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ABSTRACT: Monomethylarginine, asymmetric dimethylarginine and symmetric dimethylarginine were separated on a Wakopak Combi ODS with an acetonitrile-100 mm potassium phosphate buffer (pH 7.0; 1:1, v/v). Dimethylarginines were derived from o-phthalaldehyde for the fluorescence detector and from 6-ferrocenyl-1-hexanethiol for the electrochemical detector. The detection limits of the dimethylarginines in spiked plasma were 0.3-0.5 pmol by electrochemical detection and 1-2 pmol by fluorescence detection. The detection limits were improved over 30 times by electrochemical detection and 10 times by fluorescence detection compared with previous reports. In previous derivatization liquid chromatography, the reaction solutions, o-phthalaldehyde, 2-mercaptethanol and dimethylarginines were unstable and required quick derivatization at 4°C. By our proposed pre-column methods, the dimethylarginines were derivatized at room temperature and the fluorescent products were stable for 6 h. The manipulation performance was greatly advanced compared with previous LC reports. This is the first report on stable and sensitive dimethylarginines by dual detection. The selectivity was also improved by dual detection. The proposed method was applied to preliminary monitoring of dimethylargines in plasma and urine. Copyright © 2012 John Wiley & Sons, Ltd.
Biomedical Chromatography 09/2012; · 1.97 Impact Factor
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ABSTRACT: Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 µg/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI) usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection.
PLoS ONE 01/2012; 7(7):e42257. · 4.09 Impact Factor
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ABSTRACT: Adenocarcinoma of the rete testis is a rare malignant tumor with a poor prognosis. About 60 cases of this adenocarcinoma have been reported in the literature. The diagnosis is often difficult and made incidentally. Herein, we report a case of adenocarcinoma of the rete testis and review the literature. Our patient was an 80-year-old man who presented with painless scrotal swelling for 2 years. Physical examination revealed an enlarged, hard mass of the left scrotum. The serum markers alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (beta-HCG), and carcinoembryonic antigen (CEA) were negative. Magnetic resonance imaging (MRI) showed a left hydrocele with central necrosis of the testis. After 4 months, the patient presented with appetite loss, general fatigue, and pain in the left scrotum. Positron emission tomography (PET) was performed in another hospital, and the patient was referred for a left testicular tumor, multiple lung metastases, and para-aorta lymph node metastasis. The patient underwent left high inguinal orchiectomy. Pathological examination revealed a hard whitish mass around the testis involving the epididymis and tunica vaginalis and spreading under the subcutaneous tissue. Histological examination revealed adenocarcinoma in the hilum of the testis, which extended to the subcutaneous tissue but not to the surface of the scrotum. The tunica albuginea was intact, and no invasion of carcinoma in the testis was seen. After the histological diagnosis of adenocarcinoma of the rete testis was confirmed, computed tomography (CT) was performed and showed multiple pulmonary nodules and para-aortica lymph node swelling of 3 cm diameter. Because the patient did not wish to receive chemotherapy or other aggressive treatment, he has been followed-up with palliative care since his diagnosis. Although local recurrence has occurred 4 months later, he is still alive for 8 months since his diagnosis.
Nippon Hinyōkika Gakkai zasshi. The japanese journal of urology 11/2010; 101(7):749-53.
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ABSTRACT: The new non-reversed transcriptase inhibitor (NRTI) drugs for treatment of acquired immunodeficiency syndrome (AIDS) are reported. An improvement in the sensitivity and selectivity of high-performance liquid chromatography was obtained by diamond electrode-electrochemical detector and fluorescence detector owing to different structural information. The four anti-retroviral NRTI drugs (abacavir, didanosine, lamivudine and zidovudine) were separated on a CapcellPak C18 UG120 column (250 mm x 4.6 mm I.D., 5 microm) with an acetonitrile-25 mM potassium dihydrogenphosphate buffer (pH 8.0; 1:9, v/v) as the mobile phase. We applied dual detection (electrochemical detection and florescence detection) for improving the peak identification and also for improved selectivity, which assisted monitoring by trace-volume samples (e.g., plasma). The electrochemical detector, equipped with a diamond electrode, was set at 2000 mV (applied voltage) and the fluorescence detector was set at excitation wavelength 275 nm and emission wavelength 315 nm. The detection limits of the four NRTIs in spiked plasma were 1-100 ng/ml by electrochemical detection and 5-10 pg/ml by fluorescence detection. The calibration graphs were linear up to 20 microg/ml by electrochemical detection and 10 microg/ml by fluorescence detection. This is the first report of LC analysis of NRTIs by electrochemical detection, also combined with fluorescence detection. The detection limits of didanosine, lamivudine and zidovudine were improved 20-fold by electrochemical detection and 500-fold by fluorescence detection compared to previous reports on UV detection. The selectivity was also improved by dual detection. The proposed method was applied to the preliminary monitoring of NRTIs in plasma.
