Blocking EP4 down-regulates tumor metabolism and synergizes with anti-PD-1 therapy to activate natural killer cells in a lung adenocarcinoma model.

Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX) pathway, is produced by tumors and surrounding stromal cells. It stimulates tumor progression, promotes angiogenesis and suppresses the anti-tumor response. Pharmacological inhibition of PGE2 synthesis has been shown to suppress tumor initiation and growth in vivo. In the current study, we demonstrated that the growth of the Ptgs2-deficient 3LL lung adenocarcinoma cell line was down-regulated in vivo through natural killer (NK) cell activation and a reduction in the population of polymorphonuclear leukocyte-myeloid-derived suppressor cells (PMN-MDSCs) and tumor-associated macrophages (TAMs). On the basis of these results, the therapeutic effect of ONO-AE3-208 (EP4i), an inhibitor of EP4 (a PGE2 receptor), combined with anti-PD-1 antibody was evaluated. EP4i, but not anti-PD-1 antibody, decreased tumor metabolism including glycolysis, fatty acid oxidation and oxidative phosphorylation. EP4i induced IFNγ production from only NK cells (not from T cells) and a shift from M2-like to M1-like macrophages in TAMs. These effects were further enhanced by anti-PD-1 antibody treatment. Although CD8 T-cell infiltration was increased, IFNγ production was not significantly altered, even with combination therapy. Tumor hypoxia was ameliorated by either EP4i or anti-PD-1 antibody treatment, which was further affected by the combination. Normalization of tumor vessels was significant only for the combination therapy. The results indicated a novel effect of EP4i for the metabolic reprogramming of tumors and revealed unique features of EP4i that can synergize with anti-PD-1 antibody to promote IFNγ production by NK cells, polarize TAMs into the M1 phenotype, and reduce hypoxia through normalization of the tumor vasculature.

Massive digital gene expression analysis reveals different predictive profiles for immune checkpoint inhibitor therapy between adenocarcinoma and squamous cell carcinoma of advanced lung cancer.

BACKGROUND: Immune checkpoint inhibitors prolong the survival of non-small cell lung cancer (NSCLC) patients. Although it has been acknowledged that there is some correlation between the efficacy of anti-programmed cell death-1 (PD-1) antibody therapy and immunohistochemical analysis, this technique is not yet considered foolproof for predicting a favorable outcome of PD-1 antibody therapy. We aimed to predict the efficacy of nivolumab based on a comprehensive analysis of RNA expression at the gene level in advanced NSCLC. METHODS: This was a retrospective study on patients with NSCLC who were administered nivolumab at the Kansai Medical University Hospital. To identify genes associated with response to anti-PD-1 antibodies, we grouped patients into responders (complete and partial response) and non-responders (stable and progressive disease) to nivolumab therapy. Significant genes were then identified for these groups using Welch's t-test. RESULTS: Among 42 analyzed cases (20 adenocarcinomas and 22 squamous cell carcinomas), enhanced expression of MAGE-A4, BBC3, and OTOA genes was observed in responders with adenocarcinoma, and enhanced expression of DAB2, HLA-DPB,1 and CDH2 genes was observed in responders with squamous cell carcinoma. CONCLUSIONS: This study predicted the efficacy of nivolumab based on a comprehensive analysis of mRNA expression at the gene level in advanced NSCLC. We also revealed different gene expression patterns as predictors of the effectiveness of anti PD-1 antibody therapy in adenocarcinoma and squamous cell carcinoma.

Heterologous expression and biochemical comparison of two homologous SoxX proteins of endosymbiotic Candidatus Vesicomyosocius okutanii and free-living Hydrogenovibrio crunogenus from deep-sea environments.

Candidatus Vesicomyosocius okutanii is a currently uncultured endosymbiotic bacterium of Phreagena okutanii, a clam that inhabits deep-sea vent environments. The genome of Ca. V. okutanii encodes a sulfur-oxidizing (Sox) enzyme complex, presumably generating biological energy for the host from inorganic sulfur compounds. Here, Ca. V. okutanii SoxX (VoSoxX), a mono-heme cytochrome c component of the Sox complex, was shown to be phylogenetically related to its homologous counterpart (HcSoxX) from a free-living deep-sea bacterium, Hydrogenovibrio crunogenus. Both proteins were heterologously expressed in Escherichia coli co-expressing cytochrome c maturation genes for comparative biochemical analysis. The VoSoxX recombinant had significantly lower thermal stability than HcSoxX, reflecting the difference in growth conditions of the source bacteria. The endosymbiont inhabits a mild intracellular environment, whereas the free-living bacterium dwells in a harsh environment. This study represents the first successful case of heterologous expression of genes from Ca. V. okutanii, allowing further biochemical studies of the molecular mechanism of sulfur oxidation in deep-sea environments.

