Comprehensive analysis of diel rhythmic expression of the medaka toll-like receptor gene family.

Several immune-related genes, including Toll-like receptors (TLR), are associated with circadian rhythms in mammals. However, information on the circadian rhythmic expression of TLRs in fish is limited. In this study, we aimed to analyze the regulation of diel oscillations in the expression of TLR genes in Japanese medaka (Oryzias latipes). The expression analysis revealed diel expression patterns of tlr1, tlr5m, tlr21, and clock genes (bmal1 and clock1) under a 12 h light:12 h dark cycle. The clock gene response element (E-box) was identified in the transcriptional regulatory regions of tlr1, tlr5m, and tlr21. Moreover, overexpressed bmal1 and clock1 enhanced expression levels of tlr1, tlr5m, and tlr21 in medaka embryo (OLHdrR-e3) cells. The expression of tlr1, tlr5m, and tlr21 was significantly decreased in OLHdrR-e3 after generating a bmal1 knockdown using a morpholino oligo. These results indicate the regulation of the diel rhythmic expression of several fish TLRs by clock genes.

Interleukin-22 induces immune-related gene expression in the gills of Japanese medaka Oryzias latipes.

The cytokine interleukin (IL)-22 has been identified in several fish species; however, its functional significance in the gills of these fish species remains unclear. In this study, we analyzed the expression of proinflammatory cytokines, antimicrobial peptides, and IL-22 binding protein in the gills of wild-type and IL-22-knockout (IL-22 KO) medaka under dextran sulfate sodium-induced inflammation. We also produced medaka recombinant IL-22 (rIL-22) and analyzed the expression of immune-related genes in rIL-22-stimulated primary cell cultures from gills. The il1b, il6, tnfa, and hamp genes were significantly upregulated in wild-type gills upon dextran sulfate sodium stimulation compared with the naïve state but not in IL-22 KO gills. il22bp transcripts were barely detectable in the IL-22 KO medaka gills. However, the expression of il1b, il6, hamp, and il22bp was upregulated in rIL-22-stimulated gill cell culture. These results suggest IL-22 could be involved in immune responses through inflammatory cytokine and antimicrobial peptide production in fish gills.

Postoperative tegafur-uracil for stage I lung adenocarcinoma: first real-world data with an exploratory subgroup analysis.

PURPOSE: The effect of postoperative tegafur-uracil on overall survival (OS) after resection of stage I adenocarcinoma has been shown in clinical trials. The purpose of this study was to investigate whether findings from randomized trials of adjuvant tegafur-uracil are reproducible in a real-world setting. METHODS: A retrospective cohort study was performed using a multi-institutional database that included all patients who underwent complete resection of pathological stage I adenocarcinoma between 2014 and 2016. Survival outcomes for patients managed with and without tegafur-uracil were analyzed using the Kaplan-Meier method and a Cox proportional hazards model for the whole patient cohort and in a selected cohort based on eligibility criteria of a previous randomized trial. Propensity score matching was used to adjust for confounding effects. RESULTS: After propensity score matching, the hazard ratios for OS were 0.57 (95% confidence interval (CI) 0.29-1.14, P = 0.11) in the whole cohort and 0.69 (95% CI 0.32-1.50, P = 0.35) in the selected cohort. CONCLUSIONS: The effects of tegafur-uracil in this retrospective study appear to be consistent with those found in randomized clinical trials. These effects may be maximized in patients aged from 45 to 75 years.

Evolutional perspective and functional characteristics of interleukin-17 in teleosts.

Interleukin (IL)-17 is a proinflammatory cytokine and plays essential roles in adaptive and innate immune responses against bacterial and fungal infections. Especially in mammalian mucosal tissues, it is well known that innate immune responses via IL-17A and IL-17F, such as the production of antimicrobial peptides, are very important for microbiota control. In contrast, interesting insights into the functions of IL-17 have recently been reported in several teleost species, although little research has been conducted on teleost IL-17. In the present review, we focused on current insights on teleost IL-17 and speculated on the different or consensus parts of teleost IL-17 signaling compared to that of mammals. This review focuses on the role of teleost IL-17 in intestinal immunity. We expect that this review will encourage a further understanding of the roles and importance of IL-17 signaling in teleosts.