Journal of chromatography. A 03/2009; 1216(15):3117-21. · 4.19 Impact Factor
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Satoru Hamada,
Masayuki Umemura,
Takeru Shiono,
Kensho Tanaka,
Ayano Yahagi,
M Dilara Begum,
Kiyotetsu Oshiro,
Yuko Okamoto,
Hisami Watanabe,
Kazuyoshi Kawakami,
Christina Roark,
Willi K Born,
Rebecca O'Brien,
Koichi Ikuta, Hiromichi Ishikawa,
Susumu Nakae,
Yoichiro Iwakura,
Takao Ohta,
Goro Matsuzaki
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ABSTRACT: IL-17A is originally identified as a proinflammatory cytokine that induces neutrophils. Although IL-17A production by CD4(+) Th17 T cells is well documented, it is not clear whether IL-17A is produced and participates in the innate immune response against infections. In the present report, we demonstrate that IL-17A is expressed in the liver of mice infected with Listeria monocytogenes from an early stage of infection. IL-17A is important in protective immunity at an early stage of listerial infection in the liver because IL-17A-deficient mice showed aggravation of the protective response. The major IL-17A-producing cells at the early stage were TCR gammadelta T cells expressing TCR Vgamma4 or Vgamma6. Interestingly, TCR gammadelta T cells expressing both IFN-gamma and IL-17A were hardly detected, indicating that the IL-17A-producing TCR gammadelta T cells are distinct from IFN-gamma-producing gammadelta T cells, similar to the distinction between Th17 and Th1 in CD4(+) T cells. All the results suggest that IL-17A is a newly discovered effector molecule produced by TCR gammadelta T cells, which is important in innate immunity in the liver.
The Journal of Immunology 10/2008; 181(5):3456-63. · 5.79 Impact Factor
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Masanobu Nanno,
Yasuyoshi Kanari,
Tomoaki Naito,
Nagamu Inoue,
Tadakazu Hisamatsu,
Hiroshi Chinen,
Ken Sugimoto,
Yasuyo Shimomura,
Hideo Yamagishi,
Tetsuo Shiohara,
Satoshi Ueha,
Kouji Matsushima,
Makoto Suematsu,
Atsushi Mizoguchi,
Toshifumi Hibi,
Atul K Bhan, Hiromichi Ishikawa
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ABSTRACT: T-cell receptor (TCR) gammadelta T cells are an important component of the mucosal immune system and regulate intestinal epithelial homeostasis. Interestingly, there is a significant increase in gammadelta T cells in the inflamed mucosa of patients with ulcerative colitis (UC). However, the role of gammadelta T cells in chronic colitis has not been fully identified.
TCRalpha-deficient mice, which spontaneously develop chronic colitis with many features of human UC including an increase in gammadelta T-cell population, represent an excellent model to investigate the role of gammadelta T cells in UC-like colitis. To identify the role of gammadelta T cells in this colitis, we herein have generated TCRgamma-deficient mice through deletion of all TCR Cgamma genes (Cgamma1, Cgamma2, Cgamma3, and Cgamma4) using the Cre/loxP site-specific recombination system and subsequently crossing these mice with TCRalpha-deficient mice.
An increase in colonic gammadelta T cells was associated with the development of human UC as well as UC-like disease seen in TCRalpha-deficient mice. Interestingly, the newly established TCRalpha(-/-) x TCRgamma(-/-) double mutant mice developed significantly less severe colitis as compared with TCRalpha-deficient mice. The suppression of colitis in TCRalpha(-/-) x TCRgamma(-/-) double mutant mice was associated with a significant reduction of proinflammatory cytokine and chemokine productions and a decrease in neutrophil infiltration.
gammadelta T cells are involved in the exacerbation of UC-like chronic disease. Therefore, gammadelta T cells may represent a promising therapeutic target for the treatment of human UC.