Molecular detection of a novel perkinsid associated with the deep-sea clam Phreagena okutanii.

Based on environmental DNA surveys, it is widely held that phylogenetically diverse protists exist in chemosynthetic ecosystems. However, knowledge regarding the protists associated with the endemic animals inhabiting these environments is still very limited. In the present study, utilizing polymerase chain reaction (PCR) techniques, we detected fragments of the small subunit ribosomal RNA (SSU rRNA) gene and the internal transcribed spacer (ITS) region of the ribosomal RNA genes from a particular protist in the gills of the vesicomyid clam Phreagena okutanii (formerly described as Calyptogena okutanii), a representative animal in chemosynthetic ecosystems. Based on the phylogeny of the SSU rRNA gene, the organism in question belongs to the genus Perkinsus, which is exclusively composed of protistan parasites infecting mollusks. Intriguingly, based on the ITS phylogeny, this protist was not related to any known Perkinsus species and was deeply branched within the radiation of this genus, thus represents an undescribed species. In addition, the protist detected by PCR was localized to the intercellular spaces in the gills of the host clam with fluorescence in situ hybridization. Although the ecological significance of this novel deep-sea perkinsid remains unclear, our present findings may provide important insights into the diversity of the genus Perkinsus.

Inhibition of mutant KRAS-driven overexpression of ARF6 and MYC by an eIF4A inhibitor drug improves the effects of anti-PD-1 immunotherapy for pancreatic cancer.

Many clinical trials are being conducted to clarify effective combinations of various drugs for immune checkpoint blockade (ICB) therapy. However, although extensive studies from multiple aspects have been conducted regarding treatments for pancreatic ductal adenocarcinoma (PDAC), there are still no effective ICB-based therapies or biomarkers for this cancer type. A series of our studies have identified that the small GTPase ARF6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) are often overexpressed in different cancers, including PDAC, and closely correlate with poor patient survival. Mechanistically, the ARF6-AMAP1 pathway drives cancer cell invasion and immune evasion, via upregulating ß1-integrins and PD-L1, and downregulating E-cadherin, upon ARF6 activation by external ligands. Moreover, the ARF6-AMAP1 pathway enhances the fibrosis caused by PDAC, which is another barrier for ICB therapies. KRAS mutations are prevalent in PDACs. We have shown previously that oncogenic KRAS mutations are the major cause of the aberrant overexpression of ARF6 and AMAP1, in which KRAS signaling enhances eukaryotic initiation factor 4A (eIF4A)-dependent ARF6 mRNA translation and eIF4E-dependent AMAP1 mRNA translation. MYC overexpression is also a key pathway in driving cancer malignancy. MYC mRNA is also known to be under the control of eIF4A, and the eIF4A inhibitor silvestrol suppresses MYC and ARF6 expression. Using a KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre), we here demonstrate that inhibition of the ARF6-AMAP1 pathway by shRNAs in cancer cells results in therapeutic synergy with an anti-PD-1 antibody in vivo; and furthermore, that silvestrol improves the efficacy of anti-PD-1 therapy, whereas silvestrol on its own promotes tumor growth in vivo. ARF6 and MYC are both essential for normal cell functions. We demonstrate that silvestrol substantially mitigates the overexpression of ARF6 and MYC in KRAS-mutated cells, whereas the suppression is moderate in KRAS-intact cells. We propose that targeting eIF4A, as well as mutant KRAS, provides novel methods to improve the efficacy of anti-PD-1 and associated ICB therapies against PDACs, in which ARF6 and AMAP1 overexpression, as well as KRAS mutations of cancer cells are biomarkers to identify patients with drug-susceptible disease. The same may be applicable to other cancers with KRAS mutations. Video abstract.

Symbiont Transmission onto the Cell Surface of Early Oocytes in the Deep-Sea Clam Phreagena okutanii.

