-08-administration significantly augmented interferon (IFN)-γ production by HSV-1 antigen from splenocytes of HSV-1-infected mice, while direct exposure of splenocytes of infected mice to AF-06 significantly elevated IFN-γ production in vitro. Thus, AF-08 might have components that are active in vivo even after oral administration and those of AF-06 might be active only in vitro. Because DTH is a major host defense for intradermal HSV-1 infection, augmentation of DTH response by AF-06 or 08, directly or indirectly, respectively, may contribute to their efficacies against HSV-1 infection. In addition, AF-06 and 07 possibly contain anti-HSV-1 components contributing to their efficacies. Such biological activities of Brazilian propolis may be useful to analyze its pharmacological actions.

Antiviral activities of diarylheptanoids isolated from Alpinia officinarum against respiratory syncytial virus, poliovirus, measles virus, and herpes simplex virus type 1 in vitro.

Alpinia officinarum has been used as a folk medicine and contains diarylheptanoids that have various biological activities. However, their antiviral activities are less elucidated. We examined the antiviral activities of nine diarylheptanoids isolated from A. officinarum against respiratory syncytial virus (RSV), poliovirus, measles virus, and herpes simplex virus type 1 (HSV-1) using a plaque reduction assay. The 50% inhibitory concentrations of seven of the nine diarylheptanoids for RSV were moderately but significantly lower than their 50% cytotoxic concentrations, as determined by a trypan blue exclusion assay. Four diarylheptanoids with anti-RSV activity also showed anti-poliovirus and anti-measles virus activities and three of the four exhibited anti-HSV-1 activity. Thus, seven of the nine diarylheptanoids examined exhibited potential antiviral activity against RSV, and most of the diarylheptanoids with anti-RSV activity, including two diarylheptanoids without anti-RSV activity, were effective against poliovirus, measles virus, and/or HSV-1 in vitro. Diarylheptanoids were suggested to have a broad spectrum of antiviral activity.

In vitro and in vivo anti-influenza virus activity of diarylheptanoids isolated from Alpinia officinarum.

BACKGROUND: Diarylheptanoids (AO-0002 [7-(4''-hydroxy-3''-methoxyphenyl)-1-phenyl-4E-hepten-3-one] and AO-0011 [(5S)-5-hydroxy-7-(4''-hydroxyphenyl)-1-phenyl-3-heptanone]) isolated from Alpinia officinarum have been reported to exhibit anti-influenza virus activity in vitro. Hence, efficacies against influenza virus infection and the mode of antiviral action were evaluated in vivo and in vitro, respectively. METHODS: In a murine influenza virus infection model, diarylheptanoids were orally administered three times daily to mice infected with influenza A/PR/8/34 virus for 6 days after infection. AO-0002 was examined for its antiviral activity against the wild types of influenza viruses A/PR/8/34 (H1N1), oseltamivir-resistant A/PR/8/34 (H1N1), A/Bangkok/93/03 (H1N1), A/Ishikawa/7/82 (H3N2), A/Fukushima/13/43 (H3N2), B/Singapore/222/79 and B/Fukushima/15/93 in plaque reduction or yield reduction assays. The mode of anti-influenza virus action was assessed by a virus adsorption assay, immunofluorescence assay of viral antigens, and inhibition of viral messenger RNA synthesis using real-time reverse transcriptase PCR. RESULTS: AO-0002 at 100 mg/kg was significantly effective in reducing the body weight loss and prolonging survival times of infected mice without toxicity, but AO-0011 was not. AO-0002 at 30 and 100 mg/kg significantly reduced virus titres in bronchoalveolar lavage fluids of the lungs on days 3 and 6 after infection. AO-0002 exhibited anti-influenza virus activity against all viruses used, including the oseltamivir-resistant strain in vitro. The compound had no effect on virus adsorption or invasion into cells, but dose-dependently suppressed the expression of viral messenger RNA and antigens. CONCLUSIONS: AO-0002 was suggested to have a different anti-influenza virus action to that of oseltamivir and was verified to show anti-influenza activity in vitro and in vivo.