The effect of breathing relaxation to improve poor sleep quality in adults using a huggable human-shaped device: a randomized controlled trial.

PURPOSE: To examine whether or not breathing relaxation, using a huggable human-shaped device, improves poor sleep quality in adults. METHODS: We conducted a randomized controlled trial using outpatients with sleep problems from two clinics in Japan. The intervention group conducted three minutes of breathing relaxation using a huggable human-shaped device before going to bed every night for four weeks. Sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI), at pre-intervention, mid-intervention (2 weeks after pre-intervention), and post-intervention (4 weeks after pre-intervention). We employed intention-to-treat analysis. RESULTS: A total of 68 participants (mean [SD] age, 41.7 [11.4] years; 64 female [95%]) were randomly assigned to the intervention group (n = 29, mean [SD] age, 43.6 [9.5] years; 28 female [97%]) and the control group (n = 36, mean [SD] age, 40.3 [12.7] years; 36 female [95%]). The intervention group showed a significant decrease in the PSQI score compared to the control group (F = 3.81, p = 0.025, effect size (η2) = 0.057). Furthermore, we found the intervention to be more effective in participants without suicide risk and with a lower number of adverse childhood experiences (effect size (η2) = 0.080 and 0.160, respectively). CONCLUSIONS: A novel psychological intervention, breathing relaxation using a huggable human-shaped device, may be effective to improve sleep quality among people with sleep problems, especially those without severe psychological symptoms. TRIAL REGISTRATION: UMIN000045262. (Registration Date: September 28th, 2021).

[A Case of Intractable Chylorrhea Following Breast Cancer Surgery with a History of Liver Cirrhosis].

We present a case of intractable chylorrhea following breast cancer surgery in a 75-year-old female. During a close examination for a mass in her left breast, which was indicated by a CT scan performed to test for nausea, cancer of the left breast and an enlarged left axillary lymph node were observed. The FNA of the axillary lymph node was unsuitable as a sample since no lymph node cell-derived components were observed. A left breast mastectomy and axillary lymph node dissection were performed for the evaluation of cT2N1M0, Stage ⅡB. On postoperative day 3, cloudy drainage was observed, leading to a diagnosis of chylorrhea. Despite management by a fat-restricted diet and peripheral infusion on postoperative day 4, chyle from the drainage remained high, with a TG of 257 mg/dL, a cell count of 525/mm3(70% lymphocytes), and a postoperative drainage volume of over 500 mL per day. On postoperative day 8, octreotide subcutaneous injection was started, and drainage could be reduced. Locally injected picibanil solution through the drain on postoperative days 12 and 17 further decreased the drainage to 20 mL/day, and the drain was removed. The patient was discharged on postoperative day 22. The occurrence of chylorrhea was a concern due to the risk of distal hepatic collateral flow, regional lymph nodes and vessels, and high hepatic flow pressure due to liver cirrhosis.

Protease inhibitory activity of secretory leukocyte protease inhibitor ameliorates murine experimental colitis by protecting the intestinal epithelial barrier.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder in the intestine, and the dysfunction of intestinal epithelial barrier (IEB) may trigger the onset of IBD. Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that has been implicated in the tissue-protective effect in the skin and lung. We found that SLPI was induced in lipopolysaccharides-treated colon carcinoma cell line and in the colon of dextran sulfate sodium (DSS)-treated mice. SLPI-deficient mice were administered DSS to induce colitis and sustained severe inflammation compared with wild-type mice. The colonic mucosa of SLPI-deficient mice showed more severe inflammation with neutrophil infiltration and higher levels of proinflammatory cytokines compared with control mice. Moreover, neutrophil elastase (NE) activity in SLPI-deficient mice was increased and IEB function was severely impaired in the colon, accompanied with the increased number of apoptotic cells. Importantly, we demonstrated that DSS-induced colitis was ameliorated by administration of protease inhibitor SSR69071 and recombinant SLPI. These results suggest that the protease inhibitory activity of SLPI protects from colitis by preventing IEB dysfunction caused by excessive NE activity, which provides insight into the novel function of SLPI in the regulation of gut homeostasis and therapeutic approaches for IBD.

