Collection and transportation of SARS-CoV-2 and influenza A virus diagnostic samples: Optimizing the usage of guanidine-based chaotropic salts for enhanced biosafety and viral genome preservation.

Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.

Virucidal Activities of Acidic Electrolyzed Water Solutions with Different pH Values against Multiple Strains of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a threat to human health. Acidic electrolyzed water (AEW) has recently been suggested to demonstrate virucidal activity. Many types of AEW with different pH values, generated by the electrolysis of different chemicals, such as sodium chloride, potassium chloride, and hydrochloric acid, are commercially available. In this study, we compared the virucidal activities of these types of AEW against SARS-CoV-2, including the ancestral strain and variant Alpha, Beta, Gamma, Delta, and Omicron strains. Virus solution (viral titer, 6.9 log10 50% tissue culture infective dose [TCID50]/mL) was mixed with AEW (free available chlorine concentration, 34.5 ppm) at mixing ratios of 1:9, 1:19, and 1:49. At mixing ratios of 1:9 and 1:19, AEW with a pH of 2.8 showed stronger virucidal activities than AEW with a pH of 4.1 to 6.5 against the SARS-CoV-2 ancestral strain in 20 s. From the strongest to the weakest virucidal activity, the AEW pH levels were as follows: pH 2.8, pH 4.1 to 5.4, pH 6.4 to 6.5. At a ratio of 1:49, the viral titers of viruses treated with all AEW solutions at pH 2.8 to 6.5 were almost below the detection limit, which was 1.25 log10 TCID50/mL. The virus inactivation efficiency of AEW was reduced in the presence of fetal bovine serum and other substances contained in the virus solution used in this study. AEW with pH values of 2.8 to 6.5 showed virucidal activity against all of the tested SARS-CoV-2 strains, including the ancestral and variant strains. These results provide useful knowledge for the effective application of AEW as a SARS-CoV-2 disinfectant. IMPORTANCE Acidic electrolyzed water (AEW) demonstrates virucidal activity against multiple viruses. Since AEW exhibits low toxicity, is inexpensive, and is environmentally friendly, it can be a useful disinfectant against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the pH values of currently available AEW products vary, the impact of different pH values on SARS-CoV-2 inactivation has not previously been evaluated in detail. In this study, we compared the virucidal activities of multiple AEW solutions with different pH values, under the same experimental conditions. We found that AEW solutions with lower pH values demonstrated more potent virucidal activity. Also, we showed that the extent of virus inactivation by the AEW was based on the balance of the abundance of free available chlorine, virus, and other organic substances in the mixture. AEW exhibited rapid virucidal activity against multiple SARS-CoV-2 strains. This study demonstrated the usefulness of AEW as a disinfectant which can be applied to the inactivation of SARS-CoV-2.

Rapid Virucidal Activity of Japanese Saxifraga Species-Derived Condensed Tannins against SARS-CoV-2, Influenza A Virus, and Human Norovirus Surrogate Viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and norovirus are global threats to human health. The application of effective virucidal agents, which contribute to the inactivation of viruses on hands and environmental surfaces, is important to facilitate robust virus infection control measures. Naturally derived virucidal disinfectants have attracted attention owing to their safety and eco-friendly properties. In this study, we showed that multiple Japanese Saxifraga species-derived fractions demonstrated rapid, potent virucidal activity against the SARS-CoV-2 ancestral strain and multiple variant strains, IAV, and two human norovirus surrogates: feline calicivirus (FCV) and murine norovirus (MNV). Condensed tannins were identified as active chemical constituents that play a central role in the virucidal activities of these fractions. At a concentration of 25 µg/mL, the purified condensed tannin fraction Sst-2R induced significant reductions in the viral titers of the SARS-CoV-2 ancestral strain, IAV, and FCV (reductions of ≥3.13, ≥3.00, and 2.50 log10 50% tissue culture infective doses [TCID50]/mL, respectively) within 10 s of reaction time. Furthermore, at a concentration of 100 µg/mL, Sst-2R induced a reduction of 1.75 log10 TCID50/mL in the viral titers of MNV within 1 min. Western blotting and transmission electron microscopy analyses revealed that Sst-2R produced structural abnormalities in viral structural proteins and envelopes, resulting in the destruction of viral particles. Furthermore, Saxifraga species-derived fraction-containing cream showed virucidal activity against multiple viruses within 10 min. Our findings indicate that Saxifraga species-derived fractions containing condensed tannins can be used as disinfectants against multiple viruses on hands and environmental surfaces. IMPORTANCE SARS-CoV-2, IAV, and norovirus are highly contagious pathogens. The use of naturally derived components as novel virucidal/antiviral agents is currently attracting attention. We showed that fractions from extracts of Saxifraga species, in the form of a solution as well as a cream, exerted potent, rapid virucidal activities against SARS-CoV-2, IAV, and surrogates of human norovirus. Condensed tannins were found to play a central role in this activity. The in vitro cytotoxicity of the purified condensed tannin fraction at a concentration that exhibited some extent of virucidal activity was lower than that of 70% ethanol or 2,000 ppm sodium hypochlorite solution, which are popular virucidal disinfectants. Our study suggests that Saxifraga species-derived fractions containing condensed tannins can be used on hands and environmental surfaces as safe virucidal agents against multiple viruses.

