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Surgical removal of tumor is the primary treatment of choice for glomus tympanicum (GT). However, because the tumor has abundant blood flow, bleeding control is crucial, and preoperative embolization may be performed. Here, we report the case of a 46-year-old female who visited our hospital with a complaint of right pulsatile tinnitus. A red pulsatile mass was found in the right tympanic cavity, and she was diagnosed with class B1 GT and subsequently underwent surgical treatment. We judged that bleeding could be controlled by intratympanic cavity manipulation alone and decided to perform transmeatal tumor resection without preoperative arterial embolization. After creating a tympanomeatal flap and performing an atticotomy, some pieces of Spongel(®) were inserted between the tumor and the tympanic wall. The Spongel(®) absorbed the blood and created a space between the tumor and tympanic wall, which allowed for the insertion of the tip of the Vesalius(®) handpiece to coagulate the tumor. The coagulation caused the tumor to shrink, thereby widening the space and allowing for further resection. Although the surgical manipulation caused bleeding, complete resection was achieved by the application of Spongel(®) and coagulation with Vesalius(®). Since the tip of the Vesalius(®) was not burned, hemostasis was successfully achieved, and the operation proceeded while maintaining a clear field of view. There was little bleeding and no postoperative complications. The patient was discharged on the sixth postoperative day. One year after surgery, pure tone audiometry showed no change in the level of bone conduction. Spongel(®) and Vesalius(®) are useful tools that allow to safely perform surgeries even in narrow spaces such as the tympanic cavity.

Gorham-Stout disease, a rare and intractable disease of unknown etiology, causes systemic bone lysis and replacement with lymphoid tissue. Here, we report a case of Gorham-Stout disease with cerebrospinal fluid leakage in a 16-year-old boy. The patient complained of nasal discharge, right ear obstruction, fever, and headache. A computed tomography scan of the head showed osteolysis around the right internal carotid artery, vestibule, and cochlea and osteolytic changes in the left parietal bone. It was suggested that the patient had bacterial meningitis owing to the leakage of cerebrospinal fluid from the fistula caused by the temporal bone osteolysis. He was treated with meropenem, and a transmeatal fistula closure and a bone biopsy of the left parietal bone were performed. Intraoperatively, osteolysis was observed on the promontory and around the internal carotid artery. The fistula was closed by dense filling and compression around the fistula, in the middle ear cavity, and in the external auditory canal. The symptoms disappeared after the surgery. Bone biopsy showed the presence of a lymphangioma, and Gorham-Stout disease was diagnosed. Prophylactic bisphosphonate therapy was initiated. A 4-year follow-up revealed no progression of the disease.

The prevalence of Alzheimer's disease (AD) has been rapidly increasing worldwide. We have developed a novel angiogenic therapy with low-intensity pulsed ultrasound (LIPUS), which is effective and safe in animal models of AD and vascular dementia. We performed two trials of LIPUS therapy for AD (mild cognitive impairment due to AD and mild AD); a roll-in open trial for safety, and a randomized, double-blind, placebo-controlled (RCT) trial for efficacy and safety. The LIPUS therapy was performed for whole brain through the bilateral temporal bones for one hour 3 times a week as one session under the special conditions (1.3 MPa, 32 cycles, 5% duty cycle) we identified. The LIPUS therapy was performed for one session in the roll-in trial, and 6 sessions in the RCT trial with 3-month intervals for 1.5 years. The primary endpoint was ADAS-J cog scores. The RCT trial was terminated prematurely due to the COVID-19 pandemic. In the roll-in trial (N = 5), no adverse effects were noted. In the RCT trial (N = 22), the worsening of ADAS-J cog scores tended to be suppressed in the LIPUS group compared with the placebo group at week 72 (P = 0.257). When responders were defined as those with no worsening of ADAS-J cog scores at week 72, the prevalence was 50% (5/10) and 0% (0/5) in the LIPUS and placebo groups, respectively (P = 0.053). No adverse effects were noted. These results suggest that the LIPUS therapy is safe and tends to suppress cognitive impairment although a next pivotal trial with a large number of subjects is warranted.

