Anti-ß2 -glycoprotein I antibody with DNA binding activity enters living monocytes via cell surface DNA and induces tissue factor expression.

Autoantibodies characteristic for anti-phospholipid syndrome (APS) and systemic lupus erythematosus (SLE) are anti-ß2 -glycoprotein I (ß2 GPI) antibodies and anti-DNA antibodies, respectively, and almost half of APS cases occur in SLE. Anti-ß2 GPI antibodies are recognized to play a pivotal role in inducing a prothrombotic state, but the precise mechanism has not been fully elucidated. In a widely accepted view, binding of anti-ß2 GPI antibodies to cell surface ß2 GPI in monocytes and endothelial cells triggers the Toll-like receptor 4-myeloid differentiation primary response 88 (TLR)-4-MyD88) signaling pathway which leads to activation of p38 mitogen-activated protein kinase (MAPK), mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinases (MEK-1/ERK) and/or nuclear factor kappa B (NF-κB) and expression of tissue factor (TF). However, resting cells do not express substantial amounts of TLR-4. Previously, we generated a mouse monoclonal anti-ß2 GPI antibody WB-6 and showed that it induced a prothrombotic state - including TF expression on circulating monocytes - in normal mice. In the current study, we aimed to clarify the mechanism of interaction between WB-6 and resting monocytes, and found that WB-6 exhibits binding activity to DNA and enters living monocytes or a monocytic cell line and, to a lesser extent, vascular endothelial cells. Treatment of the cells with DNase I reduced the internalization, suggesting the involvement of cell surface DNA in this phenomenon. Monocytes harboring internalized WB-6 expressed TF and tumor necrosis factor (TNF)-α which, in turn, stimulated endothelial cells to express intercellular adhesion molecule 1 (ICAM-I) and vascular cell adhesion molecule 1 (VCAM-I). These results suggest the possibility that a subset of anti-ß2 GPI antibodies with dual reactivity to DNA possesses ability to stimulate DNA sensors in the cytoplasm, in addition to the cell surface receptor-mediated pathways, leading to produce proinflammatory and prothrombotic states.

High-speed AFM of human chromosomes in liquid.

Further developments of the previously reported high-speed contact-mode AFM are described. The technique is applied to the imaging of human chromosomes at video rate both in air and in water. These are the largest structures to have been imaged with high-speed AFM and the first imaging in liquid to be reported. A possible mechanism that allows such high-speed contact-mode imaging without significant damage to the sample is discussed in the context of the velocity dependence of the measured lateral force on the AFM tip.

Imaging of human metaphase chromosomes by atomic force microscopy in liquid.

Human metaphase chromosomes were observed using an intermittent contact mode of atomic force microscopy (AFM) in a phosphate-buffered saline solution to clarify their conformation close to that in the physiological state. In the AFM images in liquid, symmetric alternating ridges and grooves were evident on their surface of the paired sister chromatids. The number of the ridges and grooves were rather specific to the type of the chromosome. The structural changes of chromosomes caused by trypsin treatment were also directly observable using AFM in liquid. These results suggest that the intermittent contact mode AFM is useful not only for analyzing the structure of chromosomes in a liquid condition but also for studying the effect of chemical treatments on chromosomes in relation to their structural changes.

Simultaneous activation of natural killer T cells and autoantibody production in mice injected with denatured syngeneic liver tissue.

Denatured syngeneic liver tissue prepared by mechanical procedures was intraperitoneally injected into adult C57BL/6 mice. In parallel with a decrease in the total number of lymphocytes in the liver, spleen, and thymus from days 1-7 after the injection, the proportion of the CD4+NK1.1+CD3(int) subset of these cells (i.e. natural killer T or NKT cells) increased in the liver. Even the absolute number of these NKT cells increased in the liver on days 14 and 21. In response to the injection of denatured liver tissue, tissue damage was induced in the liver, as shown by elevated levels of serum transaminases and hepatocyte degeneration observed by electron microscopy. Sera obtained on days 7 and 14 contained autoantibodies including anti-DNA antibodies. The proportion of CD1d(high)B cells in the liver was found to decrease on days 1-7. In other words, denatured liver tissue stimulated both NKT cells and certain B cells in the liver. These results suggest that liver lymphocytes might contain not only autoreactive T cells (e.g. CD3(int) or NKT cells) but also some B cells (e.g. B-1 cells) which produce autoantibodies and that the denatured tissue had the potential to stimulate these lymphocytes and to evoke an autoimmune-like state.

Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells.

Proteolytic cleavage of the cohesin subunit Scc1 is a consistent feature of anaphase onset, although temporal differences exist between eukaryotes in cohesin loss from chromosome arms, as distinct from centromeres. We describe the effects of genetic deletion of Scc1 in chicken DT40 cells. Scc1 loss caused premature sister chromatid separation but did not disrupt chromosome condensation. Scc1 mutants showed defective repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently failed to complete metaphase chromosome alignment and showed chromosome segregation defects, suggesting aberrant kinetochore function. Notably, the chromosome passenger INCENP did not localize normally to centromeres, while the constitutive kinetochore proteins CENP-C and CENP-H behaved normally. These results suggest a role for Scc1 in mitotic regulation, along with cohesion.

Three-dimensional structure of G-banded human metaphase chromosomes observed by atomic force microscopy.

The structure of G-bands in human metaphase chromosomes was analyzed by comparison between light microscopic and atomic force microscopic (AFM) images of the same chromosomes. G-bands of the chromosomes were made by trypsin treatment followed by staining with a Giemsa solution. The banded chromosomes examined by light microscopy were dried either in air or in a critical point-drier, and observed by non-contact mode AFM. Air-dried chromosomes after G-band staining showed alternating ridges and grooves on their surface, which corresponded to light-microscopically determined G-positive and G-negative bands, respectively. At high magnification, the G-positive ridges were composed of densely packed chromatin fibers, while the fibers were loose in the G-negative grooves. Fibers bridging the gap between sister chromatids of a mitotic pair were often found, especially in the G-positive portions. These findings suggest that the G-banding pattern reflects the high-order structure of human metaphase chromosomes.

Scanning electron microscopic studies on the route of neutrophil extravasation in the mouse after exposure to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP).

The present study was performed to demonstrate three-dimensionally the process of neutrophil extravasation induced by the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) in mice. Thirty to 40 min after the injection of fMLP to the mouse lip, the tissues were fixed with glutaraldehyde and examined by scanning electron microscopy (SEM) as well as by transmission electron microscopy (TEM). Observation of fMLP-injected tissues showed many neutrophils adhering to the inner wall of postcapillary venules. Some of these adherent neutrophils attached to each other to form groups of two to six. There were also many neutrophils migrating through the endothelium. Most of these neutrophils took a transcellular route, in that they penetrated the cytoplasm of the endothelial cells. The junction of two neighboring endothelial cells did not open, and endothelial pores free from migrating neutrophils were scarcely observed. There were bulging portions on the vascular wall which were probably produced by the presence of underlayed neutrophils. Thus, the present study gave direct evidence of neutrophil migration via a transcellular route in response to fMLP. Our findings also indicate that the pores of the endothelium close quickly after neutrophil extravasation.

Selective uptake of intraluminal dextran sulfate sodium and senna by macrophages in the cecal mucosa of the guinea pig.

