Induced Pluripotent Stem Cell-Derived Hematopoietic Embryoid Bodies Improve Mouse Status in Septic Peritonitis.

We examined the efficacy of embryoid bodies from 6-day induced pluripotent stem cells an in vivo sepsis model. Injection of embryoid bodies to septic mice improved the condition of their lungs and significantly increased their survival rate. Although embryoid bodies secretedsphingosine-1-phosphate in vitro, its serum levels in mouse plasma were significantly reduced compared to that in the control (untreated mice receiving PBS). Low concentrations of sphingosine-1-phosphate protected endothelial cells, while high concentrations disrupted endothelial barrier integrity. Therefore, exogenous sphingosine-1-phosphate secreted by embryoid bodies during early stage of sepsis might down regulate endogenous production of sphingosine-1-phosphate. Inhibition of excessive sphingosine-1-phosphate release protects against endothelial injury and suppresses a vicious cycle of inflammatory reactions. The obtained results open new prospects in induced pluripotent stem cells-based therapy for sepsis.

Induced Pluripotent Stem Cell-Derived Hematopoietic Embryoid Bodies Secrete Sphingosine-1-Phosphate and Revert Endothelial Injury.

The possibility of sphingosine-1-phosphate production by induced pluripotent stem cells is examined to assess their potential in treatment of sepsis. The hematopoietic embryoid bodies were derived from the culture of 6-day-old differentiated induced pluripotent stem cells. These embryoid bodies secreted sphingosine-1-phosphate, an important bioactive lipid that regulates integrity of the pulmonary endothelial barrier, prevents elevation of its permeability, and impedes the formation of stress fibers in human endotheliocytes derived from umbilical vein. The data attest to potentiality of induced pluripotent stem cells in treatment of sepsis.

Ethanol attenuates vasorelaxation via inhibition of inducible nitric oxide synthase in rat artery exposed to interleukin-1ß.

Nitric oxide produced by inducible nitric oxide synthase (iNOS) regulates sepsis-induced hypotension. During septic shock, interleukin (IL)-1ß is synthesized in endothelial cells and smooth muscle cells by endotoxin. Ethanol (EtOH) suppresses endotoxin-induced hypotension. The present study aimed to elucidate the effect of EtOH on gradual relaxation and iNOS expression induced by IL-1ß in isolated rat superior mesenteric arteries (SMAs). Exposure to IL-1ß-induced contraction in SMA rings, followed by a gradual relaxation of phenylephrine precontracted tone. Contraction was abolished by indomethacin (IM), cycloheximide (Chx), and endothelium denudation. In contrast, the gradual relaxation was abolished by NOS inhibitors, Chx, endothelium denudation, and inhibited by EtOH (50 and 100 mM). However, IM had no effect on relaxation. Western blot analysis demonstrated that iNOS expression was induced by IL-1ß and was inhibited by EtOH and endothelium denudation. Furthermore, messenger RNA expression of iNOS, but not endothelial NOS, was inhibited by EtOH. These data suggest that IL-1ß-induced contraction is mediated by thromboxane A2, whereas IL-1ß-induced relaxation occurs via NO derived from iNOS. The endothelium plays an important role in vasorelaxation. Taken together, EtOH inhibits IL-1ß-mediated vasorelaxation by suppressing endothelium iNOS expression. This study provides the first evidence of EtOH -induced inhibition of IL-1ß-mediated vasorelaxation.

Three-dimensional surface figure measurement of high-accuracy spherical mirror with nanoprofiler using normal vector tracing method.

Processing technology using an extreme ultraviolet light source, e.g., next-generation lithography, requires next-generation high-accuracy mirrors. As it will be difficult to attain the degree of precision required by next-generation high-accuracy mirrors such as aspherical mirrors through conventional processing methods, rapid progress in nanomeasurement technologies will be needed to produce such mirrors. Because the measuring methods used for the surface figure measurement of next-generation mirrors will require high precision, we have developed a novel nanoprofiler that can measure the figures of high-accuracy mirrors without the use of a reference surface. Because the accuracy of the proposed method is not limited by the accuracy of a reference surface, the measurement of free-form mirrors is expected to be realized. By using an algorithm to process normal vectors and their coordinate values at the measurement point obtained by a nanoprofiler, our measurement method can reconstruct three-dimensional shapes. First, we measured the surface of a concave spherical mirror with a 1000-mm radius of curvature using the proposed method, and the measurement repeatability is evaluated as 0.6 nm. Sub-nanometer repeatability is realized, and an increase in the repeatability would be expected by improving the dynamic stiffness of the nanoprofiler. The uncertainty of the measurement using the present apparatus is estimated to be approximately 10 nm by numerical simulation. Further, the uncertainty of a Fizeau interferometer is also approximately 10 nm. The results obtained using the proposed method are compared with those obtained using a Fizeau interferometer. The resulting profiles are consistent within the range of each uncertainty over the middle portions of the mirror.