Symbiotic associations with beneficial microorganisms endow a variety of host animals with adaptability to the environment. Stable transmission of symbionts across host generations is a key event in the maintenance of symbiotic associations through evolutionary time. However, our understanding of the mechanisms of symbiont transmission remains fragmentary. The deep-sea clam Phreagena okutanii harbors chemoautotrophic intracellular symbiotic bacteria in gill epithelial cells, and depends on these symbionts for nutrition. In this study, we focused on the association of these maternally transmitted symbionts with ovarian germ cells in juvenile female clams. First, we established a sex identification method for small P. okutanii individuals, and morphologically classified female germ cells observed in the ovary. Then, we investigated the association of the endosymbiotic bacteria with germ cells. We found that the symbionts were localized on the outer surface of the cell membrane of primary oocytes and not within the cluster of oogonia. Based on our findings, we discuss the processes and mechanisms of symbiont vertical transmission in P. okutanii.

Identification of cells expressing two peptidoglycan recognition proteins in the gill of the vent mussel, Bathymodiolus septemdierum.

In symbiotic systems in which symbionts are transmitted horizontally, hosts must accept symbionts from the environment while defending themselves against invading pathogenic microorganisms. How they distinguish pathogens from symbionts and how the latter evade host immune defences are not clearly understood. Recognition of foreign materials is one of the most critical steps in stimulating immune responses, and pattern recognition receptors (PRRs) play vital roles in this process. In this study, we focused on a group of highly conserved PRRs, peptidoglycan recognition proteins (PGRPs), in the deep-sea mussel, Bathymodiolus septemdierum, which harbours chemosynthetic bacteria in their gill epithelial cells. We isolated B. septemdierum PGRP genes BsPGRP-S and BsPGRP-L, which encode a short- and a long-type PGRP, respectively. The short-type PGRP has a signal peptide and was expressed in the asymbiotic goblet mucous cells in the gill epithelium, whereas the long-type PGRP was predicted to include a transmembrane domain and was expressed in gill bacteriocytes. Based on these findings, we hypothesize that the secreted and transmembrane PGRPs are engaged in host defence against pathogenic bacteria and/or in the regulation of symbiosis via different cellular localizations and mechanisms.

Morphological and functional characterization of hemocytes from two deep-sea vesicomyid clams Phreagena okutanii and Abyssogena phaseoliformis.

Deep-sea vesicomyid clams harboring intracellular symbiotic sulfur-oxidizing bacteria are often dominant in chemosynthetic animal communities. Although they are known to have erythrocytes, little is known about other hemocytes. To investigate the types and roles of various hemocytes in vesicomyid clams, we performed morphological, histochemical and functional characterization of the hemocytes in two species, Phreagena okutanii, collected from 873 to 978 m depth, and Abyssogena phaseoliformis, from 5199 to 5355 m. Both were found to have three types of hemocytes: erythrocytes (ERCs), eosinophilic granulocytes (EGs), and basophilic granulocytes (BGs). The ERCs contain hemoglobin in the cytoplasm, with basophilic vacuoles containing acid polysaccharide, neutral lipids, and peroxidase. The EGs were found to contain acid polysaccharides and eosinophilic granules containing lysosomal enzymes, acid and alkaline phosphatases, chloroacetate esterase, and peroxidase. Although BGs had some basophilic granules with alkaline phosphatase, they lacked acid phosphatase and acid polysaccharides. The EGs and BGs were shown to have phagocytic ability, while the ERCs exhibited no phagocytosis. The EGs showed higher phagocytic activity as well as a higher phagosome-lysosome fusion rate than BGs. The hemocytes of the two vesicomyid species differed in the intracellular structures. In A. phaseoliformis, ERCs additionally contained neutral polysaccharides in vacuoles and had vesicles with acinus-like acidic mucus in the cytoplasm, neither of which were observed in P. okutanii. The eosinophilic granules in the EGs had heteromorphically-elongated shapes containing homogeneously electron-dense material in P. okutanii, but were more spherical and composed of fibrous structures in A. phaseoliformis. The difference in hemocytes between the two clams seems to be reflective of phylogenetically differentiated lineages adapting to differing conditions in their respective deep-sea environments, such as dissolved oxygen, hydrogen sulfide concentration, and hydrostatic pressure. In the view of phylogeny of veneroida clams including two vesicomyids, their hemocytes appear to be categorizable into three basic types, with the first containing ERCs and agranulocytes, the second including EGs, and the third comprised of BGs, small eosinophilic granulocytes, and other granulocytes. The present data showed no phagocytic activity of ERCs and a lack of agranulocytes in both vesicomyid species, and when combined with previous reports that other veneroid clams show low or no phagocytic activity, this suggests that ERCs have become evolutionarily differentiated from agranulocytes in the ancestral vesicomyid clam.