Objectives Whether there are prognostic links between coronary morphologies and coronary functional abnormalities was examined in ischemia and nonobstructive coronary artery disease (INOCA) patients. Background Although INOCA has attracted much attention, little is known about the prognostic impact of coronary morphologies in this disorder. Methods A total of 329 consecutive INOCA patients were enrolled and underwent spasm provocation testing combined with lactate sampling for diagnosis of epicardial and microvascular spasm (MVS). On the basis of the functional tests, the patients were classified into 4 groups: a control group without epicardial spasm or MVS (n = 32), MVS alone (n = 51), diffuse spasm in ≥2 coronary segments (n = 204), and focal spasm in 1 segment (n = 42). In this population, optical coherence tomography imaging of the left anterior descending coronary artery was performed for evaluation of adventitial vasa vasorum (AVV) and intraplaque neovessels (IPN). Index of microcirculatory resistance was also measured. Results MVS frequently coexisted with diffuse (70%) and focal spasm (68%) with a good correlation between AVV and index of microcirculatory resistance (R = 0.353; p = 0.022). For a median follow-up of 1,043 days, focal spasm showed the worst prognosis (log rank p = 0.005), for which IPN was a significant prognostic factor. By contrast, diffuse spasm showed the greatest AVV with an intermediate prognosis. The prognostic value of INOCA was significantly enhanced by adding AVV and IPN to the physiological indices (area under the curve = 0.88 vs. 0.76; p = 0.048). Conclusions These results provide the first evidence that there are important prognostic links between coronary morphologies (evaluated by optical coherence tomography) and coronary functional abnormalities in patients with INOCA, indicating the importance of both evaluations in this population.

Objective Few reports discuss the characteristics of repeated recidivism of cholesteatoma. We describe the clinical characteristics of patients with cholesteatoma who experienced at least two recidivism episodes after initial surgery for cholesteatoma requiring canal wall reconstruction. Methods We reviewed the medical records of 11 patients who underwent surgery for cholesteatoma with canal wall reconstruction at our department between April 2008 and March 2018 and subsequently experienced two relapses that necessitated revision surgery involving tympanomastoidectomy with canal reconstruction. Patient age at the time of the first surgery ranged from 6 to 56 (mean, 25.7) years. Seven (63.6%) of the 11 patients were male. These 11 patients were classified according to the type of recidivism, and their characteristics (pathology, operation date, operation method, pattern of relapse, and position of recurrence) were investigated. Results Four cases involved secondary residual cholesteatoma, with the mean interval between the first revision surgery and the second revision surgery being 23.8 (range, 11–39) months. Secondary residual sites included the anterior tympanic cavity, tympanic sinus, and anterior end of the reconstructed cartilage of the canal wall. The other seven cases involved secondary recurrence, with the mean interval between the first and the second revision surgery being 26.1 (range, 12–57) months. The sites of recurrence were at the edges of the reconstructed cartilage. One notable case involved the cartilage junction, leading us to hypothesize that retraction of the temporal muscle flap and the patulous Eustachian tube was the underlying cause. Conclusion For residual cholesteatoma, strict measures are necessary to maintain the operation under clear view, and more careful follow-up is necessary in patients who have had previous surgery at another hospital. For recurrent cholesteatoma, it was recognized that Eustachian tube function must be ascertained in advance, and careful observation of the reconstructed cartilage edge is necessary.

A 75-year-old female patient presented with a suspected recurrence of a clival chordoma. The tumor was resected using the infratemporal fossa type B and anterior petrosal approach with the help of a neurosurgeon. During cauterization of the trigeminal nerve, the patient developed cardiac arrest for approximately 10 seconds because of the trigemino-cardiac reflex (TCR). After several sternal compressions, there was return of spontaneous circulation. The operation was resumed after the circulatory dynamics stabilized. Subsequently, the surgery was completed with partial resection of the tumor without the recurrence of cardiac arrest. The pathological diagnosis was chondrosarcoma, and postoperative treatment with radiotherapy was started. Stimulation of the sensory branches of the trigeminal nerve induces TCR. There are reports of TCR developing in approximately 10% of skull base surgery cases in the absence of atropine administration. We report a rare case of TCR during the surgical procedure for the treatment of a skull base chondrosarcoma.

[Background] Postoperative hearing results of type IV tympanoplasty have been shown to have poorer results than other reconstruction techniques. There are numerous reports evaluating the factors for hearing improvement. This preliminary study aimed to analyze and determine the factors that affect hearing results. [Methods] A total of 80 patients who underwent type IV tympanoplasty were evaluated to participate in this study. The medical records of the candidate patients were collected retrospectively. Fifty out of the 80 recruited patients were excluded due to the following reasons: they could not be followed-up for more than a year after the final operation, their initial surgery was not performed in our department, or they needed a revision surgery. The pre-, intra-, and postoperative factors were evaluated and analyzed using EZR software. Cases were divided into two groups according to postoperative hearing results, and each factor was analyzed univariately. The explanatory variables included in the multivariate analysis were the variables that satisfied P < 0.1 in the univariate analysis. Furthermore, all cases were divided into two groups according to the qualitative variables that showed significant difference in the multivariate analysis, and the background factors were evaluated. [Results] The results of univariate analysis showed P < 0.1 for ‘Age’ and ‘Material of external auditory canal (EAC) packing’. Multivariate analysis showed P < 0.05 for both. The comparison between the two packing material groups showed that the gauze group was more likely to have improved hearing than Spongel? group, and the ossicular chain condition of the gauze group was maintained better. [Conclusion] ‘Age’ and ‘Material of EAC packing’ were considered to be significant factors affecting the postoperative hearing results. The selection and use of packing materials that provide stability should be considered to obtain better postoperative hearing results in type IV tympanoplasty.

