Marrow stromal cells and osteoclast precursors differentially contribute to TNF-alpha-induced osteoclastogenesis in vivo

The marrow stromal cell is the principal source of the key osteoclastogenic cytokine receptor activator of NF-kappaB (RANK) ligand (RANKL). To individualize the role of marrow stromal cells in varying states of TNF-alpha-driven osteoclast formation in vivo, we generated chimeric mice in which wild-type (WT) marrow, immunodepleted of T cells and stromal cells, is transplanted into lethally irradiated mice deleted of both the p55 and p75 TNFR. As control, similarly treated WT marrow was transplanted into WT mice. Each group was administered increasing doses of TNF-alpha. Exposure to high-dose cytokine ex vivo induces exuberant osteoclastogenesis irrespective of in vivo TNF-alpha treatment or whether the recipient animals possess TNF-alpha-responsive stromal cells. In contrast, the osteoclastogenic capacity of marrow treated with lower-dose TNF-alpha requires priming by TNFR-bearing stromal cells in vivo. Importantly, the osteoclastogenic contribution of cytokine responsive stromal cells in vivo diminishes as the dose of TNF-alpha increases. In keeping with this conclusion, mice with severe inflammatory arthritis develop profound osteoclastogenesis and bone erosion independent of stromal cell expression of TNFR. The direct induction of osteoclast recruitment by TNF-alpha is characterized by enhanced RANK expression and sensitization of precursor cells to RANKL. Thus, osteolysis attending relatively modest elevations in ambient TNF-alpha depends upon responsive stromal cells. Alternatively, in states of severe periarticular inflammation, TNF-alpha may fully exert its bone erosive effects by directly promoting the differentiation of osteoclast precursors independent of cytokine-responsive stromal cells and T lymphocytes.     

In vitro cytotoxicity assay to evaluate the toxicity of an electrophilic reactive metabolite using glutathione-depleted rat primary cultured hepatocytes

Glutathione plays an important role as not only a scavenger of reactive oxygen species but also in the conjugation or detoxification of electrophilic reactive metabolites, which has been thought to be one of the causes for idiosyncratic drug toxicity (IDT). Therefore, toxic responses to the reactive metabolites have been expected to be expressed more strongly in a glutathione-depleted condition. In the present study, we attempted to establish an in vitro cytotoxicity assay method to evaluate the toxicity of the reactive metabolite using rat primary cultured hepatocytes with cellular glutathione depletion by l-buthionine-S,R-sulfoximine. Also, we investigated whether the IDT risk is predictable by comparing the cytotoxic sensitivity between glutathione-depleted hepatocytes and untreated hepatocytes. Consequently, 10 drugs of 42 approved drugs, which were classified into 4 IDT categories (Withdrawn, Black box warning, Warning, and Safe), demonstrated higher cytotoxic sensitivity in the glutathione-depleted hepatocytes. Furthermore, a correlation was observed between the incidence of drugs with higher cytotoxic sensitivity in the glutathione-depleted hepatocytes and the IDT risk. The incidence was 50% in the Withdrawn category, 38% in the Black box warning category, 22% in the Warning category, and 8% in the Safe category. These results suggest that the IDT risk of some drugs may be predicted by comparing the cytotoxic sensitivity between them. Additionally, this method may be useful as a screening in the early stage of drug development where leads/candidates are optimized.     

Endothelin-1, an ulcer inducer, promotes gastric ulcer healing via mobilizing gastric myofibroblasts and stimulates production of stroma-derived factors

Endothelin (ET)-1 is a potent inducer of peptic ulcers. The roles of ET-1 in ulcer healing, however, have remained unclear, and these were investigated in mice. Gastric ulcers were induced in mice by serosal application of acetic acid. Three days later, mice were given a neutralizing ET-1 antibody or nonimmunized serum. The ulcer size, amount of fibrosis and myofibroblasts, and localization of ET-1 and ET(A/B) receptors were analyzed. To elucidate the mechanisms underlying the effects of ET-1, we examined the proliferation, migration, and release of growth and angiogenic factors in gastric myofibroblasts with or without ET-1. The expression of prepro-ET-1 (an ET-1 precursor) and ET-converting enzyme-1 was examined in gastric myofibroblasts using RT-PCR. Immunoneutralization of ET-1 delayed gastric ulcer healing. The areas of fibrosis and myofibroblasts were smaller in the anti-ET-1 antibody group than in the control. ET-1 was expressed in the gastric epithelium, myofibroblasts, and other cell types. ET(A) receptors, but not ET(B) receptors, were present in myofibroblasts. ET-1 increased proliferation and migration of gastric myofibroblasts. ET-1 stimulated the release of hepatocyte growth factor, VEGF, PGE(2), and IL-6 from gastric myofibroblasts. mRNA for prepro-ET-1 and ET-converting enzyme-1 was also expressed. ET-1 promotes the accumulation of gastric myofibroblasts and collagen fibrils at gastric ulcers. ET-1 also stimulates migration and proliferation of gastric myofibroblasts and enhances the release of growth factors, angiogenic factors, and PGE(2). Thus ET-1 has important roles not only in ulcer formation but also in ulcer healing via mobilizing myofibroblasts and inducing production of stroma-derived factors.     

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.     

