Diesel exhaust (DE)-induced cytokine expression in human bronchial epithelial cells: a study with a new cell exposure system to freshly generated DE in vitro

We devised a new in vitro cell exposure system to freshly generated diesel exhaust (DE), different from conventional in vitro culture systems, to examine the effects of DE on human epithelial cells. Using this system, we investigated the effects of DE on cytokine gene expressions in BET-1A human bronchial epithelial cells. DE significantly decreased [(3)H]thymidine incorporation into BET-1A cells. DE had a significant stimulatory effect on interleukin (IL)-8 release to a marked degree. IL-8 and transforming growth factor (TGF)-beta1 messenger RNA (mRNA) expression were induced by DE in a time-dependent manner. The gas obtained by filtration of DE alone did not show a sustained increase in IL-8 protein levels and showed no induction of IL-8 mRNA, suggesting that DE particles (DEPs) play an important role in the induction of IL-8 at both mRNA and protein levels. Antioxidants, pyrrolidine dithiocarbamate, and N-acetyl-L-cysteine significantly inhibited IL-8 mRNA and protein levels by BET-1A cells. These results indicate that freshly generated DEPs may be important in the induction of cytokines such as IL-8 and TGF-beta1 relevant to allergic airway inflammation.     

Experimental haemarthrosis in rhesus monkeys: morphometric, biochemical and metabolic analyses

The effects of a single episode of massive haemarthrosis in rhesus monkeys were studied. Autologous whole blood was injected into a femorotibial joint of 16 anaesthetized monkeys, equally divided into four groups and killed 7 days, 2, 3 and 6 months post-injection (PI). Synovial membrane and femoral articular cartilage were analysed morphometrically and articular cartilage was further analysed biochemically and metabolically. At 7 days PI, morphometric evaluation revealed a significant increase (P less than 0.05) in synovial membrane cellularity and synovial intimal thickness of injected joints versus control joints. This change was no longer evident 2 months PI. There was also an overall (n = 16) significant increase (P less than 0.05) in femoral articular cartilage cellularity in injected joints. The average chondrocyte lacuna area of injected joints was not statistically different from the control joints. Biochemical analyses of femoral articular cartilage revealed a significant decrease in hexosamine concentration (P less than 0.05) of injected joints. There was no significant difference between the injected and control joints in hydroxyproline or total protein concentration. Metabolic analyses revealed a significant increase (P less than 0.05) in cartilage collagenous protein production by injected joints compared with control joints. There were no significant differences in cartilage or secreted total protein production between injected and control joints. There were also no significant differences in cartilage or secreted proteoglycan production between joints. Morphometric evaluation of articular tissues following massive haemarthrosis has quantified a temporary hyperplastic reaction. A significant decrease in cartilage hexosamine concentration in haemarthrotic joints suggests this is a crucial biochemical event in the pathogenesis of blood-induced cartilage destruction.     

Immunohistological analysis in the distribution of cells in the fetal porcine adenohypophysis

Adenohypophyses of porcine fetuses from 25 to 110 days of gestation were studied by immunohistochemical staining to ascertain the ontogeny of specific cell types and their spatial distribution in the pars distalis. No hormonecontaining cells were found before 30 days of gestation. ACTH cells were observed first at 40 days, while GH and LH cells appeared first at 60 days. PRL cells were initially detected at 105 days. ACTH immunoreactive cells were also observed in the pars intermedia at 40 days. Blood capillaries were interposed between cell cords of the pars distalis after 40 days of gestation. ACTH cells were evenly distribution in all areas of the pars distalis except the rostal area (sex zone). GH cells were densely distributed in lateral wings of the pars distalis and immediately anterior to Rathke's lumen. PRL cells resembled GH cells in their distribution pattern, but PRL cells were fewer in number. LH cells were scattered in the sex zone of the pars distalis from 60 to 80 days of gestation. After 90 days, they became scattered throughout the pars distalis but were more numerous in the sex zone than in other areas. The inductive elements of adenohypophysial cells from Rathke's pouch epithelia are discussed. We hypothesize that cell cords of specific areas facing Rathke's lumen may differentiate into specific cell types of the pars distalis during fetal life. © 1992 Wiley-Liss, Inc.     

Genetic susceptibility testing for hereditary breast and ovarian cancer--approach from a commercial clinical laboratory

Genetic testing for hereditary cancers and other common diseases are still considered as the research testing, not for the clinical testing in Japan. One of the major reason of this situation is related to the guidelines regarding the human genetic testing issued successively in the spring of 2001, one by joint work of the eight learned societies and the other by the Japan clinical laboratories association. Both of these guidelines warn the condition of the clinical application of genetic testing after research stage must have the evidence data for clinical utility. We describe the situation of the genetic testing in the U.S. focusing the social background of increasing breast cancer cases and the contribution of Myriad Genetic Laboratories, Inc. for the genetic testing industry. We also describe the Japanese situation of the genetic testing and problems to be solved before spreading widely.     