Association of novel promoter single nucleotide polymorphisms in vasopressin V1a receptor gene with essential hypertension in nonobese Japanese.

We studied the association between four novel single nucleotide polymorphisms (SNPs) in the promoter region of V1aR gene and essential hypertension in 620 Japanese subjects (365 hypertensives and 255 healthy). A significant association was found between one of the genotypes and alleles at SNP -6951 and hypertension in a subsample of nonobese individuals. This association demonstrated an independent risk for nonobese hypertension.

Elevation of both cyclooxygenase-2 and prostaglandin E2 receptor EP3 expressions in rat placenta after uterine artery ischemia-reperfusion.

Intrauterine growth restriction (IUGR) has a multifactorial pathogenesis and is an important cause of perinatal mortality. The relationship between fetal weight and placental blood flow in an animal model of IUGR has been investigated, showing that fetal growth is regulated by placental blood flow. The aim of the present study was to determine whether ischemia-reperfusion (I/R) injury stimulates the prostaglandin E2 (PGE2) system or the vascular endothelial growth factor (VEGF) system in the placenta of a rat IUGR model. COX-2 is reported to be involved in ischemic damage in many organs. There are 4 types of PGE2 receptor (EP1, EP2, EP3 and EP4). It is well known that EP1 and EP3 is associated with vasoconstriction. In the present study, vessels were occluded in the right uterine horn on day 17 of pregnancy in rats, and the clamps were removed after 30 min of ischemia. At 24h, 48 h, and 5 days after I/R injury, the live fetuses and placentas were obtained by cesarean section. This study revealed that I/R injury caused IUGR 5 days after the treatment. COX-2 expression and EP3 receptor expression were significantly elevated at 24h after I/R injury, but VEGF mRNA expression was not altered in the placenta from the ischemic horn compared with the non-ischemic horn. These results suggested that induction of the COX-2-EP3 system in the placenta may be one of the causes of IUGR induced by uterine ischemia, because the EP3 receptor and PGE2 are well known to mediate vasoconstriction in many organs.

IgM secretion and absorption in the materno-fetal interface of a viviparous teleost, Neoditrema ransonneti (Perciformes; Embiotocidae).

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. 820k, 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.

Neoadjuvant chemotherapy with cisplatin, aclacinomycin A, and mitomycin C for cervical adenocarcinoma--a preliminary study.

Between 1989 and 2002, 28 patients with locally advanced cervical adenocarcinoma (bulky IB-IIIB) were recruited for a pilot study aimed at evaluation of the effectiveness of neoadjuvant chemotherapy with cisplatin, aclacinomycin-A, and mitomycin-C (PAM), followed by radical surgery. This regimen was administrated intra-arterially or intravenously. In addition to patients treated with PAM, we retrospectively analyzed the prognoses of 26 patients in stage I and II, who had been treated between 1975 and 1981 with radical surgery with/without radiation therapy. Twenty-eight patients received PAM therapy as neoadjuvant chemotherapy, and 75.0% of the 16 intra-arterially infused patients showed a response, as did 66.7% of the 12 intravenously infused patients. There was a significant difference in the 5-year prognosis of stage II (PAM group, 72.9%; without-PAM group, 36.4%). The results suggest that, as the free space in the parametrium is widened by neoadjuvant chemotherapy with PAM, it is possible that the tumor could be completely resected by radical hysterectomy. Thus, neoadjuvant chemotherapy with PAM is expected to improve the survival rate of patients with advanced cervical adenocarcinoma by the preliminary study. However, the survival rates of stage II with lymph node metastasis in the without-PAM group seem low, and we must also consider that the various technologies to evaluate and treat the cervical adenocarcinomas, e.g. computed tomography, magnetic resonance imaging, and surgical equipments, had improved during 1989-2002 than was the scenario during 1975-1981, and these improvements contributed to better prognosis. A prospective-randomized study is needed to assess the value of this approach compared with standard management.

Imaging studies in patients with malignant melanoma in the female genital tract.