Randomized Trial of Perfusion-Based Circulatory Management in Infants of Very Low Birth Weight.

OBJECTIVE: To establish the superiority of blood flow (BF)-based circulatory management over conventional blood pressure (BP)-based management strategies used for preventing intraventricular hemorrhage (IVH) in infants of very low birth weight (VLBW). STUDY DESIGN: We conducted a nonblinded, single-centered randomized trial with the aim to prevent IVH by managing BF. Infants with VLBW were assigned randomly to a BF-based group or BP-based (BP group) circulatory management group. The incidence of IVH was the outcome of interest. The IVH also data were compared among healthy patients and patients responsive and unresponsive to the intervention. RESULTS: A total of 219 and 220 infants with VLBW were assigned to the BF and BP groups, respectively. The IVH incidence rate was lower in the BF group, but the difference was not statistically significant (BF group, 6.8% vs BP group, 10.9%; P = .14). In 21% of patients of the BP group and 20% of the BF group, the intervention failed. In BF group, the IVH incidence rate was significantly greater in infants with unsuccessful intervention when compared with healthy individuals (6% vs 23%, P = .001). Multivariate logistic regression analysis revealed a correlation between low blood flow and IVH (aOR 3.24; 95% CI 1.49-7.08, P = .003) but not between low BP and IVH (P = .73). CONCLUSIONS: The BF management protocol did not significantly decrease the incidence of IVH. However, after further optimization, we speculate the treatment strategy holds promise in decreasing the incidence of IVH. Trial registration UMIN-CTR: UMIN000013296.

A simple scoring method to predict augmented renal clearance in haematologic malignancies.

WHAT IS KNOWN AND OBJECTIVE: Augmented renal clearance (ARC; hyperfiltration with over 130 mL/min/1.73 m2 of creatinine clearance (CLcr )) commonly occurs in critically ill patients. Recent reports indicate that ARC also occurs in haematologic malignancies. However, the risk factors for ARC in haematologic malignancies remain unknown, and there is no established method to predict ARC in haematologic malignancies. Our objective was to explore the risk factors for ARC retrospectively and develop a scoring method to predict ARC. METHODS: A single-centre, retrospective, observational cohort study was conducted at the Sendai Medical Center (Sendai, Japan); 133 patients (April 2017-March 2019) and 41 patients (April-November 2019) with haematopoietic tumours who were administered vancomycin were enrolled in the analysis and validation cohorts, respectively. To define ARC, we calculated the vancomycin serum concentration when CLcr  = 130 mL/min/1.73 m2 using a one-compartment model. Patients with ARC were defined as those whose actual concentration of vancomycin remained lower than the calculated concentration. Using the analysis cohort, we explored risk factors of ARC and developed a scoring method to predict ARC in haematologic malignancies. The reproducibility of the scoring system was demonstrated using the validation cohort. RESULTS AND DISCUSSION: Through multivariate analysis, young age (P < .001), leukaemia (P = .001) and low serum creatinine (P < .001) were identified as risk factors. According to this result, we established the ARC detection method: age ≤ 50 years = 3 points, 50 years < age ≤65 years = 1 point, leukaemia = 2 points, low SCr = 2 points; patients scoring ≥ 5 points represent the ARC high-risk group. Using this scoring system, we could detect ARC with a sensitivity and specificity of 60.0% and 89.7% in the analysis cohort and 90.0% and 90.9% in the validation cohort, respectively. WHAT IS NEW AND CONCLUSION: Our scoring method could predict ARC in haematologic malignancies and is useful as a simple screening tool for ARC.

[One Case of Accessory Breast Cancer Complicated by Contralateral Breast Cancer].