Epidermal growth factor receptor tyrosine kinase inhibitors as first-line treatment for postoperative recurrent EGFR-mutated lung adenocarcinoma: a multi-institutional retrospective study.

OBJECTIVES: The aim of this study was to analyse the long-term survival outcomes and prognostic factors of patients receiving epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) as first-line treatment for postoperative recurrent EGFR-mutated lung adenocarcinoma. METHODS: Using a multi-institutional database, we performed a retrospective chart review to identify all patients who had undergone complete resection of stage I-III EGFR-mutated lung adenocarcinoma at 11 acute care hospitals between 2009 and 2016 and had received first-line EGFR-TKI treatment for postoperative recurrence. Adverse events, progression-free survival (PFS) and overall survival (OS) were investigated. Survival outcomes were assessed using Kaplan-Meier analysis. Cox proportional hazards models were used to calculate the hazard ratios (HRs) and 95% confidence intervals (CIs) for PFS and OS. RESULTS: The study sample comprised 154 patients with a median age of 69. The total numbers of events were 101 for PFS and 60 for OS. The median PFS and OS were 26.1 and 55.4 months, respectively. In the multivariable analysis, EGFR ex 21 L858R mutation (HR: 1.71, 95% CI: 1.15-2.55) and shorter disease-free intervals (HR: 0.98, 95% CI: 0.96-0.99) were significantly associated with shorter PFS. Age (HR: 1.03, 95% CI: 1.00-1.07), smoking history (HR: 2.31, 95% CI: 1.35-3.94) and pathological N2 disease at the initial surgery (HR: 2.30, 95% CI: 1.32-4.00) were significantly associated with shorter OS. CONCLUSIONS: First-line EGFR-TKI treatment was generally associated with favourable survival outcomes in patients with postoperative recurrent EGFR-mutated lung adenocarcinoma. EGFR ex 21 L858R mutation may be an important prognostic factor for shorter PFS.

Diel rhythm of the inflammatory cytokine il1b in the Japanese medaka (Oryzias latipes) regulated by core components of the circadian clock.

In recent years, studies on circadian control in immunity have been actively conducted in mammals, but little is known about circadian rhythms in the field of fish immunology. In this study, we aimed to analyse the regulation of the diel oscillation of inflammatory cytokine interleukin-1ß (il1b) gene expression by core components of the circadian clock in Japanese medaka (Oryzias latipes). The expression of il1b and clock genes (bmal1 and clock1) in medaka acclimated to a 12:12 light (L): dark (D) cycle showed diel rhythm. Additionally, higher expression of il1b was detected in medaka embryo cells (OLHdrR-e3) overexpressing bmal1 and clock1. A significant decrease in il1b expression was observed in OLHdrR-e3 cells after bmal1 knockdown using morpholino oligos. These changes may be mediated by transcriptional regulation via clock proteins, which target the E-box sequence in the cis-element of il1b as identified using luciferase reporter assays. Moreover, LPS stimulation and pathogenic bacterial infection at different zeitgeber time (ZT) under LD12:12 conditions affected the degree of il1b expression, which showed high and low responsiveness to both immuno-stimulations at ZT2 and ZT14, respectively. These results suggested that fish IL-1ß exhibited diel oscillation regulated by clock proteins, and its responsiveness to immune-stimulation depends on the time of day.

Inhibition of lysozyme lytic activity by Ivy derived from Photobacterium damselae subsp. piscicida.