Aberrant PD-L1 expression through 3'-UTR disruption in multiple cancers.

Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3' region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3'-untranslated region (UTR). Disruption of the Pd-l1 3'-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3'-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.

Application of Copper Iodide Nanoparticle-Doped Film and Fabric To Inactivate SARS-CoV-2 via the Virucidal Activity of Cuprous Ions (Cu+).

As a result of the novel coronavirus disease 2019 pandemic, strengthening control measures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an urgent global issue. In addition to antiviral therapy and vaccination strategies, applying available virucidal substances for SARS-CoV-2 inactivation is also a target of research to prevent the spread of infection. Here, we evaluated the SARS-CoV-2 inactivation activity of a copper iodide (CuI) nanoparticle dispersion, which provides Cu+ ions having high virucidal activity, and its mode of actions. In addition, the utility of CuI-doped film and fabric for SARS-CoV-2 inactivation was evaluated. The CuI dispersion exhibited time-dependent rapid virucidal activity. Analyses of the modes of action of CuI performed by Western blotting and real-time reverse transcription-PCR targeting viral proteins and the genome revealed that CuI treatment induced the destruction of these viral components. In this setting, the indirect action of CuI-derived reactive oxygen species contributed to the destruction of viral protein. Moreover, the CuI-doped film and fabric demonstrated rapid inactivation of the SARS-CoV-2 solution in which the viral titer was high. These findings indicated the utility of the CuI-doped film and fabric as anti-SARS-CoV-2 materials for the protection of high-touch environmental surfaces and surgical masks/protective clothes. Throughout this study, we demonstrated the effectiveness of CuI nanoparticles for inactivating SARS-CoV-2 and revealed a part of its virucidal mechanism of action. IMPORTANCE The COVID-19 pandemic has caused an unprecedented number of infections and deaths. As the spread of the disease is rapid and the risk of infection is severe, hand and environmental hygiene may contribute to suppressing contact transmission of SARS-CoV-2. Here, we evaluated the SARS-CoV-2 inactivation activity of CuI nanoparticles, which provide the Cu+ ion as an antiviral agent, and we provided advanced findings of the virucidal mechanisms of action of Cu+. Our results showed that the CuI dispersion, as well as CuI-doped film and fabric, rapidly inactivated SARS-CoV-2 with a high viral titer. We also demonstrated the CuI's virucidal mechanisms of action, specifically the destruction of viral proteins and the genome by CuI treatment. Protein destruction largely depended on CuI-derived reactive oxygen species. This study provides novel information about the utility and mechanisms of action of promising virucidal material against SARS-CoV-2.

Non-competitive fluorescence polarization immunoassay for detection of H5 avian influenza virus using a portable analyzer.

Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 µL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.

Comparison of the SARS-CoV-2-inactivating activities of the differently manufactured hypochlorous acid water products with various pH.