We experienced a case of right sided accessory breast cancer complicated by contralateral breast cancer. A 50-year-old woman came to us for an examination because a tumor in her left breast was pointed out at breast cancer screening. A breast MRI confirmed a tumor in her left breast and a tumor continuing from the skin to the subcutis of the right axilla. A skin biopsy for the tumor in the right axilla and a core needle biopsy(CNB)for the tumor in the left breast were performed. The pathological result of the CNB for the left breast indicated an invasive ductal carcinoma of the tubular formative scirrhous type. Although the tumor of the right axilla was poorly differentiated adenocarcinoma demonstrating cord-like arrays, it was examined by skin biopsy and therefore no deep part of the tissue was included. We conducted immunostaining, in consideration of the possibility of metastasis from the left sided breast cancer. ER, PgR, mammaglobin, GATA 3 were positive, strongly suggesting that the tumor in the right axilla was also derived from a mammary gland. We also performed a wide local excision of the right axilla plus axillary dissection(level Ⅰ)in addition to conducting a left mastectomy plus sentinel lymph node biopsy, in consideration of the possibility of primary right sided accessory breast cancer. The pathological result following surgery confirmed a difference in the histologic features between both sides, residual normal accessory mammary glands around the tumor on the right side, and the presence of rich DCIS and a lobular replacement image, leading to a definitive diagnosis of primary invasive ductal carcinoma of the accessory breast on the right side.

Investigation of Appropriate Inhalation Technique for Mometasone Furoate Dry Powder Inhaler.

The aim of this study was to establish an appropriate inhalation method with a mometasone furoate dry powder inhaler (MF-DPI). Utilizing a tone-based inhalation training device, we investigated the maximum peak inspiratory flow rate time (Tmax PIFR) and peak inspiratory flow rate (PIFR) to determine whether either had an influence on lung deposition with use of an MF-DPI. A low tone indicated a PIFR of 28 L/min and a high tone that of 40 L/min, while 60 L/min was considered to be the standard. We established an inhalation profile in consideration of a human inhalation pattern, in which Tmax PIFR was set at 0.5 s (Tmax PIFR 0.5 s) and 2.5 s (Tmax PIFR 2.5 s). The reference cut-off value derived with a cascade impactor test was used for evaluation of the rate of delivered dose in the lung, which was the amount of drug from stage 3 to 7 at all PIFRs. We then investigated the relationship of the fine particle fraction (FPF) with the claimed dose at Tmax PIFR of 0.5 s and PIFR. There were no differences among the Tmax PIFR values for the doses emitted from the device or for the rate of delivered doses in stages 3-7. However, FPF for the claimed dose at 40 L/min was significantly lower than that at 60 L/min, which was dependent on PIFR. Our results showed that PIFR but not Tmax PIFR has an effect on lung deposition after inhalation with an MF-DPI.

Repeated folding stress-induced morphological changes in the dermal equivalent.

BACKGROUND/PURPOSE: Repeated mechanical stresses applied to the same region of the skin are thought to induce morphological changes known as wrinkle. However, the underlying mechanisms are not fully understood. To study the mechanisms, we examined effects of repeated mechanical stress on the dermal equivalent. METHODS: We developed a novel device to apply repeated folding stress to the dermal equivalent. After applying the mechanical stress, morphological changes of the dermal equivalent and expression of several genes related to extracellular matrix turn over and cell contraction were examined. RESULTS: The repeated folding stress induced a noticeable decrease in the width of the dermal equivalent. The mechanical stress altered orientations of collagen fibrils. Hydroxyproline contents, dry weights and cell viability of the dermal equivalents were not affected by the mechanical stress. On the other hand, Rho-associated coiled-coil-containing kinase (ROCK) specific inhibitor Y27632 completely suppressed the decrease in the width of the dermal equivalent. CONCLUSION: The present results revealed that either degradation of collagen or changes in the number of cells were not responsible for the decrease in the width of the dermal equivalent and indicate that the repeated mechanical stress induces unidirectional contraction in the dermal equivalent through the RhoA-ROCK signaling pathway.

Considerations for non-clinical safety studies of therapeutic peptide vaccines.