A pseudotuberculosis pathogen, Photobacterium damselae subsp. piscicida (Pdp), has caused enormous economic damage to yellowtail aquaculture in Japan. The Ivy gene has been discovered in plasmid of Pdp, and it has been proposed that it may help bacteria evade lysozyme-mediated lysis during interaction with an animal host. However, the lysozyme-inhibiting activity of Pdp-derived Ivy (Ivy-Pdp) is unknown, and it is unclear whether it acts as a virulence factor for host biophylaxis. In this study, the inhibitory effect of Ivy-Pdp on lysozyme was evaluated by expressing and purifying the recombinant Ivy-Pdp protein (rIvy-Pdp). The rIvy-Pdp protein inhibited hen egg white lysozyme activity in an rIvy-Pdp-concentration-dependent manner, and its inhibitory effect was similar under different temperature and pH conditions. The serum and skin mucus of the yellowtail (which is the host species of Pdp), Japanese flounder, and Nile tilapia showed bacteriolytic activity. In contrast, the addition of rIvy-Pdp inhibited the lytic activity in the serum of these fish species. In particular, it significantly inhibited lytic activity in the serum and skin mucus of Nile tilapia. On the basis of these results, we suggest that Ivy-Pdp is a temperature- and pH-stable lysozyme inhibitor. Additionally, Ivy-Pdp inhibited the lytic activity of lysozyme, which is involved in host biophylaxis. In summary, we inferred that Ivy-Pdp is an important factor that diminishes the sterilization ability of C-type lysozyme when Pdp infects the host.

Vessel invasion as a predictive factor for recurrence after surgery in stage I lung adenocarcinoma.

BACKGROUND: Patients with early-stage lung cancer who underwent R0 resection often encounter disease recurrence, especially during the early phase; thus, it is deemed vital to determine the predictive factors for recurrence after surgery. In this study, we aimed to identify the independent variables associated with recurrence after complete surgical resection of pathological stage I lung adenocarcinoma. METHODS: We retrospectively reviewed the medical records of 169 patients who underwent pulmonary resection for primary lung adenocarcinoma pathological stage I with curative intent lung cancer surgery from 2015 to December 2018 at our institution for information on the recurrence of the disease. RESULTS: Per the multivariate analysis, the presence of micropapillary pattern and vessel invasion were found to be independent predictors of disease recurrence after surgery (odds ratio [OR]: 9.36, 95% confidence interval [CI]: 2.42-36.2, P = 0.0012; and OR: 4.50, 95% CI: 1.52-13.4, P = 0.0068, respectively). Vessel invasion was also found to be an independent predictor of disease recurrence after surgery within a year (OR 11.4, 95% CI 3.08-42.5, P = 0.0003). CONCLUSIONS: The presence of vessel invasion may help in distinguishing patients with the highest risk of early-phase disease recurrence after surgery. Patients with stage I adenocarcinoma with vessel invasion should undergo intensive surveillance after surgery.

Interleukin-22 Deficiency Contributes to Dextran Sulfate Sodium-Induced Inflammation in Japanese Medaka, Oryzias latipes.

Mucosal tissue forms the first line of defense against pathogenic microorganisms. Cellular damage in the mucosal epithelium may induce the interleukin (IL)-22-related activation of many immune cells, which are essential for maintaining the mucosal epithelial barrier. A previous study on mucosal immunity elucidated that mammalian IL-22 contributes to mucus and antimicrobial peptides (AMPs) production and anti-apoptotic function. IL-22 has been identified in several teleost species and is also induced in response to bacterial infections. However, the roles of IL-22 in teleost immunity and mucus homeostasis are poorly understood. In this study, Japanese medaka (Oryzias latipes) was used as a model fish. The medaka il22, il22 receptor A1 (il22ra1), and il22 binding protein (il22bp) were cloned and characterized. The expression of medaka il22, il22ra1, and il22bp in various tissues was measured using qPCR. These genes were expressed at high levels in the mucosal tissues of the intestines, gills, and skin. The localization of il22 and il22bp mRNA in the gills and intestines was confirmed by in situ hybridizations. Herein, we established IL-22-knockout (KO) medaka using the CRISPR/Cas9 system. In the IL-22-KO medaka, a 4-bp deletion caused a frameshift in il22. To investigate the genes subject to IL-22-dependent regulation, we compared the transcripts of larval medaka between wild-type (WT) and IL-22-KO medaka using RNA-seq and qPCR analyses. The comparison was performed not only in the naïve state but also in the dextran sulfate sodium (DSS)-exposed state. At the transcriptional level, 368 genes, including immune genes, such as those encoding AMPs and cytokines, were significantly downregulated in IL-22-KO medaka compared that in WT medaka in naïve states. Gene ontology analysis revealed that upon DSS stimulation, genes associated with cell death, acute inflammatory response, cell proliferation, and others were upregulated in WT medaka. Furthermore, in DSS-stimulated IL-22-KO medaka, wound healing was delayed, the number of apoptotic cells increased, and the number of goblet cells in the intestinal epithelium decreased. These results suggested that in medaka, IL-22 is important for maintaining intestinal homeostasis, and the disruption of the IL-22 pathway is associated with the exacerbation of inflammatory pathology, as observed for mammalian IL-22.