The use of effective disinfectants is a key method of controlling the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hypochlorous acid water (HAW) has a broad spectrum of virucidal activities. We previously reported that acidic electrolyzed water, one of the HAW products, had potent SARS-CoV-2-inactivating activity and showed promise as a disinfectant. However, different manufacturing methods have produced several HAW products with various pH values. Here, we compared the SARS-CoV-2-inactivating activities of various HAW products. At sufficiently high volume and residual chlorine concentration (RCC), the HAW products inactivated SARS-CoV-2 efficiently regardless of pH or manufacturing method. However, although HAW products at pH 5.0-6.4 maintained high RCC and sustained virucidal activity for 21 days, the RCC rapidly decreased in HAW products at pH ≤ 3.0. Our results may guide in choosing appropriate HAW products for different usage situations.

Usefulness of a target sample check illuminator in the detection of target specimens in endoscopic ultrasound-guided fine-needle biopsy samples: Multicenter prospective study.

OBJECTIVES: Endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) uses a thin needle, rendering unclear whether the collected sample contains pathological evidence. We examined the usefulness of our target sample check illuminator (TSCI) through a multicenter prospective trial. METHODS: We included 52 consecutive patients. After assessing EUS-FNB samples by conventional (visual observation) and TSCI methods, we evaluated consistency with the histopathological diagnosis. We compared the target sample confirmation rate between conventional and TSCI methods and evaluated the diagnostic ability separately. RESULTS: Comparison between the conventional and TSCI methods revealed the following: (i) for all cases: sensitivity, 51.0% (25/49) vs. 95.9% (47/49) (P = 0.001); specificity, 100% (3/3) vs. 66.7% (2/3); positive predictive value (PPV), 100% (25/25) vs. 97.9% (47/48); and negative predictive value (NPV), 11.1% (3/27) vs. 50.0% (2/4) (P = 0.002); (ii) for pancreatic masses: sensitivity, 28.0% (7/25) vs. 96.0% (24/25) (P < 0.001); specificity, 100% (2/2) vs. 100% (2/2); PPV, 100% (7/7) vs. 100% (24/24); and NPV, 10.0% (2/20) vs. 66.7% (2/3) (P < 0.001) (the TSCI method showed significantly better sensitivity and NPV than the conventional method); and (iii) for lymph node tumors: sensitivity, 75.0% (18/24) vs. 95.8% (23/24) (P = 0.025); specificity, 100% (1/1) vs. 0% (0/1); PPV, 100% (18/18) vs. 95.8% (23/24); and NPV, 14.3% (1/7) vs. 0% (0/1). CONCLUSIONS: The TSCI improved the sensitivity, NPV, and accuracy of target sample confirmation for pancreatic mass EUS-FNB. Although the proportion of samples not including a target region was quite low, which could strongly influence our results, the TSCI method can be used in EUS-FNB when rapid on-site evaluation cannot be performed. (A multicenter prospective study for the utility of a target sample check illuminator, Clinical Trial ID: UMIN000023349).

Severe Acute Respiratory Syndrome Coronavirus-2 Inactivation Activity of the Polyphenol-Rich Tea Leaf Extract with Concentrated Theaflavins and Other Virucidal Catechins.

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is producing a large number of infections and deaths globally, the development of supportive and auxiliary treatments is attracting increasing attention. Here, we evaluated SARS-CoV-2-inactivation activity of the polyphenol-rich tea leaf extract TY-1 containing concentrated theaflavins and other virucidal catechins. The TY-1 was mixed with SARS-CoV-2 solution, and its virucidal activity was evaluated. To evaluate the inhibition activity of TY-1 in SARS-CoV-2 infection, TY-1 was co-added with SARS-CoV-2 into cell culture media. After 1 h of incubation, the cell culture medium was replaced, and the cells were further incubated in the absence of TY-1. The viral titers were then evaluated. To evaluate the impacts of TY-1 on viral proteins and genome, TY-1-treated SARS-CoV-2 structural proteins and viral RNA were analyzed using western blotting and real-time RT-PCR, respectively. TY-1 showed time- and concentration-dependent virucidal activity. TY-1 inhibited SARS-CoV-2 infection of cells. The results of western blotting and real-time RT-PCR suggested that TY-1 induced structural change in the S2 subunit of the S protein and viral genome destruction, respectively. Our findings provided basic insights in vitro into the possible value of TY-1 as a virucidal agent, which could enhance the current SARS-CoV-2 control measures.

