Identification of nonsynonymous polymorphisms in the superantigen-coding region of IDDMK1,2 22 and a pilot study on the association between IDDMK1,2 22 and type 1 diabetes.

To investigate the possible involvement of IDDMK1,2 22/HERV-K18 in childhood type I diabetes mellitus, we identified two nonsynonymous A/G polymorphisms in the superantigen-coding region of IDDMK1,2 22 at the 290- and 461-nucleotide (nt) positions from the initial methionine codon and compared their frequencies in 74 Japanese patients with type 1 diabetes and in 54 nondiabetic controls. Although the G substitution was observed more frequently at either site in the patients than it was in the controls (7% vs. 4% at 290 nt, and 29% vs. 20% at 461 nt), the differences were not statistically significant. A weak significance of difference in the frequency of 461G was obtained only in an early-onset group of patients manifesting the disease at 5 years of age or less (n = 24) when compared with controls (38% vs. 20%; P = 0.03). However, in addition to the common absence of a particular allele among the expected four alleles, remarkable differences in allele frequencies were present between Japanese and European populations. This first trial investigating the association of IDDMK1,12 22 with type 1 diabetes presents intriguing suggestions for the role of this region in the etiology of autoimmune and infectious diseases.

Enhanced neutrophilic granulopoiesis in rheumatoid arthritis. Involvement of neutrophils in disease progression.

OBJECTIVE: To investigate enhanced granulopoiesis in bone marrow of patients with rheumatoid arthritis (RA), and the role of neutrophils in RA pathogenesis. METHODS: Aspirated bone marrow cells and peripheral blood leukocytes from patients with RA and non-RA patient controls were analyzed morphologically and by 2 color flow cytometry. Thirteen iliac bones (8 RA, 5 non-RA) were examined by light and transmission electron microscope (TEM). RESULTS: The percentage of CD15+CD16- cells (immature neutrophils) in RA bone marrow (64.3+/-13.4%, mean +/- SD) increased significantly compared to that of non-RA controls (43.2+/-14.3%), whereas the fraction of CD15+CD]6+ cells (mature neutrophils)was greatly decreased (RA 21.8+/-10.1%; non-RA 38.1+/-8.9%). The absolute number of CD15+CD16- cells also increased markedly in RA bone marrow. The ratio of immature cells to the total granulocytes (% CD15+CD16- to % CD15+) correlated with the Lansbury Index score (R = 0.76, p<0.0001). TEM observations revealed that abundant immature neutrophils adhered closely to the trabeculae of the iliac bone. Margins of trabeculae were mostly irregular, especially in severe RA, and collagenous fibers frequently disappeared in those trabeculae with ragged margins. CONCLUSION: In RA bone marrow, immature neutrophils (CD15+CD16-) were markedly increased in number; by contrast, no changes were found for mature cells. Augmented production of immature neutrophils (at the promyelocyte-to-myelocyte stage) might lead to the destruction of collagenous fibers in RA bone trabeculae, as revealed by TEM. Generalized bone destruction in RA might, at least in part, be caused by enhanced production of immature neutrophils.

[Pathophysiology and strategy of shock in terms of tissue oxygen metabolism].

In both septic shock and hypovolemic shock, global oxygen consumption is characterized by the supply dependency phenomenon, which suggests tissue hypoxia. The conventional measurements of blood lactate level, oxygen delivery/oxygen consumption, and mixed venous oxygen saturation may not provide reliable information on the adequacy of tissue oxygenation. The intestinal mucosal villus is particularly vulnerable to a reduction in oxygen delivery because of the countercurrent mechanism. Intestinal mucosal hypoxia may have important clinical ramifications as it has been implicated in the etiology of bacterial translocation and cytokine synthesis. Gastric tonometry has been proposed as a simple, relatively noninvasive technique to detect occult tissue hypoxia. Since gastric intramucosal PCO2 is directly related to arterial PCO2, the gradient between gastric and arterial PCO2 is recommended as a sensitive and specific index of intestinal hypoperfusion. Although the effects of therapeutic interventions are far from consistent, dobutamine, but not dopamine, appears to increase intestinal mucosal blood flow in critically ill patients the most consistently. Further studies are needed to demonstrate the effects of other agents, such as angiotensin-converting enzyme inhibitor and vasodilator prostaglandins. Plasma volume expansion with colloid solution may improve the splanchnic microvascular blood flow. Older stored-blood transfusion, however, may lead to tissue hypoxia.