The involvement of macrophages in the passage of intraluminal substances into the lamina propria was examined in the large intestine of the guinea pig. Dextran sulfate sodium (DSS) and senna, which, experimentally, induce ulcerative colitis and melanosis coli, respectively, were chosen for examination, since these substances are visible under the microscope without any special treatment. DSS (MW 50,000) and senna were orally administered to guinea pigs. In tissue sections of the intestine, the presence of DSS was demonstrated by toluidine blue staining, while senna was visible under the light microscope as brown pigment. In the large intestine of guinea pigs, macrophages were most numerous in the cecum, decreasing in number towards the rectum. Metachromatic reaction due to DSS was first recognized in the epithelium of the cecum, and was subsequently incorporated by macrophages. The presence of DSS, either in the epithelium or in macrophages, was not recognized in the small intestine or the distal colon. Senna pigmentation was also limited to the cecum and proximal colon, in which pigmented macrophages aggregated in the lamina propria. The two different substances administered orally were taken up in the cecum, and partly also in the proximal colon; the substances passed through the epithelium and were incorporated by macrophages. This finding suggests the existence of a weak point in the intestinal barrier in this particular portion of the intestine.

Nitric oxide synthase in rat nasal mucosa; immunohistochemical and histochemical localization.

The localization of nitric oxide synthase (NOS) and its cofactor, nicotinamide-adenine dinucleotide hydrogen phosphate (NADPH)-diaphorase, was examined in the nasal mucosa of the rat by immunohistochemical and histochemical methods. In addition to cryostat sections, whole mount preparations were used to examine the distribution of nerves. Both in the nasal mucosa and in associated ganglia, the distribution of NOS-immunoreactive nervous structures essentially corresponded to that of NADPH-diaphorase-positive ones. The NOS-immunopositive nerve fibers in the respiratory area of the nasal mucosa were distributed around blood vessels and in submucosal glands. Part of the respiratory area was supplied with intraepithelial arborizations of the immunopositive fibers. The epithelial cells in the respiratory area were NADPH-diaphorase positive but NOS immunoreactivity negative. In the olfactory area, the NADPH-diaphorase- and NOS-positive nerve fibers were restricted to blood vessels located deep in the submucosa. Throughout the nasal mucosa, arterial endothelium was NADPH-diaphorase positive but NOS immunoreactivity negative. Both NOS immunoreactivity and NADPH-diaphorase activity were found in major populations of neuronal somata in the sphenopalatine ganglion. The present study provides the direct evidence supporting the notion that nitric oxide is richly produced in autonomic nerves of the nasal mucosa derived from the sphenopalatine ganglion.

Regional differences of CGRP-immunoreactive nerve fibers in nasal epithelium of the rat.

The regional distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the epithelium throughout the nasal cavity was determined using semiserial cryostat sections and whole mount preparations. Both respiratory and olfactory epithelia displayed, in their basal portion, an extensive nerve plexus projecting beaded nerve fibers toward the luminal surface. However, the density of the intraepithelial CGRP nerve fibers conspicuously varied according to regions. They were abundant on the septal mucosa, the ventromedial side of the nasoturbinates and the dorsal surface of the maxilloturbinates, while less numerous on the lateral side of the naso- and maxilloturbinates and on the lateral nasal wall. Another difference in the regional distribution of nerves was recognized between the anterior and posterior portions of the respiratory area: the anterior portion received a denser supply of intraepithelial CGRP fibers than the posterior portion. Characteristically, the transepithelial CGRP-immunoreactive nerve fibers were densest on the anteroventral aspect of the nasoturbinates and on the anterodorsal surface of the maxilloturbinates. Some of them appeared to penetrate through the epithelium to come into contact with the lumen of the nasal cavity. These results suggest that the CGRP fibers in the epithelium display a region-specific distribution, apparently disposed more densely over the areas which are more directly exposed to inhaled air, possibly that containing irritants and toxicants.

Topographical relation between serotonin-containing paraneurons and peptidergic neurons in the intestine and urethra.