Inactivation Activities of Ozonated Water, Slightly Acidic Electrolyzed Water and Ethanol against SARS-CoV-2.

This study aimed to compare the SARS-CoV-2-inactivation activity and virucidal mechanisms of ozonated water (OW) with those of slightly acidic electrolyzed water (SAEW) and 70% ethanol (EtOH). SARS-CoV-2-inactivation activity was evaluated in a virus solution containing 1%, 20% or 40% fetal bovine serum (FBS) with OW, SAEW or EtOH at a virus-to-test solution ratio of 1:9, 1:19 or 1:99 for a reaction time of 20 s. EtOH showed the strongest virucidal activity, followed by SAEW and OW. Even though EtOH potently inactivated the virus despite the 40% FBS concentration, virus inactivation by OW and SAEW decreased in proportion to the increase in FBS concentration. Nevertheless, OW and SAEW showed potent virucidal activity with 40% FBS at a virus-to-test solution ratio of 1:99. Real-time PCR targeting the viral genome revealed that cycle threshold values in the OW and SAEW groups were significantly higher than those in the control group, suggesting that OW and SAEW disrupted the viral genome. Western blotting analysis targeting the recombinant viral spike protein S1 subunit showed a change in the specific band into a ladder upon treatment with OW and SAEW. OW and SAEW may cause conformational changes in the S1 subunit of the SARS-CoV-2 spike protein.

Acidic electrolyzed water potently inactivates SARS-CoV-2 depending on the amount of free available chlorine contacting with the virus.

Alcohol-based disinfectant shortage is a serious concern in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Acidic electrolyzed water (EW) with a high concentration of free available chlorine (FAC) shows strong antimicrobial activity against bacteria, fungi, and viruses. Here, we assessed the SARS-CoV-2-inactivating efficacy of acidic EW for use as an alternative disinfectant. The quick virucidal effect of acidic EW depended on the concentrations of contained-FAC. The effect completely disappeared in acidic EW in which FAC was lost owing to long-time storage after generation. In addition, the virucidal activity increased proportionately with the volume of acidic EW mixed with the virus solution when the FAC concentration in EW was same. These findings suggest that the virucidal activity of acidic EW against SARS-CoV-2 depends on the amount of FAC contacting the virus.

Rapid detection of anti-H5 avian influenza virus antibody by fluorescence polarization immunoassay using a portable fluorescence polarization analyzer.

A rapid, facile and selective detection of anti-H5 subtype avian influenza virus (AIV) antibody in serum by fluorescence polarization immunoassay (FPIA) was achieved. A fragment of recombinant H5 subtype AIV hemagglutinin was produced and labeled with fluorescein to use it as a labeled antigen in FPIA. This labeled antigen was mixed with anti-AIV sera (H1-H16 subtypes) and FP of the mixture was measured using a portable FP analyzer on a microdevice. It was found that FP increased in proportion to the concentration of anti-H5 AIV antibody (serum) and was significantly higher than FP obtained with the other sera. The selective detection of anti-H5 subtype AIV antibody was confirmed. The required volume of original sample was 2 µL and analysis time was within 20 min. This detection system realizes an efficient on-site diagnosis and surveillance of AIV.

Chicken anemia virus in northern Vietnam: molecular characterization reveals multiple genotypes and evidence of recombination.