Effect of functional magnetic particles on radiofrequency capacitive heating.

Magnetic particles (magnetite) were used to make radiofrequency (RF) capacitive hyperthermia effective to a specific site. In an agar phantom experiment, a magnetite-containing agar piece was buried in a large agar phantom and heated by an 8 MHz-RF capacitive heating device. The magnetite-containing agar piece was heated more than the magnetite-free agar phantom, and the specific adsorption rate in the phantom was increased 1.5 times by the magnetite particles. The temperature distribution in the large agar phantom showed that the highest temperature was obtained at the center of the magnetite-containing piece. The rate of temperature increase was approximately proportional to the magnetite concentration to the power 0.8. This method was applied to an in vivo experiment using a pig. Magnetite was prepared as a colloidal material dispersed in a carboxymethylcellulose solution (CMC-Mag) and intramuscularly injected in the pig femur. As a result of 8 MHz-RF heating, the temperature at the CMC-Mag-injected point increased to over 43 degrees C after 7 min, while the temperature at a point without magnetite was under 40 degrees C at the same time. The specific adsorption rate in the magnetite-containing tissue was twice that of the magnetite-free tissue. In addition, the time required to reach a temperature of over 43 degrees C was only 7 min, while it was over 15 min in the case without the CMC-Mag.

DNA fragmentation is not the primary event in glucocorticoid-induced thymocyte death in vivo.

Thymocyte death has been recognized as one of the best models for studying apoptosis. Our recent study, however, indicated that most thymocytes die without DNA fragmentation and become terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling-positive (TUNEL+) only after being phagocytosed by macrophages. In this study, we used histological techniques using the TUNEL method, histochemistry, immunohistochemistry, and transmission electron microscopy as well as flow cytometry to examine in vivo the effect of glucocorticoid (GC), a well-known agent for inducing thymocyte apoptosis in vitro, on thymocyte death to determine whether or not DNA fragmentation was the first event of GC-induced thymocyte death. At 2 h and 4 h after GC injection, a large number of cortical thymocytes were TUNEL+. Most TUNEL+ cells were aggregated to form clusters. Double staining of the section showed that the TUNEL+ thymocytes were phagocytosed by acid phosphatase+ and Mac-2+ macrophages. An ultrastructural study indicated that a far greater number of small pyknotic thymocytes were present in the cortex of the GC-treated thymus than were observed in the control thymus, that all those pyknotic thymocytes were TUNEL-, and moreover, that at the electron microscopic level, TUNEL+ cells were all phagocytosed by macrophages. Flow cytometric analysis did not detect a single TUNEL+ thymocyte even 4 h after the GC treatment, suggesting that virtually no free dead thymocytes were present after DNA fragmentation. These results indicate that, consistent with our previous findings with normal thymocyte death and B cell death in the germinal centers, DNA fragmentation is not involved in the cell death process of the GC-induced rapid thymocyte death in vivo.

Increased radiosensitivity of p16 gene-deleted human glioma cells after transfection with wild-type p16 gene.

