Biomedical subjects
Publications and source records attributed to Y Yuki.
We present the MR and histopathologic findings of fibrolipomatous hamartoma (FLH) of the ulnar nerve in a 54-year-old woman, a lipomatous process that rarely affects the ulnar nerve. The case illustrated is further unusual as a local soft tissue recurrent mass developed over a remarkably long course of the disease.
Cholera toxin (CT), a major enterotoxin produced by Vibrio cholerae, elicits mucosal adjuvant activities by inducing antigen-specific CD4+ T cells secreting T helper type 2 (Th2) cytokines. Experimental autoimmune encephalomyelitis (EAE) is induced by Th1 cells specific for myelin-derived antigens. We induced EAE in C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG) 35-55 and CT was nasally administered as an immunomodulator on day 7 following MOG challenge. Clinical severity in the CT-treated mice was milder when compared to PBS-treated mice, while the levels of expression of interleukin (IL)-12 and interferon (IFN)-gamma in the central nervous system (CNS) of CT-treated mice were lower than PBS-treated mice. Thus, nasal administration of the mucosal immunomodulator CT ameliorated the severity of EAE, which was associated with the suppression of Th1 cell responses.
The three-amino-acid loop extension (TALE) homeodomain proteins are highly conserved transcription regulators. Since cooperative function among members of this growing family is critical for regulating transcription, we have tried to explore novel members to understand their regulatory mechanisms in cellular proliferation and differentiation. Here we report identification of PKNOX2, a novel TALE homeodomain protein that shows distinct homology with PKNOX1, a stable partner of PBX proteins. PKNOX2 is composed of 460 amino acids and contains HR1, HR2, and homeodomain, which are highly similar to PKNOX1, suggesting that PKNOX2 may also interact with PBX proteins as well as the same DNA sequence as PKNOX1. Genomic organization of PKNOX2 also showed high similarity to PKNOX1, though PKNOX2 lies on a different chromosomal region, 11q24. Unlike PKNOX1, which was broadly expressed in many tissues, PKNOX2 showed a more restricted pattern of mRNA expression. Nuclear localization of PKNOX2 was confirmed by transfection of epitope-tagged cDNA. Taken together, these data indicate that PKNOX2 is a novel PKNOX-related protein and may interact with PBX proteins and play a tissue-specific regulation of transcription.
Mucosal administration of experimental autoimmune encephalomyelitis (EAE)-specific autoantigens can reduce the onset of disease. To examine whether cholera toxin-B-subunit (CTB)-conjugated EAE-specific T-cell epitope can reduce development of the autoimmune disease in mice, we produced a recombinant hybrid molecule of CTB fusion protein linked with proteolipid-protein (PLP)-peptide139-151(C140S) at levels up to 0.1 gram per liter culture media in Bacillus brevis as a secretion-expression system. Amino acid sequencing and GM1-receptor binding assay showed that this expression system produced a uniformed recombinant hybrid protein. EAE was induced in SJL/J mice by systemic administration with the PLP-peptide. When nasally immunized 5 times with 70 microg rCTB PLP-peptide hybrid protein, mice showed a significantly suppressed development of ongoing EAE and an inhibition of both the PLP-peptide-specific delayed-type hypersensitivity (DTH) responses and leukocyte infiltration into the spinal cord. In contrast, all mice given the PLP-peptide alone or the PLP-peptide with the free form of CTB did not suppress the development of EAE and DTH responses. These results suggest that nasal treatment with the recombinant B. brevis-derived hybrid protein of CTB and autoantigen peptide could prove useful in the control of multiple sclerosis.