Chicken anemia virus (CAV) has a ubiquitous and worldwide distribution in the chicken production industry. Our group previously reported a high seroprevalence of CAV in chickens from northern Vietnam. In the present study, tissue samples collected from a total of 330 broiler and breeder commercial chickens in eleven provinces of northern Vietnam were tested for CAV infection. All samples were collected from clinically suspected flocks and diseased birds. The CAV genome was detected in 157 out of 330 (47.58%) chicken samples by real-time PCR. The rate of CAV genome detection in young chickens at 2-3 weeks of age (61.43%), which had not been previously reported in Vietnam, was significantly higher than that in older chickens at 4-11 (44.83%) and 12-28 (35.71%) weeks of age. For nine representative CAV strains from broiler chickens, analysis of the entire protein-coding region of the viral genome was conducted. Phylogenetic analysis of the VP1 gene indicated that the CAVs circulating in northern Vietnam were divided into three distinct genotypes: II, III, and V. Only one of the nine Vietnamese CAV strains clustered with a vaccine strain (Del-Ros), whereas the other eight strains did not cluster with any vaccine strains. Among the three genotypes, genotype III was most widely found in northern Vietnam and this included three sub-genotypes (IIIa, IIIb, and IIIc). The Vietnamese CAV strains were closely related to the Chinese, Taiwanese, and USA strains. One strain was defined to be of genotype V, which is a newly reported CAV genotype. Moreover, recombination analysis suggests that this novel genotype V was generated by recombination between genotype II and sub-genotype IIIc.

Olive-Derived Hydroxytyrosol Shows Anti-inflammatory Effect without Gastric Damage in Rats.

Hydroxytyrosol (HT) is a simple phenol compound present in olive oil. In a previous in vitro study, we showed that HT downregulated lipopolysaccharide-mediated expression of inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), tumor necrosis factor alpha, and interleukin-1ß, resulting in reduced nitric oxide and prostaglandin E2 production. In the present study, we aimed to determine whether HT suppresses COX-2-induced inflammation in a carrageenan-induced rat paw edema model. Additionally, we compared its activity with those of the selective COX-2 inhibitor, celecoxib for a comparative control, and a representative nonsteroidal anti-inflammatory drug (NSAID), indomethacin for a positive control. HT, celecoxib, and indomethacin significantly suppressed swelling in carrageenan-injected rat paws. Although HT was less effective than celecoxib and indomethacin, it had a delayed onset of action. Moreover, we evaluated whether HT aggravates gastric damage, which is a typical adverse effect associated with NSAIDs and COX-2 inhibitors under low dose aspirin (LDA) treatment, in an aspirin-induced gastric damage rat model. Unlike celecoxib and indomethacin, HT did not cause gastric damage when co-administered with aspirin. Our results indicate that HT exerts a delayed but sustained anti-inflammatory effect against COX-2-mediated inflammation. Finally, the combination of short-acting conventional anti-inflammatory drugs and long-acting HT can be considered a new, safe, and effective anti-inflammatory treatment modality even when continuously administered for a long period under LDA treatment.

Vaccine immunotherapy with ARNAX induces tumor-specific memory T cells and durable anti-tumor immunity in mouse models.

Immunological checkpoint blockade therapies benefit a limited population of cancer patients. We have previously shown that vaccine immunotherapy with Toll-like receptor (TLR)3-adjuvant and tumor antigen overcomes anti-programmed death ligand-1 (PD-L1) resistance in mouse tumor models. In the present study, 4 different ovalbumin (OVA)-expressing tumor cell lines were implanted into syngeneic mice and subjected to anti-tumor immunotherapy using ARNAX and whole OVA protein. ARNAX is a TLR3-specific agonist that does not activate the mitochondrial antiviral-signaling protein (MAVS) pathway, and thus does not induce systemic inflammation. Dendritic cell priming and proliferative CTL were induced by ARNAX + OVA, but complete remission was achieved only in a PD-L1-low cell line of EG7. Addition of anti-PD-L1 antibody to the ARNAX + OVA therapy brought complete remission to another PD-L1-high subline of EG7. Tumor shrinkage but not remission was observed in MO5 in that regimen. We analyzed tumor cells and tumor-infiltrating immune cells to identify factors associated with successful ARNAX vaccine therapy. Tumors that responded to ARNAX therapy expressed high levels of MHC class I and low levels of PD-L1. The tumor-infiltrating immune cells in ARNAX-susceptible tumors contained fewer immunosuppressive myeloid cells with low PD-L1 expression. Combination with anti-PD-L1 antibody functioned not only within tumor sites but also within lymphoid tissues, augmenting the therapeutic efficacy of the ARNAX vaccine. Notably, ARNAX therapy induced memory CD8+ T cells and rejection of reimplanted tumors. Thus, ARNAX vaccine + anti-PD-L1 therapy enabled permanent remission against some tumors that stably present antigens.