Deficiency of interleukin-17 receptor A1 induces microbiota disruption in the intestine of Japanese medaka, Oryzias latipes.

The mutual relationship between the intestinal immune system and the gut microbiota has received a great deal of attention. In mammals, interleukin-17A and F (IL-17A/F) are inflammatory cytokines and key regulators of the gut microbiota. However, in teleosts, the function of IL-17A/F in controlling the gut microbiota is poorly understood. We attempted to elucidate the importance of teleost IL-17 signaling in controlling gut microbiota. We previously established a knockout (KO) of IL-17 receptor A (RA) 1, a receptor for IL-17A/F, in the Japanese medaka (Oryzias latipes) using the CRISPR-Cas9 system and performed 16S rRNA-based metagenomic analyses using the anterior and posterior sections of the intestinal tract. The number of observed OTUs in the anterior intestine was significantly decreased in IL-17RA1 KO medaka compared to that in the wild-type (WT). Furthermore, ß-diversity analysis (weighted UniFrac) revealed considerably different bacterial composition in the anterior intestine of IL-17RA1 KO compared to WT, with similar findings in α-diversity. Notably, the pathogen Plesiomonas shigelloides was significantly increased in the posterior intestine of IL-17RA1 KO medaka. These findings indicate that signaling via IL-17RA1 is required to maintain a healthy gut microbiota in teleosts and mammals. The involvement of IL-17RA1 in controlling the gut microbiota has been demonstrated, resulting in microbiome dysbiosis in IL-17RA1 KO medaka.

Identification of two interleukin 17 receptor C (IL-17RC) genes and their binding activities to three IL-17A/F ligands in the Japanese medaka, Oryzias latipes.

In mammals, interleukin (IL)-17 receptor C (IL-17RC) and IL-17RA mediate IL-17A and IL-17F signaling to produce mucin, antimicrobial peptides, and maintain healthy intestinal flora. However, IL-17RC signaling in fish remains unclear. In this study, three il17rc transcripts (il17rca1, il17rca2, and il17rcb) from the Japanese medaka (Oryzias latipes) were cloned; il17rca1 and il17rca2 mRNAs were alternatively spliced from il17rca pre-mRNA as transcript variants. The il17rca and il17rcb genes were located on chromosomes 7 and 5, respectively. Teleost clades containing medaka il17rca and il17rcb clustered separately from the tetrapod clade. In adult tissues, il17rca1 expression was significantly higher than il17rca2 and il17rcb. Conversely, il17rcb expression was significantly higher in embryos and larvae. These expression patterns changed following infection with Edwardsiella piscicida and Aeromonas hydrophila. Furthermore, an immunoprecipitation assay using recombinant IL-17RCs and rIL-17A/Fs suggested that, in teleosts, three ligands could function in signaling through two IL-17RCs.

A defective interleukin-17 receptor A1 causes weight loss and intestinal metabolism-related gene downregulation in Japanese medaka, Oryzias latipes.