Guidelines for non-clinical studies of prophylactic vaccines against infectious diseases have been published widely, but similar guidelines for therapeutic vaccines, and especially therapeutic peptide vaccines, have yet to be established. The approach to non-clinical safety studies required for therapeutic vaccines differs from that for prophylactic vaccines due to differences in the risk-benefit balance and the mechanisms of action. We propose the following guidelines for non-clinical safety studies for therapeutic peptide vaccines. (i) Since the main safety concern is related to the immune response that might occur at normal sites that express a target antigen, identification of these possible target sites using in silico human expression data is important. (ii) Due to the strong dependence on HLA, it is not feasible to replicate immune responses in animals. Thus, the required non-clinical safety studies are characterized as those detecting off-target toxicity rather than on-target toxicity.

Quality Culture and Knowledge Management in the Japanese Pharmaceutical Industry-A Cross-Sectional Study and Case Report.

In the past few years, there have been several instances of illicit pharmaceutical manufacturing in Japan. Insufficient good manufacturing practice compliance and lack of quality culture in some pharmaceutical companies have been suggested as the underlying reasons for such cases. We aimed to focus on knowledge management and fostering of quality culture in pharmaceutical companies in Japan to understand their current situation and find a strategy for the availability of high-quality reliable pharmaceutical products. A wide-ranging questionnaire survey was conducted to understand the issues related to knowledge management and fostering of quality culture across pharmaceutical companies in Japan. A published investigation report on an illicit manufacturing case was closely examined by organizing the available facts using the diagram. Based on 395 responses to the questionnaire survey, we found that although pharmaceutical companies understand the importance of knowledge management and quality culture, issues exist in their operational methods. A total of 94% of the respondents agreed that they mentioned "knowledge management" as an enabler of the Pharmaceutical Quality System of ICH Q10, and 98% of the respondents accepted that insufficient fostering of quality culture leads to corporate risk. However, the survey revealed that many companies are struggling with this approach. Based on a report on an illicit manufacturing case, we analyzed the direct causes of misconduct and prepared a systematic summary that can be easily comprehended. Comparison of the illicit manufacturing case report with our questionnaire results suggests that many pharmaceutical companies do not regard the misconduct case as a situation that could occur in their company. With the revision of the Pharmaceuticals and Medical Devices Act and good manufacturing practice Ministerial Ordinance, we advocate the need for all employees of pharmaceutical companies to reconsider the priorities of their companies from the patient perspective.

Differences in clinical features and morphology between differentiated and undifferentiated gastric cancer after Helicobacter pylori eradication.

BACKGROUND/AIMS: Although undifferentiated gastric cancer (UGC) diagnosed after Helicobacter pylori eradication (HPE) carries a poor prognosis, characteristics of post-HPE UGC have not been evaluated in detail because of its low incidence. Therefore, we compared the clinicopathologic characteristics of UGC and differentiated gastric cancers (DGC) diagnosed after successful HPE. METHODS: GC lesions from patients who had successfully completed HPE and who had undergone upper gastrointestinal endoscopy between January 2004 and March 2016 were analyzed. Tumors were divided into DGC and UGC groups. Clinicopathologic factors of background and tumor characteristics were compared using univariate and multiple logistic analyses. RESULTS: A total of 129 tumors from 115 patients were evaluated; 113 tumors were in the DGC group and 16 in the UGC group. Depressed-type tumors (P = 0.024) and sub-submucosal invasion (P<0.001) were significantly higher in the UGC group. The UGC group had larger tumor diameters (25.9±7.3 mm) than the DGC group (13.2±10.2 mm) (P<0.001). Multivariate analysis showed that female sex (odds ratio [OR] 3.24, 95%CI:1.02-10.37; P = 0.047) and absent follow-up (OR 4.99, 95%CI:1.60-15.57; P = 0.006) were significant independent risk factors for UGC. The DGC group showed a gradually decreasing temporal trend by trend test (P = 0.015), while the UGC group showed a relatively constant incidence over time, although the number of cases was small. CONCLUSION: UGC was diagnosed even after long time spans following HPE, although the number of cases was small. Female sex, and especially absent follow-up, were risks for post-HPE UGC, suggesting that diligent long-term follow-up after HPE is essential.