Toll-like receptor 3 signal augments radiation-induced tumor growth retardation in a murine model.

Radiotherapy induces anti-tumor immunity by induction of tumor antigens and damage-associated molecular patterns (DAMP). DNA, a representative DAMP in radiotherapy, activates the stimulator of interferon genes (STING) pathway which enhances the immune response. However, the immune response does not always parallel the inflammation associated with radiotherapy. This lack of correspondence may, in part, explain the radiation-resistance of tumors. Additive immunotherapy is expected to revive tumor-specific CTL facilitating radiation-resistant tumor shrinkage. Herein pre-administration of the double-stranded RNA, polyinosinic-polycytidylic acid (polyI:C), in conjunction with radiotherapy, was shown to foster tumor suppression in mice bearing radioresistant, ovalbumin-expressing Lewis lung carcinoma (LLC). Extrinsic injection of tumor antigen was not required for tumor suppression. No STING- and CTL-response was induced by radiation in the implant tumor. PolyI:C was more effective for induction of tumor growth retardation at 1 day before radiation than at post-treatment. PolyI:C targeted Toll-like receptor 3 with minimal effect on the mitochondrial antiviral-signaling protein pathway. Likewise, the STING pathway barely contributed to LLC tumor suppression. PolyI:C primed antigen-presenting dendritic cells in draining lymph nodes to induce proliferation of antigen-specific CTL. By combination therapy, CTL efficiently infiltrated into tumors with upregulation of relevant chemokine transcripts. Batf3-positive DC and CD8+ T cells were essential for therapeutic efficacy. Furthermore, polyI:C was shown to stimulate tumor-associated macrophages and release tumor necrosis factor alpha, which acted on tumor cells and increased sensitivity to radiation. Hence, polyI:C treatment prior to radiotherapy potentially induces tumor suppression by boosting CTL-dependent and macrophage-mediated anti-tumor responses. Eventually, polyI:C and radiotherapy in combination would be a promising therapeutic strategy for radiation-resistant tumors.

Vaccine adjuvant ARNAX promotes mucosal IgA production in influenza HA vaccination.

Adjuvant stimulates pattern-recognition receptors (PRRs) expressed by dendritic cells, which causes immune-enhancing of T lymphocytes. Adjuvant also induces innate immune response in whole-body cells via PRRs to evoke cytokinemia. A cytokine-mediated immune response is important for the systemic protection of a host from microbial infections. Using an influenza subcomponent vaccine in a mouse model, we intranasally administered a TLR3-specific adjuvant ARNAX + HA split vaccine to mice. ARNAX efficiently induced mucosal IgA and systemic IgG production by nasal drop. Moreover, ARNAX + HA simultaneously induced CD8 and CD4 T cell activation. We have previously shown that ARNAX does not induce harmful systemic cytokine production. Thus, our findings indicate that the ARNAX + HA vaccine is a harmless prophylactic vaccine for flu that induces HA-specific T cell activation and IgA/IgG production. These results suggested that ARNAX + antigen enhanced the immune response without inducing inflammatory toxicity for vaccination against infectious diseases.

Adjuvant immunotherapy for cancer: both dendritic cell-priming and check-point inhibitor blockade are required for immunotherapy.