In the intestine, the host must be able to control the gut microbiota and efficiently absorb transiently supplied metabolites, at the risk of enormous infection. In mammals, the inflammatory cytokine interleukin (IL)-17A/F is one of the key mediators in the intestinal immune system. However, many functions of IL-17 in vertebrate intestines remain unclarified. In this study, we established a gene-knockout (KO) model of IL-17 receptor A1 (IL-17RA1, an IL-17A/F receptor) in Japanese medaka (Oryzias latipes) using genome editing technique, and the phenotypes were compared to wild type (WT) based on transcriptome analyses. Upon hatching, homozygous IL-17RA1-KO medaka mutants showed no significant morphological abnormality. However, after 4 months, significant weight decreases and reduced survival rates were observed in IL-17RA1-KO medaka. Comparison of gene-expression patterns in WT and IL-17RA1-KO medaka revealed that various metabolism- and immune-related genes were significantly down-regulated in IL-17RA1-KO medaka intestine, particularly genes related to mevalonate metabolism (mvda, acat2, hmgcs1, and hmgcra) and genes related to IL-17 signaling (such as il17c, il17a/f1, and rorc) were found to be decreased. Conversely, expression of genes related to cardiovascular system development, including fli1a, sox7, and notch1b in the anterior intestine, and that of genes related to oxidation-reduction processes including ugp2a, aoc1, and nos1 in posterior intestine was up-regulated in IL-17RA1-KO medaka. These findings show that IL-17RA regulated immune- and various metabolism-related genes in the intestine for maintaining the health of Japanese medaka.

Inflammasomes in Teleosts: Structures and Mechanisms That Induce Pyroptosis during Bacterial Infection.

Pattern recognition receptors (PRRs) play a crucial role in inducing inflammatory responses; they recognize pathogen-associated molecular patterns, damage-associated molecular patterns, and environmental factors. Nucleotide-binding oligomerization domain-leucine-rich repeat-containing receptors (NLRs) are part of the PRR family; they form a large multiple-protein complex called the inflammasome in the cytosol. In mammals, the inflammasome consists of an NLR, used as a sensor molecule, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) as an adaptor protein, and pro-caspase1 (Casp1). Inflammasome activation induces Casp1 activation, promoting the maturation of proinflammatory cytokines, such as interleukin (IL)-1ß and IL-18, and the induction of inflammatory cell death called pyroptosis via gasdermin D cleavage in mammals. Inflammasome activation and pyroptosis in mammals play important roles in protecting the host from pathogen infection. Recently, numerous inflammasome-related genes in teleosts have been identified, and their conservation and/or differentiation between their expression in mammals and teleosts have also been elucidated. In this review, we summarize the current knowledge of the molecular structure and machinery of the inflammasomes and the ASC-spec to induce pyroptosis; moreover, we explore the protective role of the inflammasome against pathogenic infection in teleosts.

Identification and expression of phospholipase A2 genes related to transcriptional control in the interleukin-17A/F1 pathway in the intestines of Japanese medaka Oryzias latipes.

Phospholipase A2 (PLA2), a phospholipid hydrolase, has recently attracted attention owing to its broad functionality. Immunological evidence has revealed increased susceptibility to infectious diseases and immunodeficiency in knockout (KO) mice of several pla2 genes. However, no progress has been made in terms of immunological research on any pla2 gene in fish. In this study, we focused on the intestinal immune responses of fish PLA2s. The full-length open reading frames of pla2g1b, pla2g3, pla2g10, pla2g12b1, pla2g12b2, and pla2g15 cDNAs were cloned in Japanese medaka (Orizias latipes), and their gene expressions were quantified by real-time PCR (qPCR) and in situ hybridization (ISH). Characterization of pla2 genes revealed a functional domain and three-dimensional structure similar to the mammalian counterparts. In addition, expression of pla2g1b, pla2g12b1, and pla2g12b2 was extremely high in Japanese medaka intestines. ISH detected strong expression of pla2g1b mRNAs in the basal muscle layer, and pla2g12b1 and pla2g12b2 mRNAs were detected in the epithelial cells. In the medaka exposed to Edwardsiella piscicida, pla2g12b1, pla2g12b2 and pla2g15 were significantly induced in the anterior and posterior intestines, and pla2g1b was upregulated in the anterior intestine. Furthermore, pla2g1b, pla2g3, pla2g10, and pla2g12b2 were significantly downregulated in the IL-17A/F1 KO medaka compared to those in wild-type medaka. These results suggest that these PLA2s are involved in intestinal immunity in teleosts.

Characterization and expression analysis of tandemly-replicated asc genes in the Japanese medaka, Oryzias latipes.