The purpose of this study was to review magnetic resonance imaging (MRI) and pathologic features of primary malignant melanoma (melanoma) in the female genital tract. We retrospectively evaluated MRI in six women with melanoma of the genital tract. The signal intensity of the tumor on T1-weighted images (WI) was compared with the amount of melanin granules in hematoxylin-eosin stained sections of resected specimen. On T1WI, four melanomas showed a high signal intensity, one intermediate, and one low. The four melanomas with a high signal intensity on T1W1 were rich in melanin granules, while the one intermediate tumor had few granules. The other one was amelanotic. We believe that a high signal on T1WI is characteristic of primary melanotic melanoma of the female genital tract. Our findings suggest that it is strongly influenced by the presence of melanin granules.

Hemorrhagic infarction of fibroma. MR imaging appearance.

We experienced a case of hemorrhagic infarction of the ovarian fibroma and that indicated the characteristic following appearance: exhibiting a high signal intensity area observed at the periphery of mass on T1-weighted MRI (magnetic resonance imaging). It was thought that this appearance developed because hemorrhagic infarction was caused by subacute ovarian torsion. This is a useful finding for suspecting hemorrhagic infarction preoperatively.

Lipid peroxidation in the rat brain after CO inhalation is temperature dependent.

We reported previously that 7-hydroperoxycholesterols, 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha-OOH and 7 beta-OOH), indicated lipid peroxidation. In the present study, we measured not only 7-hydroperoxycholesterols but also oxysterols (7 alpha- and 7 beta-hydroxycholesterol, 7 alpha-OH, and 7 beta-OH) and 3 beta-hydroxycholest-5-en-7-one (7-keto) in the brains of rats that underwent either a sham operation (control), hypoxia, or CO inhalation (1005 ppm) at 37 degrees C for 90 min followed by 48 h of recovery. The levels of 7-hydroperoxycholesterols, 7 beta-OH, and 7-keto were low in the hypoxia group, while the levels were unaltered in the CO group compared with the controls. Among the three groups of CO inhalation, these levels were high in the hyperthermia group (39 degrees C), and the 7-hydroperoxycholesterols were low in the hypothermia group (32 degrees C), compared with the control group. The blood O(2) saturation was almost normal in the hypothermia group, while it was similarly low in the hyperthermia and normothermia groups. The temperature-dependent lipid peroxidation in the brain after CO inhalation and recovery can not be explained by hypoxia due to CO-hemoglobin formation, but may contribute to the delayed neuronal death following CO inhalation. Hypothermia may be applicable to treat patients after CO inhalation.

Heart 7-hydroperoxycholesterol and oxysterols are elevated in chronically ethanol-fed rats.

Recently, cholesterol hydroperoxides have been shown to be sensitive pathogenic markers of reactive oxygen species (ROS)-mediated damage though they have never been measured in heart tissue. We hypothesized that cholesterol hydroperoxides and oxysterols, putative cardiotoxic products of cholesterol oxidation, are elevated in the hearts of alcoholics as a consequence of ROS-mediated reactions. To test this, we measured 7alpha- and 7beta-hydroperoxycholest-5-en-3beta-ol (7alpha-OOH and 7beta-OOH) by HPLC with postcolumn chemiluminescence as well as 7alpha- and 7beta-hydroxycholesterol (7alpha-OH and 7beta-OH) and 3beta-hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto) by HPLC-UV in cardiac muscle of alcohol-fed rats. Alcohol feeding was carried out using a pair-feeding protocol with 35% of total dietary energy as ethanol; controls were pair-fed isocaloric glucose. After 6-7 wk treatment with alcohol, heart 7alpha-OOH, 7beta-OOH and 7beta-OH were significantly greater than in controls. Levels of heart phospholipid 16:0 and 18:1 were lower than in controls, while 18:0 and 18:2 were greater. This is the first report of the presence of 7alpha-OOH, 7beta-OOH and 7alpha-OH in cardiac tissue. The elevations in 7alpha-OOH and 7beta-OOH as well as 7beta-OH are evidence of increased oxidative stress and possible membrane changes. Alterations in the proportions of 16:0, 18:1, 18:2 and 18:0 in heart phospholipids provide further evidence of an altered membrane domain.

Specific detection of circulating tumor cells by reverse transcriptase-polymerase chain reaction of a beta-casein-like protein, preferentially expressed in malignant neoplasms.