It has been reported that the degree of anxiety-like behavior differs between inbred strains of mice, and that this phenomenon was linked to the expression levels of the oxidative stress-related genes glyoxalase 1 (Glo1) and glutathione reductase 1 (Gsr) in the brain. Therefore, we investigated whether antioxidative activity in the brain affects the Glo1 and Gsr mRNA expressions and strain-dependent anxiety-like behavior using mice fed different amounts of vitamin E. First, we measured brain Glo1 and Gsr mRNA levels and evaluated the anxiety-like behaviors presented by C57BL/6J (B6) and DBA/2C (D2) mice. We demonstrated that D2 mice presented both significantly elevated Glo1 and Gsr mRNA levels as well as more prominent anxiety-like behavior in elevated plus-maze and open field tests. Next, we fed mice from these two strains either a control, vitamin E-free, or vitamin E-supplemented diet for four weeks. Plasma, liver, and brain α-tocopherol concentrations changed in a dose-dependent manner. However, neither brain Glo1 and Gsr mRNA levels nor anxiety-like behavior were affected by dietary vitamin E intake. These results demonstrated that while strain-dependent anxiety-like behavior in mice was related to oxidative stress-related gene expression, the regulatory mechanisms for these genes and anxiety-like behaviors were independent of antioxidative activity in the brain. Strain-dependent differences of the anxiety in mice are probably related to the anxiolytic effects of methylglyoxal, a substrate for Glo1 and Gsr.

A 40-year-old man suffered from short bowel syndrome. Since a large amount of intestinal output and watery diarrhea hampered his quality of life, we tried to control the intestinal output by reducing the secretion of gastric acid with lansoprazole. Because the small intestine was only 10 cm in length and effective absorption of oral lansoprazole was doubtful, we monitored his intragastric pH for 24 hours and confirmed that the holding time above pH 3.0 was 14.5 hours (60.4%). He spent his home life eating porridge during the day, and receiving total parenteral nutrition of 1,100 mL/day at night while taking lansoprazole as an oral tablet (30 mg) once a day.

Backgrounds We demonstrated that coronary adventitial inflammation plays important roles in the pathogenesis of drug-eluting stent (DES)-induced coronary hyperconstricting responses in pigs in vivo. However, no therapy is yet available to treat coronary adventitial inflammation. We thus developed the low-intensity pulsed ultrasound (LIPUS) therapy that ameliorates myocardial ischemia by enhancing angiogenesis. Aims We aimed to examine whether our LIPUS therapy suppresses DES-induced coronary hyperconstricting responses in pigs in vivo, and if so, what mechanisms are involved. Methods Sixteen normal male pigs were randomly assigned to the LIPUS or the sham therapy groups after DES implantation into the left anterior descending (LAD) coronary artery. In the LIPUS group, LIPUS (32 cycles, 193 mW/cm2) was applied to the heart at 3 different levels (segments proximal and distal to the stent edges and middle of the stent) for 20 min at each level for every other day for 2 weeks. The sham therapy group was treated in the same manner but without LIPUS. At 4 weeks after stent implantation, we performed coronary angiography, followed by immunohistological analysis. Results Coronary vasoconstricting responses to serotonin in LAD at DES edges were significantly suppressed in the LIPUS group compared with the sham group. Furthermore, lymph transport speed in vivo was significantly faster in the LIPUS group than in the sham group. Histological analysis at DES edges showed that inflammatory changes and Rho-kinase activity were significantly suppressed in the LIPUS group, associated with eNOS up-regulation and enhanced lymph-angiogenesis. Conclusions These results suggest that our non-invasive LIPUS therapy is useful to treat coronary functional abnormalities caused by coronary adventitial inflammation, indicating its potential for the novel and safe therapeutic approach of coronary artery disease.