The immune system eliminates advanced cancer when treated with programmed cell death protein-1 (PD-1) or its ligand (PD-L1) blockade, but PD-1 therapy is effective in only ∼20% of patients with solid cancer. The PD-1 antibody mainly acts on the effector phase of cytotoxic T lymphocytes (CTLs) in tumors but induces no activation of the priming phase of antigen-presenting dendritic cells (DCs). It is reasonable that both DC-priming and PD-1/L1 blocking are mandatory for efficient CTL-mediated tumor cytolysis. For DC-priming, a therapeutic vaccine containing Toll-like receptor (TLR) agonists, namely a priming adjuvant, is a good candidate; however, a means for DC-targeting by TLR adjuvant therapy remains to be developed. TLR adjuvants usually harbor cytokine toxicity, which is a substantial barrier against drug approval. Here, we discuss the functional properties of current TLR adjuvants for cancer immunotherapy and introduce a TLR3-specific adjuvant (ARNAX) that barely induces cytokinemia in mouse models.

Efficacy and safety of pancreatic juice cytology by using synthetic secretin in the diagnosis of pancreatic ductal adenocarcinoma.

BACKGROUND AND AIM: Pancreatic ductal adenocarcinoma (PDAC) is difficult to detect in its early stages with the poorest prognosis of all cancers. To improve the prognosis, a precise diagnosis is needed when we suspect PDAC. Although endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA) is a widely accepted modality for the diagnosis of PDAC, its sensitivity is 85-89%, and approximately 10% of PDAC cases cannot be diagnosed. The main causes that interrupt the diagnosis of PDAC by using EUS-FNA are tumor size, presence of a vessel or the main pancreatic duct along the puncture route, and difficulty in withdrawing anticoagulant. Pancreatic juice cytology (PJC), the sensitivity of which is 33.3-65.8%, is a method for the diagnosis of PDAC cases in which carrying out of EUS-FNA is difficult. To diagnose PDAC appropriately, we need to improve the diagnostic ability of PJC. METHODS: We examined PJC using synthetic secretin for 138 cases of pancreatic tumor and pancreatic non-cancerous diseases. RESULTS: Sensitivity of PJC improved from 50.9% to 74.0% as a result of synthetic secretin loading, and 13 PDAC cases that had not been able to be diagnosed with EUS-FNA could be diagnosed pathologically by PJC. Although there were 12 patients with mild pancreatitis (8.7%) as a complication, all were relieved with conservative treatment. CONCLUSION: Adding synthetic secretin to PJC is useful for cases in which it is difficult to carry out EUS-FNA for PDAC.

The TLR3/TICAM-1 signal constitutively controls spontaneous polyposis through suppression of c-Myc in Apc Min/+ mice.

BACKGROUND: Intestinal tumorigenesis is promoted by myeloid differentiation primary response gene 88 (MyD88) activation in response to the components of microbiota in Apc Min/+ mice. Microbiota also contains double-stranded RNA (dsRNA), a ligand for TLR3, which activates the toll-like receptor adaptor molecule 1 (TICAM-1, also known as TRIF) pathway. METHODS: We established Apc Min/+ Ticam1 -/- mice and their survival was compared to survival of Apc Min/+ Myd88 -/- and wild-type (WT) mice. The properties of polyps were investigated using immunofluorescence staining and RT-PCR analysis. RESULTS: We demonstrate that TICAM-1 is essential for suppression of polyp formation in Apc Min/+ mice. TICAM-1 knockout resulted in shorter survival of mice compared to WT mice or mice with knockout of MyD88 in the Apc Min/+ background. Polyps were more frequently formed in the distal intestine of Apc Min/+ Ticam1 -/- mice than in Apc Min/+ mice. Infiltration of immune cells such as CD11b+ and CD8α+ cells into the polyps was detected histologically. CD11b and CD8α mRNAs were increased in polyps of Apc Min/+ Ticam1 -/- mice compared to Apc Min/+ mice. Gene expression of inducible nitric oxide synthase (iNOS), interferon (IFN)-γ, CXCL9 and IL-12p40 was increased in polyps of Apc Min/+ Ticam1 -/- mice. mRNA and protein expression of c-Myc, a critical transcription factor for inflammation-associated polyposis, were increased in polyps of Apc Min/+ Ticam1 -/- mice. A Lactobacillus strain producing dsRNA was detected in feces of Apc Min/+ mice. CONCLUSION: These results imply that the TLR3/TICAM-1 pathway inhibits polyposis through suppression of c-Myc expression and supports long survival in Apc Min/+ mice.