The A1235 and T98 cell lines derived from human gliomas have homozygous deletions in their p16 genes and are radiosensitive and radioresistant, respectively, with respect to other established glioma cell lines. These differences in radiosensitivity may be due to variations to some extent among cell lines, rather than genetically defined resistance or sensitivity. We examined the effect on radiation sensitivity of introducing a wild-type p16 gene into both p16-deficient glioma cell lines. The plasmid pOPMTS containing human wild-type p16 cDNA and a neomycin resistance gene, or the control plasmid pOPRSV1, were transfected into these cells. Clones from both cell lines, which expressed wild-type p16 mRNA constitutively after transfection with pOPMTS, were more radiosensitive than the parental cells and clones obtained after transfection with the negative control plasmid.

Analysis of mitogenic substances in Bupleurum chinense by ESR spectroscopy.

The polyphenolic substance(s) in the hot water extract of Bupleurum chinense (PSF) showed strong mitogenic activity. In this paper, we analyzed PSF by using ESR spectroscopy, and found that i) PSF showed a strong ESR signal on g = 2.005 which was similar to the commercially available lignin; ii) Sho-saiko-to, which contains an extract of B. chinense, also showed similar signals on ESR; iii) Powdered B. chinense also showed similar signals on g = 2.005. Peroxidase activity, essential for producing polyphenolic substances, was detected in the cold water extract of B. chinense. In addition, the signal intensity of the ESR spectrum of B. chinense was increased after boiling. The data of the ESR spectra of the model reactions using lignin, arginine, proline and maltose also strongly suggested that a certain chemical modification proceeded during the hot water extraction to increase the percentage of the stable free radical. These facts strongly suggested that the mitogenic substance in B. chinense is a polyphenolic substance extracted by hot water, and the structure was modified during the extraction to increase the stable free radical components.

Exposure to magnetic field (5 mT at 60 Hz) does not affect cell growth and c-myc gene expression.

We designed and manufactured equipment for long-term and low-density (0 to 9 mT) exposures of cultured cells to extremely low frequency magnetic fields (ELF-MF), and examined the effects of ELF-MF on cell growth and c-myc mRNA expression in Chinese hamster ovary (CHO) cells. The ELF-MF equipment consists of a CO2 incubator with a built-in magnet generator using Helmholtz coils being 250 mm in inner diameter, 160 mm in distance and 128 turns, a slide regulator and a thermocontroller. No significant difference in the growth rate and the c-myc expression of CHO cells was observed with 5 mT ELF-MF exposure, sham-exposure and incubation in a conventional incubator.

Death of germinal center B cells without DNA fragmentation.

During the selection of B cells within germinal centers (GC) on the basis of their affinity for T-dependent antigen, B cells not positively selected are eliminated within GC. This process of B cell death has been considered to be apoptosis. In a recent study, we have reported that, although a substantial number of thymocytes were considered to be dead because of their extremely small cell size and heavy chromatin condensation even though they were not yet phagocytosed (pyknosis), they were devoid of DNA fragmentation, the most characteristic feature for apoptosis. In this study, we examined in vivo the mechanism of B cell death within GC by using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method to detect DNA double-strand breaks. TUNEL+ B cells were scattered throughout the upper dark and the light zones of GC. Double staining of the sections by the TUNEL method and acid phosphatase (AcP) activities showed that all the TUNEL+ B cells were phagocytosed by macrophages. Light microscopic and ultrastructural studies revealed the presence of small unphagocytosed B cells within the light zone. These cells are undoubtedly dead because they were much smaller than surrounding lymphoid cells and have a heavy chromatin condensation. Furthermore, ultrastructural detection of DNA fragmentation confirmed that these small unphagocytosed B cells were TUNEL-, implying that DNA fragmentation is not primarily involved in the cell death process of these small dead B cells. These results indicate that most B cells, not positively selected and thus destined to be eliminated, die within GC without DNA fragmentation, and are subsequently phagocytosed by macrophages and become TUNEL+. Typical apoptosis, characterized by DNA fragmentation in situ, is not the predominant type of cell death that occurs during the selection of B cells in GC.

Increase in hypoxanthine-guanine phosphoribosyl transferase gene mutations by exposure to high-density 50-Hz magnetic fields.