Cysteine dioxygenase and cysteine sulfinate decarboxylase genes of the deep-sea mussel Bathymodiolus septemdierum: possible involvement in hypotaurine synthesis and adaptation to hydrogen sulfide.

It has been suggested that invertebrates inhabiting deep-sea hydrothermal vent areas use the sulfinic acid hypotaurine, a precursor of taurine, to protect against the toxicity of hydrogen sulfide contained in the seawater from the vent. In this protective system, hypotaurine is accumulated in the gill, the primary site of sulfide exposure. However, the pathway for hypotaurine synthesis in mollusks has not been identified. In this study, we screened for the mRNAs of enzymes involved in hypotaurine synthesis in the deep-sea mussel Bathymodiolus septemdierum and cloned cDNAs encoding cysteine dioxygenase and cysteine sulfinate decarboxylase. As mRNAs encoding cysteamine dioxygenase and cysteine lyase were not detected, the cysteine sulfinate pathway is suggested to be the major pathway of hypotaurine and taurine synthesis. The two genes were found to be expressed in all the tissues examined, but the gill exhibited the highest expression. The mRNA level in the gill was not significantly changed by exposure to sulfides or thiosulfate. These results suggests that the gill of B. septemdierum maintains high levels of expression of the two genes regardless of ambient sulfide level and accumulates hypotaurine continuously to protect against sudden exposure to high level of sulfide.

Increases of heat shock proteins and their mRNAs at high hydrostatic pressure in a deep-sea piezophilic bacterium, Shewanella violacea.

When non-extremophiles encounter extreme environmental conditions, which are natural for the extremophiles, stress reactions, e.g., expression of heat shock proteins (HSPs), are thought to be induced for survival. To understand how the extremophiles live in such extreme environments, we studied the effects of high hydrostatic pressure on cellular contents of HSPs and their mRNAs during growth in a piezophilic bacterium, Shewanella violacea. HSPs increased at high hydrostatic pressures even when optimal for growth. The mRNAs and proteins of these HSPs significantly increased at higher hydrostatic pressure in S. violacea. In the non-piezophilic Escherichia coli, however, their mRNAs decreased, while their proteins did not change. Several transcriptional start sites (TSSs) for HSP genes were determined by the primer extension method and some of them showed hydrostatic pressure-dependent increase of the mRNAs. A major refolding target of one of the HSPs, chaperonin, at high hydrostatic pressure was shown to be RplB, a subunit of the 50S ribosome. These results suggested that in S. violacea, HSPs play essential roles, e.g., maintaining protein complex machinery including ribosomes, in the growth and viability at high hydrostatic pressure, and that, in their expression, the transcription is under the control of σ(32).

Phagocytic activities of hemocytes from the deep-sea symbiotic mussels Bathymodiolus japonicus, B. platifrons, and B. septemdierum.

Deep-sea mytilid mussels harbor symbiotic bacteria in their gill epithelial cells that are horizontally or environmentally transmitted to the next generation of hosts. To understand the immune defense system in deep-sea symbiotic mussels, we examined the hemocyte populations of the symbiotic Bathymodiolus mussel species Bathymodiolus japonicus, Bathymodiolus platifrons, and Bathymodiolus septemdierum, and characterized three types of hemocytes: agranulocytes (AGs), basophilic granulocytes (BGs), and eosinophilic granulocytes (EGs). Of these, the EG cells were the largest (diameter, 8.4-10.0 µm) and had eosinophilic cytoplasm with numerous eosinophilic granules (diameter, 0.8-1.2 µm). Meanwhile, the BGs were of medium size (diameter, 6.7-8.0 µm) and contained small basophilic granules (diameter, 0.3-0.4 µm) in basophilic cytoplasm, and the AGs, the smallest of the hemocytes (diameter, 4.8-6.0 µm), had basophilic cytoplasm lacking granules. A lectin binding assay revealed that concanavalin A bound to all three hemocyte types, while wheat germ agglutinin bound exclusively to EGs and BGs. The total hemocyte population densities within the hemolymph of all three Bathymodiolus mussel species were similar (8.4-13.3 × 10(5) cells/mL), and the percentages of circulating AGs, BGs, and EGs in the hemolymph of these organisms were 44.7-48.5%, 14.3-17.6%, and 34.3-41.0%, respectively. To analyze the functional differences between these hemocytes, the phagocytic activity and post-phagocytic phagosome-lysosome fusion events were analyzed in each cell type using a fluorescent Alexa Fluor(®) 488-conjugated Escherichia coli bioparticle and a LysoTracker(®) lysosomal marker, respectively. While the AGs exhibited no phagocytic activity, both types of granulocytes were phagocytic. Of the three hemocyte types, the EGs exhibited the highest level of phagocytic activity as well as rapid phagosome-lysosome fusion, which occurred within 2 h of incubation. Meanwhile, the BGs showed lower phagocytic activity and lower rates of phagosome-lysosome fusion than the EGs. These findings indicate that the two types of granulocyte play distinct roles in the defense system.