ASC is a component of the inflammasome playing crucial roles in the inflammatory response. In mammals, ASC induces pyroptosis and inflammatory cytokine production. In this study, three asc genes (asc1, asc2, and asc3) from the Japanese medaka (Oryzias latipes) were identified and characterized. These asc genes were tandem replicates on chromosome 16, and their exon-intron structures differed between them. All three ASCs conserved the pyrin and caspase-recruitment domains, which are important for inflammasome formation. In phylogenetic analysis, all ASCs clustered with those of other teleosts. The asc1 expression levels were significantly higher in several organs than those of asc2 and asc3, suggesting that asc1 may act as a dominant asc in the Japanese medaka. Expression of the three asc genes showed different patterns during Aeromonas hydrophila and Edwardsiella piscicida infections. Furthermore, their expression was adequately down-regulated in the medaka fin-derived cells stimulated with ATP for 12 h, while asc2 expression was statistically up-regulated after nigericin stimulation for 24 h. Moreover, the expression of asc2 and asc3 was significantly higher in the skin of ASC-1-knockout medaka than in that of the wild type medaka during A. hydrophila infection.

Diel Variation in CC Chemokine Gene Expression in the Japanese Pufferfish Takifugu rubripes.

CC chemokines are key molecules in the regulation of leukocyte trafficking to the site of injury, infection, or inflammation. In recent years, some mammalian chemokines have been shown to exhibit rhythmic expression, regulated by clock genes. However, the rhythmic expression of chemokines in teleost fish remains unknown. In the present study, the diel variation of teleost CC chemokine genes was investigated using the model fish, Fugu (Takifugu rubripes). Diel variation analysis revealed that clock (bmal1, clock1, per2, rorα, and rev-erbß) and CC chemokine (ccl18l, ccl19, and ccl25l) genes show diel expression under 12:12 light-dark cycle (LD12:12) conditions. CC chemokine genes, which exhibit diel expression, contain RORE (ccl18l, ccl19, ccl25l) and/or E-box (ccl25l) motifs in their transcription regulatory region. Moreover, in vitro head kidney stimulation with lipopolysaccharide (LPS) at different zeitgeber times (ZT) under LD12:12 conditions affected the degree of ccl18l, ccl19, and ccl25l expression; high and low responsiveness to LPS stimulation at ZT12 and ZT0 (ccl25l), and ZT16 and ZT4 (ccl18l and ccl19), respectively, were observed. These results suggest that the expression of some fish CC chemokines is affected by the diel variation regulated by clock proteins, and that responsiveness against bacterial infection depends on the time zone.

Circadian clock components Bmal1 and Clock1 regulate tlr9 gene expression in the Japanese medaka (Oryzias latipes).

Currently, circadian regulation of immune molecules in lower vertebrates, particularly, diurnal oscillation in the immune status of a fish, is not well understood. In this study, the diurnal oscillation of toll-like receptor (Tlr) 9, which plays a role in pathogen recognition, was investigated in the Japanese medaka fish (Oryzias latipes). We confirmed the expression of tlr9 and clock genes (bmal1 and clock1) in the central and peripheral tissues of medaka. These genes were expressed in a diurnal manner in medaka acclimated to a 12-h:12-h light-dark (12:12 LD) cycle. In addition, increased tlr9 expression was detected in medaka embryo cells (OLHdrR-e3) overexpressing both bmal1 and clock1 genes; however, this result was not obtained when only one or neither of the genes was overexpressed. This suggests that the increase in expression was mediated by the Bmal1 and Clock1 proteins together. In vitro stimulation of the head kidney with CpG-oligodeoxynucleotides (CpG-ODNs) at different zeitgeber times (ZTs; ZT0 = light on, ZT12 = light off) affected the degree of tlr9 gene expression, showing high and low responsiveness to CpG-ODN stimulation at ZT6/10 and ZT18/22, respectively. Similarly, bacterial infection at different ZT points induced a difference in the expression of Tlr9 signaling pathway-related genes (tlr9 and myd88). These results suggested that fish tlr9 exhibits diurnal oscillation, which is regulated by clock proteins, and its responsiveness to immune-stimulation/pathogen infection depends on the time of the day.