Exposure to extremely low frequency magnetic field (ELFMF) of 50 Hz and 400 mT induced mutations in the hypoxanthine-guanine phosphoribosyl transferase gene of human melanoma MeWo cells. The mutant frequency was enhanced both by increasing the exposure period and the induced current intensity. Mutations induced by X-rays were enhanced by ELFMF exposure. No significant increase in mutant frequency occurred when DNA replication was inhibited during ELFMF exposure. DNA replication error is suspected of causing the mutations produced by ELFMF exposure.

A case of non-Hodgkin's lymphoma with macroglobulinemia.

A case of non-Hodgkin's lymphoma with macroglobulinemia is reported. A 48-year-old man consulted our hospital with complaints of dysphagia and sleep apnea. On the first examination, degree III hypertrophy of tonsils, Bence-Jones protein of K-type on urinalysis, and an M-peak in the gamma-glb. Beta-glb areas on fractionation of protein were found. Of serum immunoglobulins, IgM was increased markedly with 3,946 mg/dl and about 30% atypical lymphocytes were noted in the bone marrow. Bilateral tonsillectomy was carried out. The diagnosis of malignant lymphoma, non-Hodgkin, follicular, medium-sized cell type, B cell (sm IgM, K) was made by immunostaining of tissues. Chemotherapy was made after operation, and IgM levels decreased gradually. At present, the patient is following a satisfactory postoperative course, showing no swelling of tonsil.

Modification of the radiosensitivity of human cells to which simian virus 40 T-antigen was transfected.

Effects of the introduction of the Simian virus 40 T-antigen (SV40 T-Ag) gene to cultured human cells were examined in relation to radiosensitivity. Two relatively radioresistant tumor cell lines (T98 and G361) became significantly radiosensitive after the introduction of SV40 T-Ag, whereas radiosensitive tumor cell lines did not show a change in radiosensitivity. In contrast, a human fibroblast cell line became radioresistant after SV40 T-Ag introduction. T98 cells which have a mutation at codon 237 in the p53 gene were unable to form a complex between p53 protein and SV40 T-Ag, whereas G361, which became radiosensitive by a SV40 T-Ag introduction, formed the complex. This indicates that the status of p53 is independent of the change in radiosensitivity in the cell lines studied.

Changes in radiation sensitivity of human osteosarcoma cells after p53 introduction.

Human osteosarcoma SAOS-2 cells, which have a deletion in p53 gene, were transfected with plasmid pMSVneop53 containing human p53 cDNA and neomycin-resistance gene. Three clones (SAOS-MC10, SAOS-MC11 and SAOS-MC43) among 60 clones expressed p53 mRNA. No p53 protein was observed in SAOS-MC10, while SAOS-MC11 and SAOS-MC43 produced p53 protein. The molecular weight of p53 protein in SAOS-MC43 was lower than that in SAOS-MC11, SAOS-MC11 and SAOS-MC43 were more sensitive and more resistant, respectively, to ionizing radiation than the parental SAOS-2. We suggest that exogenous p53 protein might be one of the factors determining cellular radiosensitivity.

Enhancement of beta-galactosidase gene expression in rat pheochromocytoma cells by exposure to extremely low frequency magnetic fields.

Exposure of PC12-VG cells to an extremely low frequency magnetic field (ELFMF) enhanced the beta-galactosidase gene expression stimulated by treatment of the cells with forskolin. The enhancing effect of the ELFMF was inhibited by treatment of the cells with a specific inhibitor of PKC, calphostin C, as well as with the Ca2+ entry blockers nifedipin and dantrolen. Enhancement appeared within the first hour of a 4h forskolin treatment when the ELFMF was given at different times during culture. We speculate that exposure of PC12-VG cells to an ELFMF during the early response to forskolin treatment affects cell signal transduction, resulting in enhanced gene expression.