Beta-casein-like protein (BCLP) is a putative protein on cervical cancer and exhibits immunological characteristics similar to those of bovine beta-casein. We evaluated if BCLP mRNA detection in the blood is useful in gynecologic malignancies. We examined 30 patients with uterine cancer, nine cultured cancer cell lines and 26 healthy women volunteers. From these study populations and samples, total RNA was obtained. Reverse transcriptase-PCR (RT-PCR) of BCLP was performed on each sample. Eighteen (60.0%) patients and 4 (15.4%) volunteers were positive for BCLP mRNA. The RT-PCR reached sensitivity and specificity of 60.0% and 84.6%, respectively. Of eight patients having diagnosed recurrence, 7 (87.5%) were positive for BCLP mRNA. In patients with recurrence, sensitivity and specificity were 87.5% and 50%, respectively. The expression of mRNA showed a correlation with recurrence, but no correlation with metastasis or histological type. BCLP was specifically expressed in cancer cells and might be an aid in clinical diagnosis.

Cyclic changes in expression of mRNA of vascular endothelial growth factor, its receptors Flt-1 and KDR/Flk-1, and Ets-1 in human corpora lutea.

OBJECTIVE: To evaluate the expression of mRNA of vascular endothelial growth factor (VEGF), its receptors Flt-1 and KDR/Flk-1, and Ets-1 in human corpora lutea. DESIGN: Prospective laboratory study. SETTING: University hospital in Japan. PATIENT(S): Women with regular menstrual cycles who underwent hysterectomy. INTERVENTION(S): Fifteen corpora lutea were obtained during hysterectomy (5 in the early luteal phase, 5 in the mid-luteal phase, and 5 in the late luteal phase). MAIN OUTCOME MEASURE(S): Expression of VEGF, Flt-1, KDR/Flk-1, and Ets-1 in human corpora lutea on northern blot analysis or immunohistochemistry. RESULT(S): Human corpora lutea in early luteal phase and mid-luteal phase had high VEGF mRNA expression. Expression of VEGF mRNA was significantly reduced in the late luteal phase. Immunohistochemistry showed that VEGF protein was expressed mainly in granulosa lutein cells and faintly in thecal lutein cells. Staining of VEGF protein was decreased in human corpora lutea in the late luteal phase. Expression of Flt-1 and KDR/Flk-1 mRNA was increased in the early luteal phase and mid-luteal phase and decreased in the late luteal phase. Immunohistochemistry showed that Flt-1 and KDR/Flk-1 proteins were expressed mainly in granulosa lutein cells and faintly in thecal lutein cells and endothelial cells in the early luteal phase and mid-luteal phase; their protein staining was reduced in the late luteal phase. Expression of Ets-1 mRNA changed similarly to VEGF and its receptor mRNA in human corpora lutea during the luteal phase. CONCLUSION(S): Levels of mRNA of VEGF and its receptors Flt-1 and KDR/Flk-1 in human luteal cells may be related to luteal function.

Two new variants of the Rhodococcus equi virulence plasmid, 90 kb type III and type IV, recovered from a foal in Japan.

This report describes the discovery of two new virulence plasmid types from a crossbred foal with Rhodococcus equi pneumonia in Kumamoto died with severe R. equi pneumonia and ulcerative enteritis. R. equi was isolated in large numbers and isolates from the foal were investigated for the presence of virulence-associated 15-17 kDa antigens (VapA) by colony blotting, using the monoclonal antibody 10G5, and by gene coding for VapA by PCR. Plasmid DNAs extracted from the isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII. The digestion patterns that resulted divided the plasmids of these isolates into two closely related types. The digestion patterns were then compared with eight representative virulence plasmid types (85 kb types I, II, III and IV, 87 kb types I and II, 90 kb types I and II), which have already been reported. None of the EcoRI and EcoT22I digestion patterns of the eight representative plasmids matched those of the two plasmid types. We tentatively designated these new plasmid types as 90 kb type III and type IV, since HindIII and BamHI digestion patterns of the two plasmid types were identical with those of a 90 kb type I plasmid. This study, demonstrated that there are at least 10 distinct but closely related plasmids present in isolates from horses in the world.

[Feasibility, activity, and change in the level of blood paclitaxel concentration after weekly paclitaxel therapy for a patient with recurrent ovarian cancer].

The patient was a 59-year-old woman with recurrent ovarian cancer. A CT scan of the abdomen showed enlargement of abdominal para-aortic lymph nodes (PAN) after the primary operation and 8 cycles of the combination chemotherapy with paclitaxel (TXL) and carboplatinum (CBDCA). As a second line chemotherapy for the patient, weekly administration of TXL (60 mg/m2/week x 3 weeks) was given. The toxicity was acceptable and less pronounced than with the standard TXL + CBDCA therapy. Peak blood TXL concentration, about 90 ng/ml, was achieved 4 hours after the administration of TXL. The blood TXL concentration was below the detectable limit 48 h after the administration of TXL. An almost 50% shrinkage in the size of the PAN was obtained after 2 cycles of treatment. Good QOL is being maintained without any repeated aggravation of the tumor.

