Heterogeneous Expression and Release of CD14 by Human Gingival Fibroblasts: Characterization and CD14-Mediated Interleukin-8 Secretion in Response to Lipopolysaccharide

To identify the role in periodontal inflammatory diseases of human gingival fibroblasts (HGF), the major constituents of gingival tissue, the expression of CD14, a possible lipopolysaccharide (LPS) receptor, and the release of soluble CD14 (sCD14) by HGF were examined. Among the HGF samples from the nine donors tested, more than 50% of the HGF from five donors expressed CD14 but less than 20% of HGF from the other four donors did so, as determined by flow cytometric analysis. The CD14 expression on the cell surface was correlated with the expression of CD14 mRNA. The HGF and skin and lung fibroblasts tested expressed no CD18, which indicates that fibroblasts do not possess other LPS receptors, such as CD11b/CD18 and CD11c/CD18. The CD14 expression by the HGF was decreased after subculturing and was highest at the confluent stage of culture. The treatment of high-CD14-expressing (CD14^high) HGF with phosphatidylinositol-phospholipase C reduced CD14 expression; this result and the increase in a 55-kDa CD14 indicate that the membrane CD14 (mCD14) on the HGF may be a 55-kDa glycosylphosphatidylinositol-anchored protein. CD14^high HGF spontaneously released 48- and 57-kDa sCD14. The total release of sCD14 by the HGF was augmented by gamma interferon and Escherichia coli LPS in accordance with the increased expression of mCD14. The CD14^high HGF secreted interleukin-8 in response to LPS, and the secretion was completely inhibited by anti-CD14 antibody. These results suggest that (i) HGF consist of populations that are heterogeneous on the basis of different levels of expression of CD14 and (ii) CD14^high HGF secrete inflammatory cytokines in response to LPS via CD14.     

Prevention of endotoxin shock by an antibody against leukocyte integrin {beta}2 through inhibiting production and action of TNF

Septic shock remains a serious disorder associated with highmortality. Accumulating evidence indicates that TNF is a majorand essential mediator of endotoxin shock. We report here thatadministration of an antibody against CD18 dramatically reducedendotoxin-induced shock inrabbits as revealed by preventionof severe hypotension, metabolic acidosis and a pathologicalchange suggestive of disseminated intravascular coagulationwith concomitant inhibition of elevation of plasma TNF activity.The anti-CD18 antibody also inhibited the hypotension inducedby administering recombinant TNF. Furthermore, an antibody againsta ligand for CD18 complexes, intercellular adhesion molecule-1,also prevented TNF-induced shock as well as endotoxin shockinrabbits. These observations suggest that adhesion of leukocytesto endothelium may be of primary importance in the action ofTNF as well as in the production of TNF in vivo and that theantibody against adhesion molecules could be of therapeuticbenefit in life-threatening septic shock in humans.     

Genetic contribution of tumor necrosis factor (TNF)-alpha gene promoter (-1031, -863 and -857) and TNF receptor 2 gene polymorphisms in endometriosis susceptibility

PROBLEM: Tumor necrosis factor (TNF)-alpha is a major cytokine involved in inflammatory and immune function. The aim of this study was to investigate whether polymorphisms at positions -1031, -863 and -857 in the TNF gene promoter region (TNFA) and TNF receptor type 2 gene (TNFR2) are responsible in part for genetic susceptibility to endometriosis. METHODS OF STUDY: TNFA and TNFR2 polymorphisms were determined in 123 patients with endometriosis and 165 fertile healthy women by the polymerase chain reaction (PCR) - preferential homoduplex formation assay and PCR-restriction fragment length polymorphism, respectively. RESULTS: The frequency of the TNFA-U01 haplotype was increased significantly in patients with endometriosis compared with controls (P = 0.045, OR = 1.45). The TNFA-U01 haplotype was strongly associated with HLA-B*0702. No difference was found in TNFR2 polymorphism between patients and controls. CONCLUSION: Our results indicated that TNFA promoter polymorphism was associated with susceptibility to endometriosis. However, this association was not independent of HLA-class I polymorphisms.     