Most thymocytes die in the absence of DNA fragmentation.

Most thymocytes are known to be depleted from the thymus during T cell development, with the process of thymocyte death considered to be apoptosis. In this study we examined the mechanism of thymocyte death in the thymus of 6-week-old mice by using terminal deoxynucleotidyl transferase to detect DNA fragmentation or double strand breaks (TUNEL method). The TUNEL positive thymocytes were scattered throughout the cortex. Double staining of the section with the TUNEL method and acid phosphatase (ACP) activity showed that all the TUNEL positive cells were phagocytosed by ACP positive macrophages. An ultra-structural study revealed the presence of a substantial number of extremely small, unphagocytosed thymocytes throughout the cortex. These small unphagocytosed thymocytes were apparently dead cells, as based on several morphological features: 1) The majority were much smaller than red blood cells; 2) the nuclei were also considerably small; and 3) the extent of chromatin condensation was enormous. Importantly, these unphagocytosed dead thymocytes were TUNEL negative. These results indicate that: 1) DNA fragmentation, which is detected by the TUNEL method, is not involved in the cell death process of small unphagocytosed dead thymocytes shown in the present study; and that 2) typical apoptosis, which is characterized by DNA fragmentation, is not the dominant type of cell death in the normal murine thymus. Processes of cell death other than typical apoptosis taking place in most thymocytes require further investigation.

[How should we treat intestinal ischemia?--II: Effects of pentoxifylline, glucagon and prostaglandin E1].

It is important to repair or ameliorate the intestinal ischemia in critically ill patients. Recent study of our suggests the superiority of dobutamine, but not dopamine, in improving the intestinal oxygenation. In this study we examined the effects of pentoxifylline (PF), glucagon (GL) and prostaglandin E1 (PGE1) during reduced blood flow of the superior mesenteric artery (SMA) in 20 anesthetized dogs. As an index of the intestinal oxygenation, tonometrically measured intestinal intramural pH (pHi) was used. A tonometer was inserted into the midjejunum through enterotomy. The SMA blood flow was measured by a transit-time flow meter. A vascular screw clamp for blood flow reduction was placed around the origin of the SMA, proximal to the flow probe. The SMA blood flow was adjusted to 70% of baseline for three hours. After two hours of decreased blood flow, pHi dropped significantly from baseline. Then, either PF (20 mg.kg-1.min-1 over 10 min, followed by 0.1 mg.kg-1.min-1), GL (1 microgram.kg-1.min-1) or PGE1 (0.05 and 0.5 microgram.kg-1.min-1) was infused intravenously for one hour. With infusions of GL and large dose of PGE1, pHi tended to decrease further, although GL increased the cardiac output. Small dose of PGE1 had no significant effect on pHi. PF treatment showed beneficial effects not only on the cardiac output and the SMA blood flow, but also on pHi. We conclude that PF therapy may restore the intestinal microvascular blood flow. Further study of the effects of PF on tissue oxygenation and blood rheology is warranted.

A glucan with immunological activities from the tuber of Alisma orientale.

A glucan, called alisman SI, was isolated from the tuber of Alisma orientale Juzepcz. It was homogeneous on electrophoresis and gel chromatography, and its molecular mass was 1.1 x 10(4). It is composed solely of D-glucose. Methylation analysis, nuclear magnetic resonance and enzymic degradation studies indicated that it has a high-branched glucan type structure mainly composed of alpha-1,4-linked D-glucopyranosyl residues with partially alpha-1,6-linked units and both 3,4- and 4,6-branching points. The polysaccharide exhibited significant reticuloendothelial system-potentiating activity in a carbon clearance test, as well as a pronounced anti-complementary activity.

[How should we treat intestinal ischemia?--1: Effects of dobutamine and dopamine].