Oral delivery of a large dose or prolonged feeding of protein Ags induce systemic unresponsiveness most often characterized as reduced IgG and IgE Ab- and Ag-specific CD4(+) T cell responses. It remains controversial whether oral tolerance extends to diminished mucosal IgA responses in the gastrointestinal tract. To address this issue, mice were given a high oral dose of OVA or PBS and then orally immunized with OVA and cholera toxin as mucosal adjuvant, and both systemic and mucosal immune responses were assessed. OVA-specific serum IgG and IgA and mucosal IgA Ab levels were markedly reduced in mice given OVA orally compared with mice fed PBS. Furthermore, when OVA-specific Ab-forming cells (AFCs) in both systemic and mucosa-associated tissues were examined, IgG AFCs in the spleen and IgA AFCs in the gastrointestinal tract lamina propria of mice given OVA orally were dramatically decreased. Furthermore, marked reductions in OVA-specific CD4(+) T cell proliferative and cytokine responses in spleen and Peyer's patches were seen in mice given oral OVA but were unaffected in PBS-fed mice. We conclude that high oral doses of protein induce both mucosal and systemic unresponsiveness and that use of mucosal adjuvants that induce both parenteral and mucosal immunity may be a better way to assess oral tolerance.
Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.
Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis in humans. EAE can be passively transferred into naive syngeneic animals by administration of MOG-specific T cell clones. Lymphocytes isolated from green fluorescent protein (GFP)-transgenic (Tg) mice can light up by emitting green fluorescence, thus making it feasible to use such animals in a passive transfer model for EAE. When MOG-sensitized splenic lymphocytes from GFP-Tg mice were adoptively transferred to irradiated, syngeneic C57BL/6 and RAG-1(-/-)mice, typical symptoms of EAE developed. Analysis of the reconstituted mice with EAE revealed prominent infiltration of fluorescing (GFP+), CD4+ T cells into the central nervous system (CNS). Real-time confocal imaging revealed these cells in the spinal cords and brains of recipient mice. This infiltration was also confirmed by anti-GFP monoclonal antibodies. Furthermore, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) evaluation indicated that the infiltrating GFP+, CD4+ T cells exclusively produced T helper type 1 (Th1) cytokines, especially interferon-gamma (IFN-gamma). These results clearly show that MOG-specific CD4+ T cells preferentially invade into the CNS and mediate the development of EAE by producing Th1-biased cytokines.
We tested the notion that the mucosal adjuvant cholera toxin (CT) could target, in addition to nasal-associated lymphoreticular tissues, the olfactory nerves/epithelium (ON/E) and olfactory bulbs (OBs) when given intranasally. Radiolabeled CT ((125)I-CT) or CT-B subunit ((125)I-CT-B), when given intranasally to mice, entered the ON/E and OB and persisted for 6 days; however, neither molecule was present in nasal-associated lymphoreticular tissues beyond 24 h. This uptake into olfactory regions was monosialoganglioside (GM1) dependent. Intranasal vaccination with (125)I-tetanus toxoid together with unlabeled CT as adjuvant resulted in uptake into the ON/E but not the OB, whereas (125)I-tetanus toxoid alone did not penetrate into the CNS. We conclude that GM1-binding molecules like CT target the ON/E and are retrograde transported to the OB and may promote uptake of vaccine proteins into olfactory neurons. This raises concerns about the role of GM1-binding molecules that target neuronal tissues in mucosal immunity.
We have developed an enzyme-linked immunoassay (ELISA) for serum complexed-antigen (prostate-specific antigen; PSA)(c-PSA) with simultaneous blocking of free-antigen (PSA)(f-PSA). The assay utilizes three different monoclonal antibodies (MAbs) recognising three distinct PSA epitopes. The detection limit was established as 0.19 microg/l (n=20, mean of zero standard+2S.D.) and the average recovery of f-PSA was 98-100%. The within-run and between-day coefficients of variation (CV) ranged from 2.1% to 3.2% and 2.8% to 6.3%, respectively. There was a good correlation between serum c-PSA measured by the present ELISA and PSA-alpha(1)-antichymotrypsin complex (PSA-ACT) concentrations (r=0.991). This method should provide a better tool for discriminating between benign and malignant prostatic disease.
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DNA base composition and DNA-DNA hybridization among the cyanobacterial genus Microcystis were determined using nine axenic Microcystis strains, including the three 'morphological' species of Microcystis aeruginosa, Microcystis viridis and Microcystis wesenbergii. These Microcystis species showed a similar DNA base composition (42.1-42.8 mol% G + C) and demonstrated more than 70% DNA relatedness, confirming their synonymy based on bacterial criteria.