Superficial siderosis is a disease in which iron from hemoglobin is deposited in the central nervous system, resulting in conditions such as progressive sensorineural hearing loss, cerebellar ataxia, dementia, and pyramidal signs. A 30-year-old man with superficial siderosis received a cochlear implant in the left ear, which had shown complete hearing loss. Good auditory responses were obtained at 14 days after implantation. The postoperative average hearing level with the cochlear implant was 56.7 dB at 3 months and 55.0 dB at 6 months. However, the patient showed gradual hearing loss, and the dynamic range changed each time the electrode parameters were adjusted. To assess residual hearing ability, single-photon emission computed tomography was performed together with an assessment of electrical auditory brainstem response, which showed a good response and increased blood flow in both the temporal lobes. Based on this result, we asked the patient to continue using the cochlear implant to see whether a perception of speech response would be obtained. However, the patient discontinued using the cochlear implant because he could not hear satisfactorily. Hearing outcomes after cochlear implant surgery for patients with superficial siderosis are not necessarily good. Therefore, the possibility of unsatisfactory results should be fully explained before recommending this surgery to patients.

Background The coronary adventitia harbors lymphatic vessels (LVs). We previously demonstrated that coronary adventitial inflammation and LV dysfunction play important roles in the pathogenesis of coronary artery spasm, including drug-eluting stent (DES)-induced coronary hyperconstricting responses, in pigs and humans. However, a direct therapeutic approach to the coronary adventitia remains to be developed. Purpose In this study, we aimed to examine whether our novel and non-invasive therapy with low-intensity pulsed ultrasound (LIPUS) ameliorates DES-induced coronary hyperconstricting responses, and if so, what mechanisms are involved. Methods An everolimus-eluting stent (EES) was implanted into the left anterior descending (LAD) coronary artery in normal male pigs. They were randomly assigned to the LIPUS or the sham therapy groups. After EES implantation, in the LIPUS group, LIPUS (32 cycles, 193 mW/cm2) was applied to the heart at 3 different levels (proximal and distal stent edges and middle portion of the stent) through X-ray fluoroscopy for 20 min at each level for every other day for 2 weeks (6 days in total) (Fig. 1A, B). The sham therapy group was treated in the same manner but without LIPUS. At 4 weeks after the procedure, we performed coronary angiography to examine coronary vasoconstricting responses to intracoronary serotonin in vivo. Finally, stented coronary vessels were harvested for immunohistochemistry of vasa vasorum (vWF), LVs (LYVE-1), vascular inflammation (CD68-positive macrophages and IL-1β expression), vascular endothelial growth factor A (VEGF-A, angiogenesis marker), VEGF-C and VEGF receptor 3 (VEGFR3, lymphangiogenesis markers). Results Coronary vasoconstricting responses to intracoronary serotonin at the DES edges in the LAD were significantly enhanced in the sham group but were significantly suppressed in the LIPUS group, while those responses were comparable at the non-DES implanted left circumflex (LCx) coronary artery between the 2 groups (Fig. 1C, D). In addition, in vivo lymph transport speed was significantly faster in the LIPUS group than in the sham group (Fig. 1E–G). In histological analysis, the number of LVs was significantly increased in the LIPUS group compared with the sham group, whereas those of CD68 and IL-1β expressions were significantly reduced in the LIPUS group compared with the sham group. In contrast, the density of vasa vasorum was comparable between the 2 groups. Mechanistically, the extents of VEGF-C and VEGFR3 expressions were increased in the LIPUS group, whereas that of VEGF-A was comparable between the 2 groups (Fig. 1G–K). Importantly, there were significant correlations among the LV-related changes and enhanced coronary vasoconstricting responses. Conclusion These results provide the first evidence that the LIPUS therapy ameliorates DES-induced coronary hyperconstricting responses in pigs in vivo through structural and functional alterations of LVs (Fig. 1L). Figure 1 Funding Acknowledgement Type of funding source: None

The effects of fish oil for improving mental health have been reported. The present study was undertaken to compare the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on anxiety-like behavior using a rat model. Experimental diets enriched in EPA or DHA as glycerides were prepared. Rats were exposed to social isolation stress and fed the experimental diet for 14 days. The results of behavioral tests revealed that rats fed the EPA-enriched diet exhibited less anxiety-like behavior than rats fed the control or DHA-enriched diets. Furthermore, EPA suppressed anxiety-like behavior only in socially isolated rats. The increase in EPA contents in the brain phospholipid fraction by feeding EPA-enriched diet was more significant than that of DHA by feeding DHA-enriched diet. These results suggest that dietary EPA is more anxiolytic than DHA in rats exposed to social isolation stress and is effective in increasing EPA content in brain membranes.