Gastroenterology 03/2008; 134(2):481-90. · 11.68 Impact Factor
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ABSTRACT: Improvement of the sensitivity and specificity of a simultaneous stress-free screening method for catechol estrogens as a potential prostate cancer marker in urine has been accomplished by HPLC with a diamond-electrode electrochemical detector and a fluorescence detector. Since taking urine samples generates less stress (or pain) than the drawing of blood, the method can readily be applied to almost any patient, and will also assist in improving the sensitivity and specificity of the prostatic specific antigen test. Catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 2-hydroxyestriol) and estrogens (estrone, estradiol, estriol) were separated on an Inertsil ODS-II column with acetonitrile-potassium dihydrogen phosphate (pH 3.0). The diamond-electrode electrochemical detector used had the great advantage of being a maintenance-free system, and could sequentially analyze hundreds of samples. Fluorescence detection improved the sensitivity 10-500 times (e. g., the LOD of 2-hydroxyestriol was improved 250 times) compared to previous electrochemical detection reports, and dual detection improved peak identification in the urine samples. The proposed method was applied to the simultaneous determination of catechol estrogens in spiked urine in a preliminary study on estrogens and PSA values in biopsy and prostate cancer patients.
Journal of Separation Science 10/2007; 30(14):2279-85. · 2.73 Impact Factor
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ABSTRACT: Immunoglobulin-A has an irreplaceable role in the mucosal defence against infectious microbes. In human and mouse, IgA-producing plasma cells comprise approximately 20% of total plasma cells of peripheral lymphoid tissues, whereas more than 80% of plasma cells produce IgA in mucosa-associated lymphoid tissues (MALT). One of the most biologically important and long-standing questions in immunology is why this 'biased' IgA synthesis takes place in the MALT but not other lymphoid organs. Here we show that IgA class-switch recombination (CSR) is impaired in inducible-nitric-oxide-synthase-deficient (iNOS-/-; gene also called Nos2) mice. iNOS regulates the T-cell-dependent IgA CSR through expression of transforming growth factor-beta receptor, and the T-cell-independent IgA CSR through production of a proliferation-inducing ligand (APRIL, also called Tnfsf13) and a B-cell-activating factor of the tumour necrosis factor (TNF) family (BAFF, also called Tnfsf13b). Notably, iNOS is preferentially expressed in MALT dendritic cells in response to the recognition of commensal bacteria by toll-like receptor. Furthermore, adoptive transfer of iNOS+ dendritic cells rescues IgA production in iNOS-/- mice. Further analysis revealed that the MALT dendritic cells are a TNF-alpha/iNOS-producing dendritic-cell subset, originally identified in mice infected with Listeria monocytogenes. The presence of a naturally occurring TNF-alpha/iNOS-producing dendritic-cell subset may explain the predominance of IgA production in the MALT, critical for gut homeostasis.
Nature 09/2007; 448(7156):929-33. · 36.28 Impact Factor
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ABSTRACT: Background: Patients with malignancy (n = 130) participated in the sperm cryopreservation program.Methods: After washing and concentrating, sperm was cryopreserved using KS-VIm cryoprotectant medium. Participant background factors such as age, marital status, underlying disease, presence or absence of previous treatment and semen findings (concentration, motility and morphology) were analyzed to determine parameters associated with the program.Results: Patients in their 20s were most common (64 cases) and 94 cases were unmarried at the first visit. The main underlying diseases were testicular tumor (53 cases), leukemia (43 cases) and malignant lymphoma (13 cases). The program was completed for 118 cases. For leukemia, all semen parameters were closer to normal in patients without previous treatment (untreated group, UG) compared with the treated group (TG). When semen findings in the UG were classified according to underlying disease, sperm concentration was lower in patients with testicular tumor compared with those who had leukemia or malignant lymphoma. Four couples underwent reproductive therapies with the cryopreserved sperm through assisted reproductive technology, and three babies were born to two couples.Conclusion: Sperm cryopreservation liberates patients with malignancy from iatrogenic infertility as a consequence of intensive therapy, allowing them to retain reproductive ability.