A 72-year-old woman who had had an endoscopic sclerotherapy for esophageal varices presented with high fever and severe cough. Chest X-ray and CT demonstrated a pneumopericardium and pericardial effusion. Esophagoscopy and esophagography revealed an esophageal perforation into the pericardial cavity and into the lung. Consequently, drainage and irrigation of the pericardial cavity and mediastinum were done for MRSA infection. However, these procedures failed to reduce the inflammation, and she expired because of liver failure soon after placing a covered stent in the esophagus. Postmortem examination revealed small cell carcinoma in the left lung invading into the esophagus and pericardium.
Two-dimensional electrophoresis (2-DE) followed by electroblotting and microsequencing is considered to be the most powerful method for the isolation and characterization of proteins. In this paper, we report the separation and determination of the N-terminal and/or internal amino acid sequences of the minor proteins of human colostral and mature milk by 2-DE and microsequencing. In order to analyze the minor proteins of human milk, we use immunoabsorbents to remove three major proteins, alpha-lactalbumin, lactoferrin and secretory immunoglobulin A. The major proteins removed by this process accounted for about 79 and 93% of the total whey proteins of mature and colostral milk, respectively. The remaining milk proteins were then separated by isoelectric focusing gel electrophoresis between pH 3 and 10, and subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Approximately 400 spots were detected in both colostral and mature milk by silver staining after 2-DE. Twenty-two major, well-resolved proteins (out of 400) were microsequenced (N-termini as well as internal). These include fatty acid binding protein, beta 2-microglobulin, complement C4, clusterin, alpha 1-antritrypsin, lysozyme C, alpha- and beta-casein, prealbumin, serotransferrin, fructose-bisphosphate aldolase A, and beta-casein fragments. No major differences in the protein patterns were observed between the minor proteins of colostrum and mature milk, indicating that the minor proteins remained relatively constant during lactation. These results suggest that the minor milk proteins are important for the health and development of breast-fed infants throughout lactation.
A 37-year-old woman presented severe cough for several years. Chest X-ray showed an abnormal shadow in the right upper lung field. That was absent but hyperlucent before onset. Chest CT scan revealed a mass shadow with mucoid impaction in the right S2. Thoracoscopic right upper lobectomy dramatically improved her complaints. Histological examination revealed the dilated bronchus containing mucoid impaction and confirmed a diagnosis of congenital bronchial atresia. It was suggested that organizing pneumonia resulting from repeated infection caused severe cough. Thoracoscopic surgery for congenital bronchial atresia should be recommended in young patients.
Oral administration of large doses of protein antigen generally induces a state of systemic unresponsiveness currently termed mucosally induced tolerance. In this study, we used human milk protein (HMP) without casein as a multi-protein antigen for the study of mucosally induced tolerance. The HMP utilized in this study mainly contained secretory (S) IgA, lactoferrin (Lf) and alpha-lactalbumin (Lact). When mice were given 1 or 25 mg of HMP orally 3 times or 25 mg orally four consecutive weeks prior to systemic immunization, antigen-specific serum IgG responses to HMP were induced by subsequent parenteral immunization with 100 microg of HMP. Analysis of IgG subclasses revealed that IgG1 followed by IgG2b accounted for the IgG responses noted. When both HMP and ovalbumin (OVA) were fed to mice, tolerance developed to OVA but not to HMP. To further investigate the nature of immune responses seen following oral gavage of HMP, we examined responses to individual protein of HMP. Brisk serum IgG1 and IgG2b responses to both S-IgA and Lf were induced by oral followed by systemic immunization with HMP. Analysis of splenic CD4+ T cells from mice given oral HMP revealed production of Th2- but not Th1-type cytokines. These results show that oral administration of HMP preferentially induces exclusive Th2-type immune responses, which may prevent the development of HMP (S-IgA and Lf)-specific mucosally induced tolerance.