ASC-deficiency impairs host defense against Aeromonas hydrophila infection in Japanese medaka, Oryzias latipes.

Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is a component of inflammasome, which plays crucial roles in the inflammatory response. In mammals, ASC regulates caspase-1 activation, thereby inducing pyroptosis and producing activated inflammatory cytokines. In addition, ASC also interacts with receptor-interacting protein kinase 2 (RIPK2) and induces nuclear factor-κB (NF-κB) activation. However, the role of ASC remains poorly understood in fish. In this study, we focused on elucidating the role of ASC in fish that were infected with Aeromonas hydrophila using Japanese medaka (Oryzias latipes) as fish model, and ASC-knockout (KO) medaka was established using CRISPR-Cas9 system. ASC-KO and wild type (WT) medakas were infected with A. hydrophila, and mortality was observed. ASC-KO medaka demonstrated higher mortality than WT. Moreover, the expression of immune-related genes in the kidney and intestine of the ASC-KO and WT medakas challenged with A. hydrophila were analyzed. Following A. hydrophila infection, the kidney of ASC-KO medaka exhibited significantly lower expression of NF-κB regulated genes (e.g., IL-1ß, IL-6, IL-8 and TNF-α) and RIPK2 gene than in WT kidney. Moreover, to investigate the immune response against A. hydrophila via ASC in the medaka, bacterial burden, superoxide anion production, and lactate dehydrogenase release in the kidney cells of ASC-KO medaka were measured. After infection, these responses in ASC-KO medaka were significantly decreased compared to those in WT. These results suggest that the medaka ASC plays a critical role against A. hydrophila infection by inducing inflammatory responses and cell death for bacterial clearance.

A case of intrapulmonary lymphangioma surrounded by pulmonary hilar structures.

A 53-year-old man was referred to our hospital for examination of a nodule in the right hilar region observed on a chest roentgenogram. Computed tomography and magnetic resonance imaging revealed a 34 × 32-mm multilocular cystic mass with partial calcification adjacent to the hilar structures in the right upper lung lobe. The mass was resected using video-assisted thoracoscopic surgery and identified as pulmonary cystic lymphangioma through postoperative histopathological analysis. Intrapulmonary lymphangioma is an uncommon benign tumor, with only few reports of surgically resected cases. We describe our surgical findings and recommend surgical resection where possible for suspected intrapulmonary lymphangioma.

Interleukin-17A/F1 from Japanese pufferfish (Takifugu rubripes) stimulates the immune response in head kidney and intestinal cells.

In mammals, interleukin (IL)-17A and IL-17F, mainly produced by Th17 cells, are hallmark inflammatory cytokines that play important roles in the intestinal mucosal immune response. In contrast, three mammalian IL-17A and IL-17F counterparts (IL-17A/F1-3) have been identified in teleosts, and most of their functions have been described in the lymphoid organs. However, their function in the intestinal mucosal immune response is poorly understood. In this study, a recombinant (r) tiger puffer fish fugu (Takifugu rubripes) IL-17A/F1 was produced and purified using a mammalian expression system, and was used to stimulate cells isolated from fugu head kidney and intestines. The gene expression levels of TNF-α, IL-1ß, IL-6, and ß-defensin-like protein-1 (BD-1) genes were evaluated at 0, 3, 6 and 12 h post-stimulation (hps). Phagocytic activity and superoxide anion production were evaluated at the same time points using an NBT assay. The rIL-17A/F1 protein was shown to induce the expression of pro-inflammatory cytokines and antimicrobial peptides in both head kidney and intestinal cells. Expression levels for IL-1ß, TNF-α, and IL-6 were all up-regulated between 3 and 12 hps. In addition, stimulation with rIL-17A/F1 enhanced phagocytic activity at 24 hps. Superoxide anion production was increased at 48 hps in the head kidney cells and moderately increased at 48 hps in intestinal cells. This study suggests that fugu IL-17A/F1 plays an important role in promoting the innate immune response and may act as a bridge between innate and adaptive immunity in the head kidney and intestine.