Reproductive Medicine and Biology 05/2007; 6(2):127 - 131.
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Chikara Nakasone,
Natsuo Yamamoto,
Masashi Nakamatsu,
Takeshi Kinjo,
Kazuya Miyagi,
Kaori Uezu,
Kiwamu Nakamura,
Futoshi Higa, Hiromichi Ishikawa,
Rebecca L O'brien,
Koichi Ikuta,
Mitsuo Kaku,
Jiro Fujita,
Kazuyoshi Kawakami
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ABSTRACT: The present study was designed to elucidate the role of Vgamma4(+) gammadelta T cells, a major subset of pulmonary gammadelta T cells, in host defense against infection with Streptococcus pneumoniae. The proportion and number of whole gammadelta T cells, identified as CD3(+) and TCR-delta(+) cells, and Vgamma4(+) gammadelta T cells, identified as CD3(+) and TCR-Vgamma4(+) cells, increased in the lungs at 3, 6 and 12h post-infection. Survival of infected mice and lung bacterial clearance were severely impaired in TCR-Vgamma4(-/-) mice compared with control wild-type (WT) mice. The impaired host protection in TCR-Vgamma4(-/-) mice correlated well with attenuated recruitment of neutrophils in lungs. MIP-2 and TNF-alpha synthesis in the infected tissues was significantly reduced in TCR-Vgamma4(-/-) mice compared with WT mice. Similar results were noted in the synthesis of TNF-alpha, but not clearly of MIP-2, by lung leukocytes stimulated with live bacteria. Our results demonstrate that Vgamma4(+) gammadelta T cells play an important role in the neutrophil-mediated host defense against S. pneumoniae infection by promoting the synthesis of TNF-alpha and possibly of MIP-2 in the lungs.
Microbes and Infection 04/2007; 9(3):251-8. · 3.10 Impact Factor
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ABSTRACT: Intradermal inoculation of cloned self-reactive alphabeta T cells into the footpads of mice induced cutaneous graft-versus-host disease (GVHD), and after recovery from GVHD, the epidermis became resistant to subsequent attempts to induce GVHD. Resistance to GVHD was not induced in the epidermis of T-cell receptor delta-deficient (TCRdelta(-/-)) mice that lacked gammadelta T cells bearing canonical Vgamma5/Vdelta1(+)gammadeltaTCRs, known as dendritic epidermal T cells (DETCs), and resistance was restored by reconstitution of these mutant mice with precursors of Vgamma5(+) DETCs. Pulmonary infection by Cryptococcus neoformans induced an increase of gammadelta T cells in the lung, and in comparison with wildtype mice, TCRdelta(-/-) mice eliminated C. neoformans more rapidly and synthesized more interferon-gamma in the lung. In the mouse small intestine, the absence of gammadelta T cells is associated with a reduction in epithelial cell turnover and downregulation of the expression of major histocompatibility complex class II molecules. The protective role of gammadelta T cells was verified in a dextran sodium sulfate-induced inflammatory bowel disease (IBD) model, whereas in a spontaneous model of IBD, gammadelta T cells were involved in the exacerbation of colitis in TCRalpha(-/-) mice. Taken together, in addition to the homeostatic regulation of epithelial tissues, gammadelta T cells appear to play a pivotal role in the modification of inflammatory responses induced in many organs containing epithelia.
Immunological Reviews 03/2007; 215:103-13. · 11.15 Impact Factor
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ABSTRACT: The alimentary tract has an epithelial layer, consisting mainly of intestinal epithelial cells (IECs), that is exposed to the exterior world through the intestinal lumen. The IEC layer contains many intestinal intraepithelial T cells (IELs), and the total number of IELs constitutes the largest population in the peripheral T-cell pool. Virtually all gammadelta-IELs and many alphabeta-IELs in the mouse small intestine are known to express CD8 alpha alpha homodimers. A wide range of evidence that supports extrathymic development of these CD8 alpha alpha(+) IELs has been collected. In addition, while several studies identified cells with precursor T-cell phenotypes within the gut epithelium, how these precursors, which are dispersed along the length of the intestine, develop into gammadelta-IELs and/or alphabeta-IELs has not been clarified. The identification of lymphoid cell aggregations named 'cryptopatches' (CPs) in the intestinal crypt lamina propria of mice as sites rich in T-cell precursors in 1996 by our research group, however, provided evidence for a central site, whereby precursor IELs could give rise to T-cell receptor-bearing IELs. In this review, we discuss the development of IELs in the intestinal mucosa and examine the possibility that CPs serve as a production site of extrathymic IELs.