Saireito, a Japanese herbal medicine, alleviates leaky gut associated with antibiotic-induced dysbiosis in mice.

Antibiotics disrupt normal gut microbiota and cause dysbiosis, leading to a reduction in intestinal epithelial barrier function. Disruption of the intestinal epithelial barrier, which is known as "leaky gut", results in increased intestinal permeability and contributes to the development or exacerbation of gastrointestinal diseases such as inflammatory bowel disease and irritable bowel syndrome. We have previously reported on a murine model of intestinal epithelial barrier dysfunction associated with dysbiosis induced by the administration of ampicillin and vancomycin. Saireito, a traditional Japanese herbal medicine, is often used to treat autoimmune disorders including ulcerative colitis; the possible mechanism of action and its efficacy, however, remains unclear. In this study, we examined the efficacy of Saireito in our animal model for leaky gut associated with dysbiosis. C57BL/6 mice were fed a Saireito diet for the entirety of the protocol (day1-28). To induce colitis, ampicillin and vancomycin were administered in drinking water for the last seven consecutive days (day22-28). As previously demonstrated, treatment with antibiotics caused fecal occult bleeding, cecum enlargement with black discoloration, colon inflammation with epithelial cell apoptosis, and upregulation of pro-inflammatory cytokines. Oral administration of Saireito significantly improved antibiotics-induced fecal occult bleeding and cecum enlargement by suppressing inflammation in the colon. Furthermore, Saireito treatment ensured the integrity of the intestinal epithelial barrier by suppressing apoptosis and inducing cell adhesion proteins including ZO-1, occludin, and E-cadherin in intestinal epithelial cells, which in turn decreased intestinal epithelial permeability. Moreover, the reduced microbial diversity seen in the gut of mice treated with antibiotics was remarkably improved with the administration of Saireito. In addition, Saireito altered the composition of gut microbiota in these mice. These results suggest that Saireito alleviates leaky gut caused by antibiotic-induced dysbiosis. Our findings provide a potentially new therapeutic strategy for antibiotic-related gastrointestinal disorders.

Association between Childhood Maltreatment History and Premenstrual Syndrome.

Childhood maltreatment history has known relationships with various mental and physical diseases; however, little is known about its association with premenstrual syndrome (PMS). In this study, we investigated the association between childhood maltreatment history and PMS among young women in Japan. In a Japanese city, we approached 3815 women aged 10-60 years who visited a gynecology clinic and one general practice clinic. A questionnaire on childhood maltreatment history and PMS was administered to them. We observed that women with histories of childhood maltreatment demonstrated a significantly increased risk of PMS compared with those without such histories (odds ratio: 1.47, 95% confidence interval: 1.20-1.81). Particularly, women with childhood physical or emotional abuse demonstrated a stronger association with PMS, whereas other forms of childhood maltreatment (emotional neglect, witnessing of intimate-partner violence, or sexual abuse) were not associated with PMS. Our results illustrate that childhood maltreatment may be a risk factor for PMS.

Conservatively Treated Femoral Intertrochanteric Fracture With Early Asymptomatic Novel Coronavirus Disease 2019 (COVID-19): A Case Report.