Widespread lipoplex-mediated gene transfer to vascular endothelial cells and hemangioblasts in the vertebrate embryo.

We report here a method that allows fast, efficient, and low-cost screening for gene function in the vascular system of the vertebrate embryo. Through intracardiac delivery of nucleic acids optimally compacted by a specific cationic lipid, we are able to induce in vivo endothelial cell-specific gain-of-function during development of the vascular network in the chick embryo. When the nucleic acids are delivered during the period of intraembryonic hematopoiesis, aortic hemangioblasts, the forerunners of the hematopoietic stem cells known to derive from the aortic endothelium, are also labeled. Similarly, we show that siRNA could be used to induce loss-of-function in vascular endothelial cells. This gene transfer technique was also applied to the mouse embryo with a high efficiency. The present method allows large-scale analysis and may represent a new and versatile tool for functional genomics.     

Effects of focus on working memory

Effects of focus on a target word during performance of the reading span test (RST) were investigated. A focus word in the sentence was defined as the most critical word with a core meaning to integrate the sentence. Two kinds of RST were compared. One was focus-RST (F-RST) in which the target word to be maintained was a focus word of the sentence. The other was a non-focus-RST (NF-RST) in which the target word was not a focus word of the sentence, although the sentence did contain a focus word. Results showed that RST scores were found to be higher for F-RST than for NF-RST. Moreover, the effect of focus was proved to be dominant for low span subjects. Intrusion errors also increased in NF-RST. Sentence length effect, however, was not found. The results showed that low span subjects had severe deficits in making and updating the focus, which is critical for sentence comprehension.     

Eicosapentaenoic acid inhibits antigen-presenting cell function of murine splenocytes.

Recently, many investigators have studied the effects of eicosapentaenoic acid (EPA)-rich fish oil on immune function and immune disease. However, effects of dietary supplementation of fish oil or EPA on the immune system are still unclear. In the present study, the effects of EPA on antigen presentation were investigated. We have used antigen-specific helper T-cell clones that proliferate in the presence of antigen [keyhole limpet haemocyanin (KLH)] and spleen cells as antigen-presenting cells (APC). Mice were divided into two groups and fed an experimental diet or a control diet for 4 weeks ad libitum. In mice fed the experimental diet, the arachidonic acid (AA) content of spleen cells was decreased and that of EPA and docosapentaenoic acid was increased markedly compared to those of the control diet. Dietary enrichment with EPA inhibited the ability of accessory cells to present antigen to murine helper T-cell clones. This effect was observed for two distinct helper T-cell clones, Th1 and Th2. We also examined the effects of EPA-TG emulsion on APC function. The direct addition of EPA-TG emulsion to a T-cell proliferation assay system suppressed APC function. The inhibition was proportional to the concentration of EPA-TG emulsion. Pretreatment of splenocytes with EPA-TG emulsion resulted in inhibition of APC function. Inhibition of antigen presentation by dietary supplementation with EPA might depress immune reactivity.     

Distribution of 5-hydroxytryptamine (5HT) in tissue of a mutant mouse deficient in mast cell (W/W v ). Demonstration of the contribution of mast cells to the 5HT content in various organs

The content of 5-hydroxytryptamine (5HT) in various tissues of mutant mouse (W/W ^v) deficient in mast cells and of control mouse (+/+) was determined by high performance liquid chromatography. The depletion of mast cells in the mutant mouse (W/W ^v) was expected to cause a decrease in the 5HT content. In the control mice, 5HT was most densely accumulated in the lung (9.66±5.23 g/g). Large intestine (6.40±2.61 g/g) and stomach (6.10±2.14 g/g) followed the lung in the rating of the 5HT content. The 5HT content ofW/W ^v mice was only 23.4% and 4.1% that of the control in the stomach (p<0.01) and the skin (p<0.01), respectively. The results were consistent with the expectation. In other organs (small intestine, caecum, large intestine, brain, lung, blood and salivary gland), the difference between theW/W ^v and normal mice was not statistically significant. The difference in the 5HT content of the stomach between the two genotypes was 4.67 g/g and was much larger than the 5HT content (0.49g/g) of normal mouse skin. With regard to the relatively small number of mast cells present in the stomach, the great difference in the 5HT content in the stomach between the two genotypes cannot be explained by the loss of mast cells. Hence, besides mast cells other cells may contribute to the high 5HT content of the stomach.This work was supported in part by grants from Takeda Science Foundation (1982), from the Ministry of Education, Science and Culture of Japan (Grant-in-Aid for Special Project Research. 1981–83) and from National Center of Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan (1981–83).To whom correspondence should be addressed     