Background We previously demonstrated that coronary adventitial inflammation plays important roles in the pathogenesis of coronary artery spasm, including drug-eluting stent (DES)-induced coronary hyperconstricting responses, in pigs and humans. Indeed, the coronary adventitia has recently attracted much attention as the important site for vascular inflammation. However, a direct therapeutic approach to the coronary adventitia remains to be developed. We have developed a non-invasive low-intensity pulsed ultrasound (LIPUS) therapy for angina, which exerts anti-inflammatory effects through improved coronary microcirculation. Purpose In this study, we aimed to examine whether our LIPUS therapy ameliorates DES-induced coronary hyperconstricting responses in pigs in vivo, and if so, what mechanisms are involved. Methods An everolimus-eluting stent (EES) was implanted into the left anterior descending (LAD) coronary artery in normal male pigs. They were randomly assigned to the LIPUS or the sham therapy groups. After EES implantation, in the LIPUS group, LIPUS (32 cycles, 193 mW/cm2) was applied to the heart at 3 different levels (proximal and distal stent edges and middle portion of the stent) through X-ray for 20 min at each level for every other day for 2 weeks (6 days in total) (Figs. 1A, 1B). The sham therapy group was treated in the same manner but without LIPUS. At 4 weeks after the procedure, we performed coronary angiography to examine coronary vasoconstricting responses to intracoronary serotonin in vivo. Finally, stented coronary vessels were harvested for immunohistochemistry of vasa vasorum, lymphatic vessels (LYVE-1), sympathetic nerve fibers (SNF), vascular inflammation (macrophages and IL-1β expression), Rho-kinase expression and activity as evaluated by phosphorylated myosin phosphatase target subunit-1 (pMYPT-1). Results Coronary vasoconstricting responses to intracoronary serotonin were significantly enhanced in the sham therapy group but were significantly suppressed in the LIPUS group at the DES edges in the LAD, whereas those responses were comparable at the non-DES implanted segments in the left circumflex (LCx) coronary arteries between the 2 groups. (Figs. 1C, 1D). Furthermore, in vivo lymph transport speed was significantly faster in the LIPUS group than in sham group (Figs. 1E–1G). Histological analysis showed that except vasa vasorum formation, the number of lymphatic vessels, adventitial inflammatory cells infiltration, Rho-kinase expression and activity were all significantly enhanced in the sham therapy group and were significantly suppressed in the LIPUS group (Figs. 1G–1K). Figure 1 Conclusion We were able to develop a non-invasive LIPUS therapy for coronary functional abnormalities caused by chronic adventitial inflammation in pigs in vivo, for which multiple beneficial effects appear to be involved (Fig. 1L).

Background: Intersigmoid hernia is a hernia of the small intestine into the intersigmoid fossa. Because the cavity of the intersigmoid fossa is so small, the preoperative detection of incarcerated intestine and/or mesenteric convergence is very difficult. We report a case of intersigmoid hernia in which the incarcerated bowel and mesenteric convergence could be visualized by oblique multiplanar reconstruction (MPR) images on multi-detector computed tomography (MDCT). Case Report: An 82-year-old man with small bowel obstruction was treated conservatively with a long intestinal tube. Axial plane images of MDCT detected only a thickening of the small bowel wall and a narrowing of the lumen in the pelvis. Since a fourteen-day waiting period did not improve the condition at all, he underwent surgery. The small bowel was herniated into the intersigmoid fossa. After surgery, we studied the preoperative images of MDCT once again. However, neither converged mesentery nor hernia orifice had been depicted. We attempted to make oblique coronal/sagittal MPR images using SYNAPSE VINCENT® and succeeded in visualizing not only the incarcerated bowels but also the hernia orifice and mesenteric convergence. Conclusion: Creating oblique MPR images from the MDCT volume data would help in making a preoperative diagnosis of sigmoid mesocolon hernias such as intersigmoid hernia with increasing confidence.

A skin mucus lectin exhibiting a homodimeric structure and an S-S bond between subunits of ~40 kDa was purified from flathead Platycephalus indicus (Scorpaeniformes). This lectin, named FHL (FlatHead Lectin), exhibited mannose-specific activity in a Ca(2+)-dependent manner. Although FHL showed no homology to any previously reported lectins, it did exhibit ~20% identity to previously discovered plasma kallikreins and coagulation factor XIs of mammals and Xenopus laevis. These known proteins are serine proteases and play pivotal roles in the kinin-generating system or the blood coagulation pathway. However, alignment analysis revealed that while FHL lacked a serine protease domain, it was homologous to the heavy-chain domain of plasma kallikreins and coagulation factor XI therefore suggesting that FHL is not an enzyme but rather a novel animal lectin. On the basis of this finding, we investigated the lectin activity of human plasma kallikrein and revealed that it could indeed act as a lectin. Other genes homologous to FHL were also found in the genome databases of some fish species, but not in mammals. In contrast, plasma kallikreins and coagulation factor XI have yet to be identified in fish. The present findings suggest that these mammalian enzymes may have originally emerged as a lectin and may have evolved into molecules with protease activity after separation from common ancestors.