INTRODUCTION: The ongoing outbreak of novel coronavirus disease 2019 (COVID-19) is a worldwide problem. Although diagnosing COVID-19 in fracture patients is important for selecting treatment, diagnosing early asymptomatic COVID-19 is difficult. We describe herein a rare case of femoral intertrochanteric fracture concomitant with early asymptomatic novel COVID-19. CASE PRESENTATION: An 87-year-old Japanese woman was transferred to our emergency room with a right hip pain after she fell. She had no fever, fatigue, or respiratory symptoms on admission and within the 14 days before presenting to our hospital, and no specific shadow was detected in chest X-ray. However, chest computed tomography (CT) was performed considering COVID-19 pandemic, and showed ground-glass opacities with consolidation in the dorsal segment of the right lower lung field. Then, qualitative real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) was carried out and turned out to be positive. She was diagnosed right femoral intertrochanteric fracture with concomitant COVID-19 infection. Conservative treatment was applied to the fracture due to infection. After admission, fever and oxygen demand occurred but she recovered from COVID-19. Throughout the treatment period, no cross-infection from the patient was identified in our hospital. CONCLUSION: This case highlights the importance of considering chest CT as an effective screening method for infection on hospital admission in COVID-19-affected areas, especially in trauma patients with early asymptomatic novel COVID-19.

[Good Laboratory Practice: Initial Development, Necessity, and Issues of Data Reliability in Basic Research].

This review describes the initial development of good laboratory practice (GLP) and follows the discoveries of quality control problems in labs that conducted tests in U.S. pharmaceutical companies. In addition to introducing the essence of the GLP standards, how the GLP ensures the reconstructability and reproducibility of study results is explained in detail. Issues in nonclinical safety studies in drug development and approaches of the Japanese Pharmaceuticals and Medical Devices Agency to overcome them are also described. It is hoped that this review is helpful not only to those who work on drug development but also to faculties and students who work in academia and are involved in basic research when they attempt to resolve problems related to ensuring the reliability of basic research and research integrity.

ESTIMATION OF ORGAN DOSES AND EFFECTIVE DOSES BASED ON IN-PHANTOM DOSIMETRY FOR INFANT DIAGNOSTIC CARDIAC CATHETERISATIONS WITH NOVEL X-RAY IMAGING TECHNOLOGY.

The present study evaluated the organ and effective doses in infant diagnostic cardiac catheterisation performed using a modern x-ray imaging unit by in-phantom dosimetry. In addition, conversion factors from dose-area product (DAP) to effective dose were determined. The organ and effective doses in 1-year old during diagnostic cardiac catheterisations were measured using radiophotoluminescence glass dosemeters implanted into an infant anthropomorphic phantom. The mean effective doses, evaluated according to the International Commission on Radiologic Protection Publication 103, were 4.0 mSv (range: 1.5-8.7 mSv). The conversion factors from DAP to effective dose were 2 and 3.5 mSv (Gy cm2)-1 for posteroanterior and lateral fluoroscopy, respectively, and 1.8 and 3.3 mSv (Gy cm2)-1 for posteroanterior and lateral cineangiography, respectively. The dose data and conversion factors evaluated in the present study may be useful for estimating radiation exposure in infants during diagnostic cardiac catheterisation.

Analysis of Multiply Charged Poly(ethylene oxide-co-propylene oxide) Using Electrospray Ionization-Ion Mobility Spectrometry-Mass Spectrometry.

Poly(ethylene oxide), poly(propylene oxide), and their copolymer (poly(EO-co-PO)) were analyzed by electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS). ESI produced multiply charged analytes of 2 to 5 Na+ additions, and they were separately observed in a 2D map of m/z value vs. drift time. The collision cross-section of the analyte polymers was almost linearly proportional to (molecular weight)0.644, except for the analytes with 2Na+ addition; a nonlinear relation called "folding" was significantly observed for the analytes with 2Na+ addition. An increase in electrostatic repulsion, because of the increase in Na+ addition, suppressed the folding of the polymer. Analyses of poly(EO-co-PO) with different EO compositions revealed that the copolymer with high EO composition tended to show folding. The separation of highly multiply charged poly(EO-co-PO)s with different EO contents by ESI-IMS-MS was successfully demonstrated.

ESTIMATION OF ORGAN DOSES AND EFFECTIVE DOSES BASED ON IN-PHANTOM DOSIMETRY FOR PAEDIATRIC DIAGNOSTIC CARDIAC CATHETERISATION.