[Prognosis of Papillary Thyroid Carcinoma with Local Invasion].

OBJECTIVE: The aim of this study was to detect prognostic factors in patients with locally advanced papillary thyroid carcinoma. PATIENTS: The study included 72 patients (T4a/T4b 72/0, N0/N1a/N1b 25/15/32, M0/M1 68/4, mean follow-up 8.1 4.4 years) who underwent initial surgical treatment at Osaka Red Cross Hospital between April 1993 and April 2011. RESULTS: Eleven patients died of PTC, 3 patients with recurrence died of unrelated disease and 10 patients are alive with recurrence. The overall 5-year survival rate was 88.3%, and the 10-year survival rate was 73.4%. The disease-specific 5-year survival rate was 91.4%, and the 10-year survival rate was 88.6%. The 5-year local control rate was 94.1%, and the 10-year local control rate was 85.4%. Patients with distant metastasis (M1), tracheal invasion and/or multiple organs invasion showed a significantly worse disease-specific survival rate based on a univariate analysis, which also revealed that tracheal invasion, laryngeal invasion, esophageal invasion and multiple organs invasion were risk factors linked to the development of distant metastasis during follow-up (recurrence as distant metastasis). The following were found to be clinically significant risk factors, based on the multivariate analysis among tracheal invasion, laryngeal invasion, esophageal invasion and recurrent laryngeal nerve invasion : Tracheal invasion was a risk factor for disease-specific survival, and tracheal invasion and laryngeal invasion were risk factors for recurrence as distant metastasis. CONCLUSIONS: In this study, distant metastasis, multiple organs invasion, tracheal invasion and/or laryngeal invasion were shown to be higher risk factors.

The structure of a deoxygenated 400 kDa haemoglobin reveals ternary- and quaternary-structural changes of giant haemoglobins.

The quaternary structures of invertebrate haemoglobins (Hbs) are quite different from those of vertebrate Hbs. The extracellular giant Hbs of molecular masses of about 400 and 3600 kDa are composed of a dome-shaped dodecameric subassembly which consists of four individual globin subunits. Several crystal structures of 400 kDa Hbs from annelids have been reported, including structures in oxygenated and partially unliganded states, but the structure of the fully deoxygenated state has not been reported. In the present study, crystal structures of V2Hb from the tube worm Lamellibrachia satsuma have been determined in both the fully oxygenated and deoxygenated states. A glycosylation site and novel metal-binding sites for divalent cations were clearly observed with no intersubunit interactions in V2Hb. A comparison of the oxygenated and the deoxygenated forms of V2Hb reveals that the ternary- and quaternary-structural changes occur in a manner that maintains the molecular D3 symmetry. These structures suggest that the mechanisms of quaternary-structural changes between the oxy and deoxy states for the giant Hbs are identical across species.

Sensing deep extreme environments: the receptor cell types, brain centers, and multi-layer neural packaging of hydrothermal vent endemic worms.