Immunological Reviews 03/2007; 215:154-65. · 11.15 Impact Factor
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ABSTRACT: A new, simple and sensitive pre-column fluorescence derivatization high-performance liquid chromatographic method for the determination of the oxidative DNA stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, was developed. Solid-phase extraction using an Oasis HLB cartridge avoided troublesome sample preparation steps, interference from charged species and frequent and essential electrode maintenance in electrochemical procedures. 8-Oxo-7,8-dihydro-2'-deoxyguanosine and other guanine compounds were selectively derivatized with glyoxal reagents (phenylglyoxal, 3,4-methylenedioxyglyoxal, 2-naphtylglyoxal and 6-methoxynaphthylglyoxal) at 40-60 degrees C. Derivatization with 6-methoxynaphthylglyoxal at 40 degrees C for 30 min gave the strongest fluorescence product. The fluorescence derivatives from reaction with 6-methoxynaphthylglyoxal were separated on a Capcell Pak C18 SG 120A column (4.6 mm i.d. x 150 mm, 5 microm) with acetonitrile-5 mM phosphate buffer (pH 6.0; 3:7, v/v) as mobile phase. The detection wavelength of the fluorescence derivative of 8-oxo-7,8-dihydro-2'-deoxyguanosine was lambda(ex) 400 nm and lambda(em) 510 nm. The detection limit of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 1 ng/mL using 50 mL of urine. The calibration graphs were linear up to 30 microg/mL for 8-oxo-7,8-dihydro-2'-deoxyguanosine. The relative standard deviation of 20 ng/mL of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 7.0%. The proposed method was compared with the enzymatic ELISA 8-oxo-7,8-dihydro-2'-deoxyguanosine analysis method (8-OH-dG Check, JaICA, Shizuoka, Japan). The correlation coefficient was 0.79 (n = 20) and y = 0.85x + 5.34. The proposed method was applied to the monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine from male heavy smokers.
Biomedical Chromatography 09/2006; 20(8):800-5. · 1.97 Impact Factor
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ABSTRACT: We explored the role of the transcription factor c-Fos in lipopolysaccharide (LPS)-induced cytokine response using mice lacking c-Fos (Fos-/- mice). Compared with wild-type controls, Fos-/- macrophages and mice showed significantly enhanced production of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12 p40, but reduced production of the anti-inflammatory cytokine IL-10. Bandshift analysis revealed that LPS-induced NF-kappaB binding activity to a functional site in the TNF-alpha promoter was significantly higher in Fos-/- than in wild-type macrophages. Using telemetry, we monitored body temperature and heart rate after LPS injection and found that Fos-/- mice undergo more severe hypothermia and bradycardia than wild-type mice. Such shock responses in Fos-/- mice were significantly reversed by neutralizing TNF-alpha. These data reveal a novel in vivo role for c-Fos as an anti-inflammatory transcription factor acting through suppression of NF-kappaB activity.
International Immunology 06/2006; 18(5):671-7. · 3.41 Impact Factor
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ABSTRACT: A new, simple and sensitive pre-column high-performance chromatographic method for the determination of diabetes marker d-glucose, 1,5-anhydro-d-glucitol and related compounds is reported. Sugars (d-glucose, d-galactose, d-mannose, sucrose and arabinose) were derivatized with benzoic acid (BA) at 80 degrees C for 60 min. l-Fucose, fructose, d-lactose, l-rhamnose, arabinose and ascorbic acid were not reacted. Sugar alcohols (xylitol, erythritol, mannitol, sorbitol myo-inositol) were also derivatized with BA at 80 degrees C for 60 min. The fluorescence derivatives were separated on a TSK amide 80 column (4.6 mm i.d. x 250 mm, 5 microm) with acetonitrile-50 mm acetate buffer (pH 5.6; 4:96, v/v) as the mobile phase. The detection wavelength of beizoic acid derivatives was lambda(ex) 275 nm and lambda(em) 315 nm. The detection limits of sugars were 10-80 microg/mL. The calibration graphs were linear up to 10 mg/mL. The relative standard deviations of 500 microg/mL sugars were 7.0-7.3%. The proposed method was compared with the enzymatic photometric glucose analysis method (Glucose B-Test II Wako). The correlation coefficient was 0.83 (n = 20) and y = 0.82x + 5.91, where y and x are concentrations in microg/mL obtained by the proposed pre-column HPLC and enzyme-photometric method, respectively. The detection limits of sugar alcohols were 100-1000 ng/mL. The calibration graphs were linear to 50 microg/mL and relative standard deviations of 10 microg/mL were 7.2-8.2%. The 1,5-AG data by the proposed method was also compared with the enzymatic photometric 1,5-AG analysis method (Rana AG 1,5-AG determination kit, Nihon Kayaku) and good correlation (r = 0.91, n = 20) was also obtained. The proposed method was applied to the simultaneous determination of d-glucose, 1,5-AG and related sugar alcohols in serum from healthy males.