As the fetus expresses paternal major histocompatibility complex molecules, viviparous vertebrates require sophisticated mechanisms to modulate maternal immunology to ensure successful pregnancy. We anticipated that ovarian cavity fluid (OCF) is likely to feature significantly in the modulation of ovarian cavity immunology. Consequently, we examined the effects of OCF upon leukocyte function in Neoditrema ransonnetii. OCF did not affect phagocytosis or superoxide production by phagocytes. However, OCF suppressed lymphocyte proliferation induced by ConA almost completely. As OCF contained PGE(2) at high levels during late pregnancy, we also investigated the effect of PGE(2) upon lymphocyte expansion. PGE(2) exhibited negative effects upon lymphocyte mitogenesis in a dose-dependent manner (10-1000 ng/ml). PGE(2) significantly suppressed lymphocyte proliferation when present at levels equivalent to that seen in OCF (30.2 +/- 16.1 approximately 185.4 +/- 107.4 ng/ml). Data indicate that PGE(2) is one of the key modulatory molecules of the maternal immune system ensuring successful pregnancy in this viviparous species.

To determine whether fish intestinal epithelial cells (IECs) contribute to mucosal immunity, we established a method for isolating IECs from the rainbow trout Oncorhynchus mykiss and examined cytokine production in these cells. Components of the intestinal epithelium were released by incubation of intestinal pieces with 1 mM dithiothreitol (DTT)/ethylenediamine tetraacetic acid (EDTA). The IEC-rich fraction (purity >90%; survival rate ∼95%) was obtained by centrifugation on a 35%/40% Percoll gradient, followed by magnetic cell sorting using an anti-trout IgM antiserum. The gene expression profiles of 14 cytokines in trout IECs were investigated after culturing the cells for 6 h with or without the pathogenic bacterium Aeromonas salmonicida . Trout IECs could produce several cytokines, of which IL-1β and TNFα2 were upregulated when the cells were stimulated with live A. salmonicida . Immunohistochemical analyses with the anti-trout TNF antibody confirmed that the TNF protein was present in the IECs of trout that were intra-anally challenged with live A. salmonicida . These results show that trout IECs are an important trigger of the intestinal immune system. Further, formalin-killed A. salmonicida , conditioned medium of this bacterium, or live nonpathogenic Escherichia coli could not upregulate the expression of these cytokines. These results indicate that the production of inflammatory cytokines by IECs is caused by the adhesion of A. salmonicida , but is not due to only simple ligand–receptor interactions between the surface molecules of IECs and the bacterium or in response to bacterial secretions.

L-rhamnose-binding lectins (RBLs) have been isolated from various kinds of fish and invertebrates and interact with various kinds of bacteria, suggesting RBLs are involved in various inflammatory reactions. We investigated the effect of RBLs from chum salmon (Oncorhynchus keta), named CSL1, 2 and 3, on the peritoneal macrophage cell line from rainbow trout (Oncorhynchus mykiss) (RTM5) and an established fibroblastic-like cell line derived from gonadal tissue of rainbow trout (RTG-2). CSLs were bound to the surface of RTM5 and RTG-2 cells and induced proinflammatory cytokines, including IL-1beta1, IL-1beta2, TNF-alpha1, TNF-alpha2 and IL-8 in both cells by recognizing globotriaosylceramide (Gb3). In addition, CSLs had an opsonic effect on RTM5 cells and this effect was significantly inhibited by L-rhamnose, indicating that CSLs enhanced their phagocytosis by binding to Gb3 on cell surfaces. This is the first finding that Gb3 plays a role in innate immunity by cooperating with natural ligands, RBLs.

Congerin, a mucosal galectin of the Japanese conger eel, provides chemical fortification through its agglutinating and opsonizing activity. Congerin is produced in the epidermis, and the epithelia of the oral cavity to the esophagus, but not in the stomach or intestine. We hypothesized that congerin secreted from the upper digestive tract can reach and function in the intestinal lumen. We found that congerin possessed marked resistance against digestion by gastric and enteric enzymes of conger eel. It was not degraded until 6h of incubation with stomach extract or intestinal digestion juice. Western blotting demonstrated that congerin essentially remained in the intestinal mucus. The mucus agglutinated rabbit erythrocytes, and the agglutination was hampered by anti-congerin antibody. Furthermore, congerin could bind to some enteric bacteria. These results support the above hypothesis.