INTRODUCTION: Deep-sea alvinellid worm species endemic to hydrothermal vents, such as Alvinella and Paralvinella, are considered to be among the most thermotolerant animals known with their adaptability to toxic heavy metals, and tolerance of highly reductive and oxidative stressful environments. Despite the number of recent studies focused on their overall transcriptomic, proteomic, and metabolic stabilities, little is known regarding their sensory receptor cells and electrically active neuro-processing centers, and how these can tolerate and function in such harsh conditions. RESULTS: We examined the extra- and intracellular organizations of the epidermal ciliated sensory cells and their higher centers in the central nervous system through immunocytochemical, ultrastructural, and neurotracing analyses. We observed that these cells were rich in mitochondria and possessed many electron-dense granules, and identified specialized glial cells and serial myelin-like repeats in the head sensory systems of Paralvinella hessleri. Additionally, we identified the major epidermal sensory pathways, in which a pair of distinct mushroom bodies-like or small interneuron clusters was observed. These sensory learning and memory systems are commonly found in insects and annelids, but the alvinellid inputs are unlikely derived from the sensory ciliary cells of the dorsal head regions. CONCLUSIONS: Our evidence provides insight into the cellular and system-wide adaptive structure used to sense, process, and combat the deep-sea hydrothermal vent environment. The alvinellid sensory cells exhibit characteristics of annelid ciliary types, and among the most unique features were the head sensory inputs and structure of the neural cell bodies of the brain, which were surrounded by multiple membranes. We speculated that such enhanced protection is required for the production of normal electrical signals, and to avoid the breakdown of the membrane surrounding metabolically fragile neurons from oxidative stress. Such pivotal acquisition is not broadly found in the all body parts, suggesting the head sensory inputs are specific, and these heterogenetic protection mechanisms may be present in alvinellid worms.

Rapid, contamination-less, and efficient environmental DNA filtration system.

Due to the sporadic distribution and trace amount of environmental DNA (eDNA) in deep-sea water, in the context of biodiversity monitoring, large volumes of filtration and multiple filtration replicates are required for eDNA metabarcoding. To address issues tied to the use of multiple filtration devices and large filtration volumes (e.g., contamination, time consumption, etc.), we have developed two systems for simple, rapid, and contamination-less filtration simultaneously that allow for the processing of multiple sample replicates from large volumes of water. First, the water from a Niskin bottle was filtered directly using a solenoid pump. Second, the pumped deep-sea water, using the siphon effect, was directly filtered by a filtration device driven by water pressure. This system can process 24 replicates simultaneously without the need for expensive equipment and active driving force. Compared with conventional filtering methods, e.g., peristaltic pumps, the proposed systems reduce filtration time, minimizing contamination, and enabling the simultaneous acquisition of multiple replicates. Overall, the systems presented here provide an effective approach for eDNA metabarcoding analysis, particularly for the filtration of large volumes of water containing small amounts of eDNA, such as deep-sea water. •The present systems reduce filtration time and contamination without water having to be transferred.•Simultaneous multiple replicates improve the efficiency and reliability of biodiversity assessments.

Exclusive localization of carbonic anhydrase in bacteriocytes of the deep-sea clam Calyptogena okutanii with thioautotrophic symbiotic bacteria.

Deep-sea Calyptogena clams harbor thioautotrophic intracellular symbiotic bacteria in their gill epithelial cells. The symbiont fixes CO2 to synthesize organic compounds. Carbonic anhydrase (CA) from the host catalyzes the reaction CO2 + H2O ↔ HCO3(-) + H(+), and is assumed to facilitate inorganic carbon (Ci) uptake and transport to the symbiont. However, the localization of CA in gill tissue remains unknown. We therefore analyzed mRNA sequences, proteins and CA activity in Calyptogena okutanii using expression sequence tag, SDS-PAGE and LC-MS/MS. We found that acetazolamide-sensitive soluble CA was abundantly expressed in the gill tissue of C. okutanii, and the enzyme was purified by affinity chromatography. Mouse monoclonal antibodies against the CA of C. okutanii were used in western blot analysis and immunofluorescence staining of the gill tissues of C. okutanii, which showed that CA was exclusively localized in the symbiont-harboring cells (bacteriocytes) in gill epithelial cells. Western blot analysis and measurement of activity showed that CA was abundantly (26-72% of total soluble protein) detected in the gill tissues of not only Calyptogena clams but also deep-sea Bathymodiolus mussels that harbor thioautotrophic or methanotrophic symbiotic bacteria, but was not detected in a non-symbiotic mussel, Mytilus sp. The present study showed that CA is abundant in the gill tissues of deep-sea symbiotic bivalves and specifically localizes in the cytoplasm of bacteriocytes of C. okutanii. This indicates that the Ci supply process to symbionts in the vacuole (symbiosome) in bacteriocytes is essential for symbiosis.

A novel alveolate in bivalves with chemosynthetic bacteria inhabiting deep-sea methane seeps.