Intestinal mucosal ischemia is one of the earliest manifestations of impaired core tissue perfusion in critically ill patients. The aim of this study was to investigate the effects of dobutamine and dopamine on intestinal ischemia in ten anesthetized dogs. Intestinal intramural pH (pHi) by tonometry is an early reliable marker of assessing the adequacy of tissue oxygenation. A tonometer was inserted into the midjejunum through enterotomy. The superior mesenteric artery (SMA) blood flow was measured by a transit-time ultrasonic flowmeter. A vascular screw clamp for blood flow reduction was placed at the origin of the SMA, proximal to the flow probe. The SMA blood flow was maintained at 70% of baseline flow for three hours. After two hours of decreased blood flow, intravenous dobutamine or dopamine was infused at a rate of 5 micrograms.kg-1.min-1 for one hour. The pHi fell significantly from 7.16 +/- 0.04 to 7.08 +/- 0.04 in dobutamine group, and from 7.18 +/- 0.04 to 7.06 +/- 0.05 in dopamine group two hours after induction of intestinal ischemia. Treatments with dobutamine and dopamine increased the SMA blood flow to near baseline levels. Dobutamine caused a significant increase in pHi to 7.11 +/- 0.04. On the contrary, dopamine tended to decrease pHi further to 7.01 +/- 0.09. These results suggest that dobutamine may improve intestinal tissue oxygenation. However, dopamine even at lower doses, may induce constriction of the intestinal mucosal arterioles.

A newly designed experimental system for exposure of mammalian cells to extremely low frequency magnetic fields.

To examine the biological effects of extremely low frequency magnetic field (ELFMF), we have designed and manufactured a new equipment for long-term and high-density exposure of cells to ELFMF. The ELFMF exposure system consists of a generator of magnets with a built-in CO2 incubator, an alternating current (AC) power supply, a gas compressor and a thermocontroller for the incubator, and a cooling unit for the magnets. The CO2 incubator made of acrylic resin is inserted into the inner-space of the silicon steel strip-cores. In this system, the temperature of the incubator is maintained at 37 +/- 0.5 degrees C. The maximum magnetic flux density on the exposure area of the incubator is 500 mT (T; tesla) at a current of 556 Arms (rms; root mean square) at 50 Hz. The long-term (up to 120 hr) exposure of 400 mT ELFMF did not affect the growth of both HL60RG and CCRF-CEM cells originated from human leukemia. The post-X-irradiation exposure of 400 mT ELFMF for 2 hr also did not affect the radiation sensitivity of GM0637 and TAT2SF cells originated from a normal human and an ataxia telangiectasia patient.

[Gastrointestinal mucosal ischemia in hemorrhagic shock--measurement with a tonometer].

Splanchnic ischemia produces the most rapid progression of mucosal injury. A reliable method for monitoring the adequacy of tissue oxygenation in the mucosal layer would be clinically valuable. In this study, we investigated the potential value of tonometric intestinal intramucosal pH (pHi) monitoring during hemorrhagic shock in twelve anesthetized dogs. The tonometer consists of a sampling tube with a silicone balloon attached to the tip that is freely permeable to CO2. Intestinal pHi was calculated by the Henderson-Hasselbalch equation and measurements of the PCO2 of the normal saline within the balloon and arterial bicarbonate. The tonometer was placed in the midpart of the small intestine through a small enterotomy. Arterial blood was removed into a heparinized bag to achieve a mean arterial blood pressure of 50-60 mmHg for two hours. Baseline pHi level was 7.28 +/- 0.02 (mean +/- SE). Following hemorrhage, pHi continued to decrease significantly during the experiments down to 6.94 +/- 0.05 at one hour and 6.82 +/- 0.12 at two hours. By treatment with an intravenous infusion of dopamine at a rate of 3 micrograms.kg-1.min-1 started at one hour after hemorrhage, pHi did not returned to baseline levels. Monitoring pHi in the small intestine using tonometer could be a useful technique to provide early detection of insufficient mucosal blood flow.