The present study evaluated the organ doses, effective doses and conversion factors from the dose-area product to effective dose in pediatric diagnostic cardiac catheterization performed by in-phantom dosimetry and Monte Carlo simulation. The organ and effective doses in 5-y-olds during diagnostic cardiac catheterizations were evaluated using radiophotoluminescence glass dosemeters implanted into a pediatric anthropomorphic phantom and PCXMC software. The mean effective dose was 3.8 mSv (range: 1.8-7.5 mSv). The conversion factors from the dose-area product to effective dose were 0.9 and 1.6 mSv (Gy cm2)-1 for posteroanterior and lateral fluoroscopy, respectively, and 0.9 and 1.5 mSv (Gy cm2)-1 for posteroanterior and lateral cineangiography, respectively. Effective doses evaluated using the pediatric dosimetry system agreed with those obtained using PCXMC software within 12%. The dose data and conversion factors evaluated may guide the estimation of exposure doses in children undergoing diagnostic cardiac catheterization.

Macrophage migration inhibitory factor (MIF) is essential for development of zebrafish, Danio rerio.

Macrophage migration inhibitory factor (MIF) was discovered as the first cytokine that inhibited the random migration of macrophages. Recently, MIF has been reported to be involved in embryonic development in higher vertebrates. In fish, however, nothing is known about the function of MIF at early life stages, although immunological functions of MIF have been reported in adult fish. To elucidate the function of MIF during embryonic development in fish, we examined expression patterns and function of the zebrafish MIF gene using antisense morpholino-mediated knockdown (morpholino oligonucleotide-MO). In whole-mount in situ hybridization analysis, zebrafish MIF mRNA was detected in developing eyes, tectum, branchial arches, pectoral fin buds, liver and gut. The onset of MIF mRNA expression coincided with the beginning of tissue differentiation during embryogenesis. MIF-MO-injected embryos (morphants) displayed malformed eyes, abnormal swelling in the tectum and fourth ventricle region, and undeveloped jaw cartilage and pectoral fins. An increased number of apoptotic cells in the eye and neural tissues were observed in MIF morphants by histological analysis and acridine orange staining. Moreover, proliferating cell nuclear antigen (PCNA)-positive cells were reduced in morphant eyes. These results suggest that MIF is essential for normal embryonic development even at the level of teleosts and that it functions as a growth factor for the proliferation and differentiation of embryonic tissues.

Expression analysis of two Eomesodermin homologues in zebrafish lymphoid tissues and cells.

Eomesodermin (Eomes) is a T-box transcription factor that is involved in mesoderm formation in most vertebrates. Eomes is also expressed in CD8+ T cells and NK cells. No information is available on the role of Eomes in the immune system of lower vertebrates to date, although developmental studies on Eomes (Eomes1) have been performed in zebrafish. Here we report the identification of a second Eomes (Eomes2) in zebrafish and compare expression of the two Eomes genes in the immune system. Zebrafish Eomes1 and Eomes2, composed of 661 and 534 amino acids, respectively, share 49.3% amino acid identity in their coding regions and 88.7% amino acid identity in their T-box regions. Conserved synteny between regions of the human and zebrafish genomes, gene organization and phylogenetic analysis all indicate that the zebrafish Eomes2 gene is a homologue of mammalian Eomes, as previously found for zebrafish Eomes1. Eomes1 mRNA was found to be expressed in the gonad, body kidney, spleen and gill, while Eomes2 mRNA was not detected in any of these tissues. However, strong expression of both Eomes mRNAs was detected in the leukocytes from the spleen, followed by those from body kidney and peripheral blood, with expression of Eomes1 always stronger than that of Eomes2. RT-PCR analysis of body kidney cells sorted by FACS revealed that Eomes1 was expressed strongly in lymphocytes, weakly in blast cells, and was not expressed in granulocytes, while Eomes2 was expressed weakly in lymphocytes. These results suggest that both Eomes genes are involved in the zebrafish immune response, particularly in lymphocyte function as has been found in mammals.