A mannose-specific lectin with agglutination activity against a yeast, Saccharomyces serevisiae, was purified from conger eel (Conger myriaster) skin mucus. The lectin, named conCL-s, has a tetrameric structure consisting of two non-covalently associated dimers whose constituent monomers with a molecular mass of about 16,000 Da are linked by a disulphide bond. conCL-s is composed of 173 amino acid residues including a 24-residue signal peptide, and belongs to the C-type lectin family, which is typically characterized by binding to sugar in a Ca2+-dependent manner. Nevertheless, conCL-s showed Ca2+-independent activity in its yeast-binding. The gene expression of the lectin was widely detected in external and internal mucosal tissues, i.e., skin, gills, tongue, buccal cavity wall and esophagus. In these tissues, mRNA of conCL-s was exclusively distributed in club cells, which are one of the major components of the epidermis and mucosal epithelium in Anguilliforme species. conCL-s showed agglutination activity to a bacterium, Escherichia coli. Furthermore, attachment of the lectin to microspheres significantly enhanced their phagocytosis in conger eel macrophages. These findings suggest that conCL-s acts as an opsonin and plays an important role in the innate immunity on the body surface in conger eels.

We investigated maternal IgM secretion in the ovary and the absorption of IgM by fetuses in a viviparous fish, Neoditrema ransonneti (Embiotocidae). Serum IgM, whose molecular weight was approx. 820 k, was purified by two steps of gel filtration chromatography. Immunohistochemistry and Western blotting revealed that IgM was secreted from the epithelia of the ovigerous lamellae of pregnant females into ovarian cavity fluid. The IgM-secreting activity of ovigerous folds showed notable changes according to the reproductive stage. In fetuses, IgM was absorbed as macromolecules by enterocytes of the hypertrophied hindgut. IgM in the fetal blood was also demonstrated, although its concentration remained low during gestation. These findings suggest that IgM was transported from the maternal tissues to embryos via a unique pathway in N. ransonneti.

Japanese conger Conger myriaster has well-developed splenic ellipsoids (sheath structure) that are thought to participate in the elimination of foreign substances and effete hemocytes from circulating blood. After injection of fluorescent latex beads (LB, 0.5 or 2.0 μm in diameter) through the aortic bulb of the Japanese conger, the spleens and kidneys were examined by light and electron microscopy. The splenic ellipsoids trapped many LB of 0.5 μm in diameter but none of 2.0 μm. This suggests that the LB were filtered by ellipsoids depending on size. Latex beads of 0.5 μm were ingested by macrophages beneath the endothelium of the arteriole in ellipsoids but not by endothelial cells. At 24 h, many 0.5 μm LB were found in the ellipsoids and splenic cord, but there were few in the melanomacrophage centers (MMC). However, most of the 0.5 μm LB had accumulated in the MMC within 48 h. Many macrophages laden with the microspheres were found around MMC. The 2.0 μm LB were detected in the splenic cord and MMC 24 h and 48 h after injection. This suggests that the macrophages take up 0.5 μm LB in the ellipsoids and migrate through the splenic cord to MMC. The process of entrapment and accumulation of particles in the kidney was also examined and discussed.

The epidermis of premetamorphic leptocephali of Japanese conger Conger myriaster consisted of only a few cell layers including congerin- (a galectin) containing club cells. The epidermis increased in thickness after metamorphosis, mainly due to a preponderant development of club cells. This suggested that the biological significance of congerins increased at this stage, which may be related to a change in life style. Epidermal mucous cells showed only limited distribution. Immuno-positive cells in the mucosal epithelia of the buccal cavity wall and oesophagus were not found in the youngest individuals. Though these cells were observed at the commencement of metamorphosis, the development of the epithelia and club cells was apparently retarded compared to that of the epidermis. No immuno-staining was found in the gills at any stages.

The localization of three l -rhamnose-binding lectins named STL1, STL2, and STL3 from eggs of steelhead trout ( Oncorhynchus mykiss ) was analyzed by indirect immunohistochemical staining with specific antisera against individual lectins. In early previtellogenic oocyte, STL1 was localized not only in the cortical vesicles, but also in the plasma membrane and germinal vesicle. On the other hand, STL2 and STL3 were localized only in the cortical vesicles. In pre-fertilization mature egg, STLs were localized in a thin layer of cortical granules at the cytoplasmic side of the plasma membrane. STLs were accumulated on the surface of cytoplasm and inner membrane 30 min after fertilization. The strong staining with anti-STL1 antiserum was observed in several tissues and cells of the steelhead trout, such as spleen, thrombocytes, and blood leukocytes, but not erythrocytes. STL1 was also identified in exocrine cells, such as goblet cells of intestine and mucous cells of gill. These results indicate that the multiple lectins found in eggs of the steelhead trout play physiological roles not only in eggs, but also in various cells related to the innate immunity.