Suppressed gap junctional intercellular communication in carcinogenesis of endometrium.

To examine whether and at which stage of endometrial carcinogenesis decreased connexin expression occurs, we investigated changes in the expression of the gap junction proteins, connexin 26 (Cx26), Cx32 and Cx43, in human endometrial hyperplasia and cancer samples. Forty-eight endometrial tissue samples (15 endometrial hyperplasias and 33 endometrial cancers) were subjected to immunofluorescence and RT-PCR analysis. In endometrial hyperplasia, Cx26 was aberrantly expressed in all samples as revealed immunohistochemically. There was weak or negative expression in 12 samples (80.0%) and diffuse expression in cytoplasm in 3 samples (20.0%). Cx32 expression in those samples was similar to that of Cx26; there was weak or negative expression in 11 samples (73.3%) and diffuse expression in 4 samples (26.7%). In endometrial cancer, Cx26 was expressed weakly or negatively in 25 samples (75.8%), diffusely in 6 samples (18.2%) and normally in 2 samples (6.1%), while Cx32 was expressed weakly or negatively in 26 samples (78.8%), diffusely in 5 samples (15.2%) and normally in 2 samples (6.1%). It was confirmed that weak staining of Cx26 and Cx32 was due to poor expression of their mRNA. All samples showed weak Cx43 protein expression as revealed by immunohistochemical analysis. In the majority of samples, concomitant expression levels of Cx26 and Cx32 protein were observed, confirming our long-term hypothesis that Cx26 and Cx32 are both abnormally regulated in a coordinated fashion in the endometrium. Our results indicate that during endometrial carcinogenesis, loss of gap junctional intercellular communication (GJIC) may occur due to the suppressed expression and the aberrant localization of connexin at relatively early stages.

Cloning and characterization of a tumor-associated antigen, beta-casein-like protein.

Monoclonal antibody (MAb) 1C5 reacts with 87% of uterine cervical adenocarcinoma and bovine beta -casein, but not with squamous cell carcinoma. To clarify the characteristics of the antigen (beta-casein-like protein; BCLP) recognized by MAb 1C5, molecular cloning was performed using 5' rapid amplification of the cDNA ends (5' RACE) and the oligo-capping method. The protein predicted from the cDNA consisting of 937 nucleotides comprises 222 amino acids. The BCLP gene and deduced amino acid sequences were novel and showed no similarity to known cancer-associated genes in the database. Northern blot analysis showed that a 1.1 kb transcript was ubiquitously expressed in cancer cell lines and was predominantly expressed in uterine cervical adenocarcinoma. To clarify the function of BCLP, BCLP cDNA was transfected into L929 cells, resulting in a significant increase in cell area, a downregulation of cell growth rate and a decrease in cell attachment. We conclude that BCLP might be associated with cell morphology and a regulation of growth pattern of tumor.

Changes in expression and subcellular localization of nuclear retinoic acid receptors in human endometrial epithelium during the menstrual cycle.

The endometrium is a uniquely dynamic tissue in that it undergoes monthly cycles of proliferation and secretory activity, and is regulated by ovarian steroid hormones. In this study, we focused on retinoic acid receptors (RAR and RXR) which are ligand-dependent transcription factors belonging to the large family of steroid hormones and are expected to affect to cell growth and differentiation in the endometrium. We analysed the expression and subcellular localization of the RA receptors in 57 samples of human endometrium by immunohistochemistry and Western blotting. In the nuclei of the endometrial epithelium, the RA receptors were expressed strongly in the proliferative phase. However, RAR were drastically reduced in the epithelial nuclei during the secretory phase in association with changes in serum oestradiol and in the expression of the oestrogen receptor. The expression of RXR was localized in the epithelial nuclei throughout the menstrual cycle. Confocal laser scanning microscopical observation clearly showed the difference in the localization between RAR and RXR in the secretory phase. Furthermore the findings of immuno-electron microscopy showed pooled RAR around the rough endoplasmic reticulum, suggesting that transport of these receptors to the nuclei is inhibited. These findings suggest that RAR and RXR work mainly in the proliferative phase and that in the endometrium RXR may play a different role to RAR during the secretory phase.