Biomedical Chromatography 06/2006; 20(5):440-5. · 1.97 Impact Factor
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ABSTRACT: A new, simple and sensitive pre-column high-performance chromatographic method for the determination of diabetes marker d-glucose, 1,5-anhydro-d-glucitol and related compounds is reported. Sugars (d-glucose, d-galactose, d-mannose, sucrose and arabinose) were derivatized with benzoic acid (BA) at 80°C for 60 min. l-Fucose, fructose, d-lactose, l-rhamnose, arabinose and ascorbic acid were not reacted. Sugar alcohols (xylitol, erythritol, mannitol, sorbitol myo-inositol) were also derivatized with BA at 80°C for 60 min. The fluorescence derivatives were separated on a TSK amide 80 column (4.6 mm i.d. × 250 mm, 5 µm) with acetonitrile–50 mm acetate buffer (pH 5.6; 4:96, v/v) as the mobile phase. The detection wavelength of beizoic acid derivatives was λex 275 nm and λem 315 nm. The detection limits of sugars were 10–80 µg/mL. The calibration graphs were linear up to 10 mg/mL. The relative standard deviations of 500 µg/mL sugars were 7.0–7.3%. The proposed method was compared with the enzymatic photometric glucose analysis method (Glucose B-Test II Wako). The correlation coefficient was 0.83 (n = 20) and y = 0.82x + 5.91, where y and x are concentrations in µg/mL obtained by the proposed pre-column HPLC and enzyme-photometric method, respectively. The detection limits of sugar alcohols were 100–1000 ng/mL. The calibration graphs were linear to 50 µg/mL and relative standard deviations of 10 µg/mL were 7.2–8.2%. The 1,5-AG data by the proposed method was also compared with the enzymatic photometric 1,5-AG analysis method (Rana AG 1,5-AG determination kit, Nihon Kayaku) and good correlation (r = 0.91, n = 20) was also obtained. The proposed method was applied to the simultaneous determination of d-glucose, 1,5-AG and related sugar alcohols in serum from healthy males. Copyright © 2005 John Wiley & Sons, Ltd.
Biomedical Chromatography 04/2006; 20(5):440 - 445. · 1.97 Impact Factor
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ABSTRACT: The crypt lamina propria of the mouse small intestine has been shown to harbor multiple tiny clusters filled with c-kit- and interleukin 7 receptor (IL-7R)-positive lympho-hemopoietic cells (cryptopatches; CPs). However, it has remained an open question whether similar lymphoid tissue are present in the gastrointesitinal tract in other animals. In the present study, we investigated whether the small intestine of rats harbored lymphoid tissues similar to mouse CPs.
Immunohistochemical and flow cytometric analyses were carried out using various antibodies, including those to c-kit and IL-7R molecules.
Lymphocyte-filled villi (LFVs), populated predominantly with c-kit- and IL-7 receptor (IL-7R)-positive cells and less with T cell receptor (TCR)-alphabeta T cells were found throughout the small intestine of young adult rats. Although LFVs were absent from fetal rat intestine, they were first detected at around 2 weeks after birth. Notably, in most LFVs that settled in the antimesenteric wall of the small intestine in young adult rats, immunoglobulin M-positive B cells were also detectable at the bottom of the LFVs. In aged rats, lymphocytes in some LFVs displayed a different phenotype, comprising a large B-cell area that included a germinal center. Thus, these clusters represent the first description of isolated lymphoid follicles (ILFs) in the rat small intestine.