BRA-2, a major C-type lectin in the hemolymph of the acorn barnacle Megabalanusrosa was detected in shell-associated proteins. Immobilized BRA-2 promoted CaCO3 crystallization as determined by an in vitro pH-drift mineralization assay. In nature, calcification occurs as wound repair in M. rosa after breakage of the shell plate. To observe this phenomenon, we inserted a rubber cube inside the mantle cavity of M. rosa through the basal plate, and kept the organisms in a tank for several weeks. The rubber cube was covered first with brownish-colored material, probably a melanin, and then with CaCO3 5 weeks after the insertion. In the CaCO3-associated proteins, BRA-3, a minor component of M. rosa hemolymph lectins, was present in addition to BRA-2. The presence of both the lectins and their ligands on the hemocyte was also observed. These results suggest that the C-type lectins of the M. rosa hemolymph participate in mineralization as well as defense in these organisms.

Congerin is a β-galactoside binding lectin (galectin) purified from the skin mucus of the Japanese conger, Conger myriaster . To clarify its tissue distribution and productive cells, several tissue samples including skin, buccal cavity wall, tang, pharynx, gills, esophagus, stomach, intestine, liver, kidney, spleen and ovary of conger were stained immunohistochemically using polyclonal rabbit anti-congerin serum. In the epidermis, a number of club cells were strongly stained. Because no agglutinating activity was detected in plasma, it appears evident that congerin is produced and secreted into mucus by those cells. In addition, congerin-positive club cells were distributed in the mucosal epithelium lining the digestive tract preceding the stomach and in the gills. These findings suggest that congerin participates in innate immunity on the intra- and the extra-body surface of the conger. The putative functions of club cells in fish and their contained lectin are discussed.

Selenocysteine lyase (SCL) (EC 4.4.1.16) is a pyridoxal 5′-phosphate-dependent enzyme that specifically catalyzes the decomposition of l-selenocysteine tol-alanine and elemental selenium. The enzyme was proposed to function as a selenium delivery protein to selenophosphate synthetase in selenoprotein biosynthesis (Lacourciere, G. M., and Stadtman, T. C. (1998) J. Biol. Chem. 273, 30921–30926). We purified SCL from pig liver and determined its partial amino acid sequences. Mouse cDNA clones encoding peptides resembling pig SCL were found in the expressed sequence tag data base, and their sequences were used as probes to isolate full-length mouse liver cDNA. The cDNA for mouse SCL (mSCL) was determined to be 2,172 base pairs in length, containing an open reading frame encoding a polypeptide chain of 432 amino acid residues (M r 47,201). We also determined the sequence of the N-terminal region of putative human SCL. These enzymes were shown to be distantly related in primary structure to NifS, which catalyzes the desulfurization of l-cysteine to provide sulfur for iron-sulfur clusters. The recombinant mSCL overproduced inEscherichia coli was a homodimer with the subunitM r of 47,000. The enzyme was pyridoxal phosphate-dependent and highly specific tol-selenocysteine (thek cat/K m value forl-selenocysteine was about 4,200 times higher than that forl-cysteine). Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that mSCL is cytosolic and predominantly exists in the liver, kidney, and testis, where mouse selenophosphate synthetase is also abundant, supporting the view that mSCL functions in cooperation with selenophosphate synthetase in selenoprotein synthesis. This is the first report of the primary structure of mammalian SCL.

We measured the amount of Se in bovine liver tRNA. tRNA was chromatographed on a BD-cellulose column and Se-rich tRNA was eluted from the column in front of a main tRNA peak. There was 0.3 mmol Se/mol of tRNA and this level is about one tenth that of Escherichia coli tRNA. This suggests the presence of an enzyme that modifies tRNA with Se in bovine liver. We isolated the activity of this enzyme (selenouridine synthase) by chromatography of bovine liver extracts on a DEAE-cellulose column. ATP and selenophosphate synthetase, as well as selenouridine synthase and tRNA, were necessary for the reaction. 75Se was used to label the reaction products, which were analyzed by TLC after digestion with ribonuclease T2. The position of the 75 Se-nucleotide on a TLC plate was identical to that of the Se-nucleotide, 5-methylaminomethyl-2-seleno-Up, prepared from 75Se-tRNA in E. coli.

Carp (cyprinus carpio) hematopoietic cells collected from the kidney-hematopoietic tissue were cultured. After 3-5 days, colonies consisting of a few to 30 small round cells were observed. The number of cells in the colonies increased steadily. Colonies having several hundreds of cells were observed later. Colonnies were formed on an adherent cell layer, which is possibly acting as feeder cells for the colony. Most cells of the adherent layer were 0.1-1.0 mm in diameter and contained 3-200 oval or round nuclei, similar in appearance to that of multinucleated giant cells.The proliferated cells in the colonies were separated from the colony into the culture medium. These floating cells were constantly produced for more than 1 month while half of the medium was renewed at 1-5 day intervals. Morphologically, the majority of the floating cells had features of immature type of cells which were mainly round cells with basophilic cytoplasm and a round nucleus. Mitotic figures were frequently observed.