Cytoplasmic aggregation of TRAF2 and TRAF5 proteins in the Hodgkin-Reed-Sternberg cells

We previously reported that ligand-independent signaling by highly expressed CD30 in Hodgkin-Reed-Sternberg (H-RS) cells is responsible for constitutive activation of NF-kappa B. In the present study, we characterize the intracellular localization of tumor necrosis factor (TNF) receptor associated factor (TRAF) proteins in H-RS cells. Confocal immunofluorescence microscopy of cell lines derived from H-RS cells and HEK293 transformants highly expressing CD30 revealed aggregation of TRAF2 and TRAF5 in the cytoplasm as well as clustering near the cell membrane. In contrast, TRAF proteins were diffusely distributed in the cytoplasm in cell lines unrelated to Hodgkin's disease (HD) and control HEK293 cells. Furthermore, the same intracellular distribution of TRAF proteins was demonstrated in H-RS cells of lymph nodes of HD, but not in lymphoma cells in lymph nodes of non-Hodgkin's lymphoma. Dominant-negative TRAF2 and TRAF5 suppressed cytoplasmic aggregation along with constitutive NF-kappa B activation in H-RS cell lines. Confocal immunofluorescence microscopy also revealed co-localization of IKK alpha, NIK, and I kappa B alpha with aggregated TRAF proteins in H-RS cell lines. These results suggest involvement of TRAF protein aggregation in the signaling process of highly expressed CD30 and suggest they function as scaffolding proteins. Thus, cytoplasmic aggregation of TRAF proteins appears to reflect constitutive CD30 signaling which is characteristic of H-RS cells.     

A murine plasmacytoma with a variant (15;16)(D2/3;B1) translocation that involves the c-myc and lambda light chain gene-carrying chromosomes

A murine plasmacytoma (MPC) with a reciprocal translocation between chromosomes 15 and 16 with breakpoints in 15D2/3 and 16B1 is reported. The breakpoint on chromosome 15 is identical to the breakpoint in the MPC-associated typical (12;15) and kappa variant (6;15) translocation. Therefore it probably involves the c-myc gene as well. Unlike the Burkitt lymphoma (BL) system, a lambda/myc variant translocation has not been described in the MPC system. Chromosome 16 is known to carry the lambda gene. Therefore, the 15;16 translocation probably represents the "missing" lambda/myc variant in MPC, suggesting that the lambda gene is localized at 16B1.     

Attenuation of antigen-induced bronchoconstriction in guinea pigs by a new xanthine derivative (HWA448).

We examined the effect of a new xanthine derivative, HWA448, on antigen-induced bronchoconstriction in actively sensitized guinea pigs. Guinea pigs were sensitized by intraperitoneal injection of bovine serum albumin (BSA) on two occasions, separated by 10 days. Two weeks after the second injection, the animal was placed in a two-chambered whole body plethysmograph and specific airway resistance (SRaw) was monitored for 10 min after an aerosol inhalation of BSA. HWA448 prevented the increase in SRaw after challenge (at 5 and 20 mg/kg i.p.). Aminophylline also prevented the increase in SRaw at 20 mg/kg, but not at a 5-mg/kg dose. The concentration of HWA448, which produced 50% relaxation of the tracheal rings constricted with 0.1 mM of histamine, was 49.9 microM as compared with 18.2 microM in aminophylline. HWA448 has a protective effect on antigen-induced bronchoconstriction in guinea pigs and may be a useful agent in the therapy of bronchial asthma.