Kinetic and growth parameters of Arthrospira (Spirulina) platensis cultivated in tubular photobioreactor under different cell circulation systems

Arthrospira platensis was cultivated in tubular photobioreactor in order to evaluate growth and biomass production at variable photosynthetic photon flux density (PPFD = 60, 120, and 240 μmol photons m(-2)s(-1)) and employing three different systems for cell circulation, specifically an airlift, a motor-driven pumping and a pressurized system. The influence of these two independents variables on the maximum cell concentration (X(m)), cell productivity (P(x)), nitrogen-to-cell conversion factor (Y(X/N) ), photosynthetic efficiency (PE), and biomass composition (total lipids and proteins), taken as responses, was evaluated by analysis of variance. The statistical analysis revealed that the best combination of responses' mean values (X(m) = 4,055 mg L(-1), P(x) = 406 mg L(-1)day(-1), Y(X/N) = 5.07 mg mg(-1), total lipids = 8.94%, total proteins = 30.3%, PE = 2.04%) was obtained at PPFD = 120 μmol photons m(-2)s(-1); therefore, this light intensity should be considered as the most well-suited for A. platensis cultivation in this photobioreactor configuration. The airlift system did not exert any significant positive statistical influence on the responses, which suggests that this traditional cell circulation system could successfully be substituted by the others tested in this work.     

Variation in stress resistance patterns among stx genotypes and genetic lineages of shiga toxin-producing Escherichia coli O157

To evaluate the relationship between bacterial genotypes and stress resistance patterns, we exposed 57 strains of Shiga toxin-producing Escherichia coli (STEC) O157 to acid, freeze-thaw, heat, osmotic, oxidative, and starvation stresses. Inactivation rates were calculated in each assay and subjected to univariate and multivariate analyses, including principal component analysis (PCA) and cluster analysis. The stx genotype was determined for each strain as was the lineage-specific polymorphism assay (LSPA6) genotype. In univariate analyses, strains of the stx(1) stx(2) genotype showed greater resistance to heat than strains of the stx(1) stx(2c) genotype; moreover, strains of the stx(1) stx(2) genotype showed greater resistance to starvation than strains of the stx(2) or stx(2c) genotypes. LSPA6 lineage I (LI) strains showed greater resistance to heat and starvation than LSPA6 lineage II (LII) strains. PCA revealed a general trend that a strain with greater resistance to one type of stress tended to have greater resistance to other types of stresses. In cluster analysis, STEC O157 strains were grouped into stress-resistant, stress-sensitive, and intermediate clusters. In stx genotypes, all strains of the stx(1) stx(2) genotype were grouped with the stress-resistant cluster, whereas 72.7% (8/11) of strains of the stx(1) stx(2c) genotype grouped with the stress-sensitive cluster. In LI strains, 77.8% (14/18) of the strains were grouped with the stress-resistant cluster, whereas 64.7% (11/17) of LII strains were grouped with the stress-sensitive cluster. These results indicate that the genotypes of STEC O157 that are frequently associated with human illness, i.e., LI or the stx(1) stx(2) genotype, have greater multiple stress resistance than do strains of other genotypes.     

Complement activation plays a key role in antibody-induced infusion toxicity in monkeys and rats

Infusion reactions are a major side effect of the administration of therapeutic Abs and are the result of a complex immune reaction. In this study, we report that substitutions of Fc amino acids in the anti-HLA-DR Ab HD8 reduce its ability to induce infusion reactions in rats and monkeys. We first showed that i.v. administration of IgG1- and IgG2-subclass HD8 Abs induces severe infusion reactions in monkeys. These Abs express strong complement-dependent cytotoxicity (CDC), and in vivo depletion of complement in rats by pretreatment with cobra venom factor abrogated the lethal infusion reactions generated by HD8-IgG1. Thus, the infusion reactions appear to be largely driven by the complement system. To reduce the CDC function of HD8-IgG1, its Fc region was modified by two amino acid substitutions at Pro(331)Ser and Lys(322)Ala. The modified Ab was incapable of expressing CDC in vitro and did not induce severe infusion reactions in rats and monkeys, even at extremely high doses. The modified Ab retained its Ab-dependent cellular cytotoxicity function as well as its antitumor activity in a tumor-bearing mouse model. In summary, complement appears to drive infusion reactions, and modifications that eliminate the CDC activity of an Ab also reduce its ability to induce infusion reactions.     

A water quality analysis system to evaluate the impact of agricultural activities on N outflow in river basins in Japan

We have developed a personal-computer-based water quality analysis system for river basins. The system estimates potential N outflow by model and calculates actual N outflow from monitoring data. For the former it uses the potential load factor method to estimate annual nitrogen load from various sources and runoff potential from each area of land in a basin. For the latter it analyzes water quality monitoring data in relation to meteorological data. We used the system to analyze N outflow in basins around Lake Kasumigaura and the Yahagi River in central Honshu, Japan. The land around Lake Kasumigaura is rather flat, and about 25% is periodically flooded for rice and lotus cultivation. The land around the Yahagi River is mountainous, and much less land is flooded. In the Yahagi River basin the actual N outflow agreed closely with the potential. However, the actual N outflow in the basin around Lake Kasumigaura was much less than the potential, suggesting that a large part of the N load is denitrified in flooded soils. This further indicates that a sequence of different land uses including flooded rice fields is an important factor determining N outflow in basins in Japan. On the basis of the above analyses, we incorporated a denitrification model into the system that enables us to estimate N balance in a designated basin; this system may be helpful in the formulation of scenarios of land use and soil management for improving water quality.     

Effect of pre-flight circadian phase-shifting approach on sleep variables after 9 time-zone eastward transition: a case report

Sleep and Biological Rhythms - The purpose of this study was to evaluate the effects of a pre-flight circadian phase-shifting approach on sleep variables after 9 time-zone eastward transition. Two...