The present study provides the first evidence for c-kit- and IL-7R-positive lymphocyte clusters in the rat small intestine. Our data also indicating that LFVs and ILFs may constitute novel organized gut-associated lymphoid tissues in lamina propria of the rat small intestine.
Journal of Gastroenterology 11/2005; 40(10):956-63. · 4.16 Impact Factor
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Nippon rinsho. Japanese journal of clinical medicine 05/2005; 63 Suppl 4:454-8.
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Yasuyuki Kai,
Ichiro Takahashi, Hiromichi Ishikawa,
Takachika Hiroi,
Tsunekazu Mizushima,
Chu Matsuda,
Daisuke Kishi,
Hiromasa Hamada,
Hiroshi Tamagawa,
Toshinori Ito,
Kazuyuki Yoshizaki,
Tadamitsu Kishimoto,
Hikaru Matsuda,
Hiroshi Kiyono
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ABSTRACT: Mice that have a truncated mutation of the common cytokine receptor gamma chain (CR gamma -/Y) are known to spontaneously develop colitis. To identify the pathologic elements responsible for triggering this localized inflammatory disease, we elucidated and characterized aberrant T cells and their enteropathogenic cytokines in CR gamma -/Y mice with colitis.
The histologic appearance, cell population, T-cell receptor V beta usage, and cytokine production of lamina propria lymphocytes were assessed. CR gamma -/Y mice were treated with anti-interleukin (IL)-6 receptor monoclonal antibody to evaluate its ability to control colitis, and splenic CD4 + T cells from the same mouse model were adoptively transferred into SCID mice to see if they spurred the appearance of colitis.
We found marked thickening of the large intestine, an increase in crypt depth, and infiltration of the colonic lamina propria and submucosa with mononuclear cells in the euthymic CR gamma -/Y mice, but not in the athymic CR gamma -/Y mice, starting at the age of 8 weeks. Colonic CD4 + T cells with high expressions of antiapoptotic Bcl-x and Bcl-2 were found to use selected subsets (V beta 14) of T-cell receptor and to exclusively produce IL-6. Treatment of CR gamma -/Y mice with anti-IL-6 receptor monoclonal antibody prevented the formation of colitis via the induction of apoptosis in IL-6-producing CD4 + T cells. Adoptive transfer of pathologic CD4 + T cells induced colitis in the recipient SCID mice.
Colonic IL-6-producing thymus-derived CD4 + T cells are responsible for the development of colitis in CR gamma -/Y mice.
Gastroenterology 04/2005; 128(4):922-34. · 11.68 Impact Factor
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ABSTRACT: Through analysis of athymic (nu/nu) mice carrying a transgenic gene encoding GFP instead of RAG-2 product, it has recently been reported that, in the absence of thymopoiesis, mesenteric lymph nodes and Peyer's patches (PP) but not gut cryptopatches are pivotal birthplace of mature T cells such as the thymus-independent intestinal intraepithelial T cells (IEL). To explore and evaluate this important issue, we generated nu/nu mice lacking all lymph nodes (LN) and PP by administration of lymphotoxin-beta receptor-Ig and TNF receptor 55-Ig fusion proteins into the timed pregnant nu/+ mice that had been mated with male nu/nu mice (nu/nu LNP- mice). We also generated nu/nu aly/aly (aly, alymphoplasia) double-mutant mice that inherently lacked all LN, PP, and isolated lymphoid follicles. Although gammadelta-IEL were slightly smaller in number than those in nu/nu mice, substantial colonization of gammadelta-IEL was found to take place in the intestinal epithelia of nu/nu LNP- and nu/nu aly/aly mice. Notably, the population size of a major CD8alphaalpha+ gammadelta-IEL subset was maintained, the use of TCR-gamma-chain variable gene segments by these gammadelta-IEL was unaltered, and the development of cryptopatches remained intact in these nu/nu LNP- and nu/nu aly/aly mice. These findings indicate that all LN, including mesenteric LN, PP, and isolated lymphoid follicles, are not an absolute requirement for the development of gammadelta-IEL in athymic nu/nu mice.
The Journal of Immunology 03/2005; 174(4):1906-12. · 5.79 Impact Factor