PURPOSE: To evaluate the MR appearance of bone marrow during and after multiple fractionated and single-dose irradiation using SE sequences, with histopathological correlation. MATERIAL AND METHODS: The left hind limbs of Wistar rats were irradiated with doses of either 2 Gy x 10 fractions or 20 Gy as a single dose. MR images of the femora were taken on days 0, 1, 3, 5, 7, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150 and 180. T1-, proton-density- and T2-weighted images were obtained with SE sequences. The rats were sacrificed on days 3, 12, 40 and 180 for histological study. RESULTS: No significant change in the T1 relaxation time was observed in the fractionated group during the first 15 days after irradiation. T2 did not change during the initial 7 days. There were significant differences in T1 and T2 between nonirradiated and fractionated, and between fractionated and single-dose irradiated marrows (p < 0.01). Sinusoidal dilatation, decrease in cellularity, and hemorrhage were observed on day 3. Fat increased on/after day 12. The fractionated group showed mild changes compared to the single-dose irradiated group. CONCLUSION: The T1 and T2 relaxation times of bone marrow receiving multiple fractionated irradiation did not change significantly on SE sequences during the early phase. However, dilatation of sinusoids, hemorrhage, decrease in cellularity, and increase in fat content were observed. Single-dose irradiation resulted in marked and early changes in T1 and T2 of the bone marrow.
We report a successful resection of an osteosarcoma in the chest wall developed 25 years after irradiation. A 74-year-old woman was admitted to our hospital for her swelling in the left chest wall at August 24, 1995. At 49-year-old, she had undergone an operation and postoperative irradiation for left breast cancer. A computed tomography demonstrated a mass in the left chest wall that destructed the first rib, extending into the pleural space and invaded into the left common carotid and subclavian arteries. We planned a radical resection of the mass after repeated CT scannings, since it was histopathologically diagnosed as a chondrosarcoma and showed a rapid growth. The tumor was completely removed with radical transmediastinal forequarter amputation of the partial chest wall and total left upper extremity. The left common carotid artery was partially replaced with 6 mm EPTFE vascular prosthesis. The chest wall was reconstructed with Marlex-mesh prosthesis and a myocutaneous flap. She was discharged uneventfully and has not shown any evidence of recurrence.
The carbohydrate-protein linkage region of a chondroitin 4-sulfate chain attached to urinary trypsin inhibitor (UTI) was isolated from human urine and characterized structurally. The chondroitin 4-sulfate chain was released from UTI by beta-elimination using alkaline NaBH4 then digested with chondroitinase ABC. These treatments resulted in only a single hexasaccharide alditol derived from the carbohydrate-protein linkage region. Chemical and enzymic analyses and 600-MHz 1H-NMR spectroscopy revealed that the hexasaccharide alditol had the following structure: delta HexA alpha 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1- 3Gal(4-sulfate) beta 1-3Gal beta 1-4Xyl-ol, where delta HexA, GlcA and Xyl-ol represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucuronic acid and D-xylitol, respectively. This structure contained the novel 4-sulfated Gal residue, which was first demonstrated in one of the three linkage hexasaccharide-serines isolated from chondroitin 4-sulfate of rat chondrosarcoma [Sugahara, K., Yamashina, I., de Waard, P., Van Halbeek, H. & Vliegenhart, J. F. G. (1988) J. Biol. Chem. 263, 10168-10174]. This disulfated structure was recently identified as the sole structural component in the linkage hexasaccharide alditol fraction isolated from inter-alpha-trypsin inhibitor (ITI) in human plasma [Yamada, S., Oyama, M., Kinugasa, H., Nakagawa, T., Kawasaki, T., Nagasawa, S., Khoo, K.-H., Morris, H.R., Dell, A. & Sugahara, K. (1995) Glycobiology 5, 335-341]. The structural uniformity in the linkage hexasaccharide structure of ITI and UTI is in marked contrast to the heterogeneity demonstrated in the linkage hexasaccharides isolated from cartilaginous chondroitin sulfate whose linkage regions are sometimes but not always phosphorylated on the Xyl residue or sulfated on the Gal residue(s). The uniform structure containing the novel 4-sulfated Gal residue in the linkage region of UTI and ITI may imply its significance in the biosynthetic mechanism of chondroitin sulfate.