It has recently been unveiled that a wide variety of microbial eukaryotes (protists) occur in chemosynthetic ecosystems, such as hydrothermal vents and methane seeps. However, there is little knowledge regarding protists associated with endemic animals inhabiting these environments. In the present study, utilizing PCR techniques, we detected fragments of the small subunit ribosomal RNA gene (SSU rRNA gene) from a particular protist from gill tissues of a significant fraction of the vesicomyid clams Calyptogena soyoae and C. okutanii complex and of the mussel Bathymodiolus platifrons and B. japonicus, all of which harbor chemosynthetic endosymbiont bacteria and dominate methane seeps in Sagami Bay, Japan. Based on the phylogeny of SSU rRNA gene, the organism in question was shown to belong to Alveolata. It is noteworthy that this protist did not affiliate with any known alveolate group, although being deeply branched within the lineage of Syndiniales, for which the monophyly was constantly recovered, but not robustly supported. In addition, the protist detected using PCR followed by sequencing was localized within gill epithelial cells of B. platifrons with whole-mount fluorescence in situ hybridization. This protist may be an endoparasite or an endocommensal of Calyptogena spp. and Bathymodiolus spp., and possibly have physiological and ecological impacts on these bivalves.

IFN-α directly promotes programmed cell death-1 transcription and limits the duration of T cell-mediated immunity.

Programmed cell death-1 (PD-1) is an inhibitory coreceptor for T lymphocytes that provides feedback inhibition of T cell activation. Although PD-1's expression on T cells is known to be activation dependent, the factors that determine the timing, intensity, and duration of PD-1 expression in immune reactions are not fully understood. To address this question, we performed a fine mapping analysis of a conserved 5'-flanking region of the PD-1 gene and identified a putative IFN stimulation response element, which was responsible for PD-1 transcription in the 2B4.11 T cell line. Consistent with this finding, activation by IFN-α enhanced both the induction and maintenance of PD-1 expression on TCR-engaged primary mouse T cells through an association IFN-responsive factor 9 (IRF9) to the IFN stimulation response element. Furthermore, PD-1 expression on Ag-specific CD8(+) T cells was augmented by IFN-α in vivo. We propose that strong innate inflammatory responses promote primary T cell activation and their differentiation into effector cells, but also cause an attenuated T cell response in sustained immune reactions, at least partially through type I IFN-mediated PD-1 transcription. Based on this idea, we demonstrate that IFN-α administration in combination with PD-1 blockade in tumor-bearing mice effectively augments the antitumor immunity, and we propose this as a novel and rational approach for cancer immunotherapy.

Active bacterial flora surrounding foraminifera (xenophyophorea) living on the deep-sea floor.

Bacteria form unique ecosystems by coexisting with large organisms. Here we present the first evidence of active flora surrounding xenophyophorea revealed through clone analyses of environmental ribosomal RNA gene sequences. The flora included eight phyla in the xenophyophorean cells with agglutinated test. The major operational taxonomic units were unique from that in the near-surface sediment. This flora appears to be formed by coexistence with xenophyophores.

[Evaluation of hearing results after tympanoplasty according to the classification of the development degree for pars flaccid cholesteatoma].

Our surgical treatment for middle ear cholesteatoma is based on the following 2 concepts: (1) Preservation of the physiological morphology and function of the middle ear, that is, maximal preservation of the posterior wall of the external auditory meatus and the middle ear mucosa and (2) Careful resection of the matrix membrane of the cholesteatoma through the continuity of the matrix membrane. In case the cholesteatoma matrix membrane is ruptured, a staged operation should be performed to prevent the development of residual cholesteatoma from the residual matrix. In this study, we classified a total of 238 cases of the pars flaccida cholesteatoma primary operated on Osaka Red Cross Hospital between January 2006 and March 2008 according to the Classification and Staging of Cholesteatoma proposed in 2010. The age of the patients ranged from 4 to 79 years (average: 49.8 years) and there were 123 males and 115 females. Follow up ranged from 1 year to 5 years with a mean follow-up period of 47.8 months. Regarding the stage, 38 (16.0%) ears had stage I cholesteatoma, 155 (65.1%) ears had stage II, and 45 (18.9%) ears had stage III. The successful outcome rate was 97.4% for stage I, 78.7% for stage II and 60.0% for stage III. The rate of the residue and the postoperative recurrence was 2.5% and 4.2%.