Luminal stimulation of enterochromaffin (EC) cells is known to cause their release of serotonin which in turn may induce a variety of reflexes via mucosal intrinsic neurons. To morphologically support this idea, the present study demonstrates a close topographical relationship between EC cells and nerves, using whole-mount preparations. Beaded nerve fibers containing vasoactive intestinal polypeptide (VIP) were observed in close proximity to serotonin-immunoreactive EC cells in the small and large intestines of the rat and guinea pig; in whole-mount preparations, the VIP nerve fibers appeared to underlie EC cells. This finding correlates with the physiological datum that the intra-arterial infusion of serotonin causes VIP release from the intestine. Canine urethral serotonin cells, a counterpart of the intestinal EC cells, were shown to contact intraepithelial nerves immunoreactive for calcitonin gene-related peptide. The neuroparaneuronal connection in the urethra may play an important role in the serotonin-evoked urethrogenital reflex. Intestinal and urethral serotonin-containing paraneurons, which are sensory in nature, may release serotonin in response to luminal stimuli, and directly activate adjacent peptidergic neurons to initiate the reflex arcs.

Lamina propria macrophages involved in cell death (apoptosis) of enterocytes in the small intestine of rats.

In the rat small intestine, apoptotic enterocytes are exfoliated at the villus tip as a whole cell, in contrast to guinea pig enterocytes which are phagocytosed by macrophages in their cell body and shed off only in their apical cortex. While macrophages gather in the lamina propria of the intestinal villi in both species, their functions seem to differ. Unlike the guinea pig, lamina propria macrophages observed in the rat small intestine did not show morphological signs of phagocytosis, revealing few cellular elements in their phagosomes. At the "shoulder" of the villus, i.e., a certain distance proximal to the villus tip, subepithelial macrophages extended a thick process deep into the epithelium; their branched terminals penetrated the cytoplasm of enterocytes, resulting in the formation of excavated spaces in the cell body. Processes of macrophages frequently reached close to the brush border. At the shoulder of the villus, a few effete cells showed typical apoptotic signs and appeared to be pushed out into the lumen; still, the shedding of apoptotic enterocytes was recognized mainly at the very top of the villus, where no intraepithelial processes of macrophages could be seen. The present findings indicate that in the rat, lamina propria macrophages do not engulf aged enterocytes, but are involved in inducing their apoptosis.

Morphological analysis of acute ulcerative colitis experimentally induced by dextran sulfate sodium in the guinea pig: some possible mechanisms of cecal ulceration.

This study in the guinea pig demonstrated that ulcerative colitis-like lesions were induced more rapidly and effectively than in other laboratory animals by the oral administration of dextran sulfate sodium (DSS). In all guinea pigs receiving 3% DSS solution, diarrhea was noted within 48h, and bleeding at 48-72h. Light microscopically, hemorrhagic and ulcerative lesions were observed in the cecum and proximal colon as early as 72h after administration. Lamina propria macrophages, gather in the subepithelial region under normal conditions, were markedly increased in number after DSS administration; by 96 h, they were approximately three times as many as in the control specimens. The cecal mucosa was also characterized, in earlier stages, by the obliteration of virtually all cryptal lumina because of the accumulation of mucous secretions, leading to the subsequent disappearance of the crypts. The obliteration of crypts, which preceded the increase of macrophages, is suggested to play a leading part in this ulceration.

[A case of bronchogenic cyst in the posterior mediastinum with high level of serum SCC antigen].

A 78-year-old man was picked up by chest X-ray findings. His CT scan film showed a posterior mediasinal tumor shadow. Serum level of SCC antigen was high and thoracotomy revealed bronchogenic cyst with high production of SCC antigen. After resection of the cyst, his serum level of SCC was within normal range. It is proposed that the elevated serum level of SCC is due to chronic inflammation or structural feature of the cystic wall.

[Two cases of cloacogenic carcinoma of the anal canal: chemotherapeutic effect of cis-dichlorodiammine-platinum].

Cloacogenic carcinoma, originating from the remnant of the cloaca, is a rare malignant tumor, comprising 2-3% of cancers of the anal canal. We reported here two cases of cloacogenic carcinoma whose pulmonary and hepatic metastases were successfully treated with CDDP administration, in spite of limited effect on primary lesions of the rectum. The promising usage of CDDP for cloacogenic carcinoma, especially for distant metastasis was discussed.