Active Cyclin A-CDK2 Complex, a Possible Critical Factor for Cell Proliferation in Human Primary Lung Carcinomas

Expression of cyclins A and E and cyclin-dependent kinase 2 (CDK2) was examined immunohistochemically in 190 cases of human lung carcinoma. Cyclin A and CDK2 were expressed in the majority of squamous cell carcinomas, small cell carcinomas, and large cell carcinomas, but in significantly fewer cases of adenocarcinomas. Cyclin E was expressed in a minority of all subtypes. In particular, well differentiated cells in squamous cell carcinoma stained positively for cyclin E; in contrast, cyclin A was expressed in the nonkeratinized proliferating areas of the tumor nests. Immunoblotting revealed that all these proteins were expressed at higher levels in tumor tissues than in adjacent normal tissues. Immunoprecipitation also revealed higher levels of cyclin A and cyclin E associated with CDK2 in tumor tissues. Furthermore, tumor tissues which exhibited higher cyclin A and CDK2 expression also had higher CDK2 kinase activity. However, cyclin E-associated kinase activity was barely detectable even in tumor samples exhibiting higher cyclin E expression. Consistent with these data, elevated expression of cyclin A correlated to shorter survival periods in contrast to expression of cyclin E, which correlated to longer survival periods. These results suggest that in human lung carcinomas, elevated expression of active cyclin A-CDK2 complexes with associated higher CDK2 kinase activity is critical for promoting cell cycle progression and unrestrained proliferation of tumor cells and can be a predictive marker for patients’ prognosis. On the other hand, immunohistochemical detection of cyclin E-CDK2 reflects accumulation of inactive forms of protein complexes, implying differentiation or senescence of the tumor and the better prognosis.     

Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice

Missense mutations of the tau gene cause autosomal dominant frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an illness characterized by progressive personality changes, dementia, and parkinsonism. There is prominent frontotemporal lobe atrophy of the brain accompanied by abundant tau accumulation with neurofibrillary tangles and neuronal cell loss. Using a hamster prion protein gene expression vector, we generated several independent lines of transgenic (Tg) mice expressing the longest form of the human four-repeat tau with the R406W mutation associated with FTDP-17. The TgTauR406W 21807 line showed tau accumulation beginning in the hippocampus and amygdala at 6 months of age, which subsequently spread to the cortices and subcortical areas. The accumulated tau was phosphorylated, ubiquitinated, conformationally changed, argyrophilic, and sarcosyl-insoluble. Activation of GSK-3beta and astrocytic induction of mouse tau were observed. Astrogliosis and microgliosis correlated with prominent tau accumulation. Electron microscopic examination revealed the presence of straight filaments. Behavioral tests showed motor disturbances and progressive acquired memory loss between 10 to 12 months of age. These findings suggested that TgTauR406W mice would be a useful model in the study of frontotemporal dementia and other tauopathies such as Alzheimer's disease (AD).     

Reduction of a vascular endothelial growth factor receptor, fetal liver kinase-1, by antisense oligonucleotides induces motor neuron death in rat spinal cord exposed to hypoxia

Vascular endothelial growth factor (VEGF) is reported to play a neuroprotective role through a VEGF receptor, fetal liver kinase-1 (Flk-1) in vitro. We investigated whether reduction of Flk-1 could induce motor neuron loss in rat spinal cord by inhibiting the expression of Flk-1 in rat spinal cord using antisense oligodeoxynucleotides (ODNs) against the Flk-1 receptor. Rat spinal cord was repetitively exposed to 12% hypoxia, and the change of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the mitogen-activated protein kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK) pathway was examined. Intrathecal infusion of Flk-1 antisense ODNs for 7 days suppressed almost completely Flk-1 expression in the lumbar segment of the spinal cord and was followed by a hypoxic challenge with 12% oxygen for 1 h that was repeated for 7 more days. In the lumbar segment, we observed that reduced Flk-1 expression and hypoxic challenge for 7 days resulted in approximately 50% loss of motor neurons, in which the activation of Akt and ERK, that is, increased levels of phosphorylated-Akt and of phosphorylated-ERK by hypoxia, was markedly inhibited. In contrast, the reduction of Flk-1 expression alone did not induce motor neuron loss. These results suggest that VEGF exerts its protective effect on motor neurons against hypoxia-induced toxicity by the Flk-1 receptor through the PI3-K/Akt and the MEK/ERK signaling pathways.     

Studies on fine structure and location of lipids in quick-freeze replicas of atherosclerotic aorta of WHHL rabbits

Summary The fine structure of intracellular and extracellular lipids in the atherosclerotic aorta of Watanabe-heritable hyperlipidemic (WHHL) rabbits was demonstrated by a quick-freeze etching technique. Many lipid droplets, with and without a membrane, were observed in the foam cells. Membrane-free droplets were observed as onionlike structure with a concentric lamellar structure surrounded by 10 nm filaments. Droplets surrounded by a limited membrane probably correspond to lipid-laden lysosomes.In the extracellular connective tissue space, marked accumulation of lipids with a vesicular structure was seen among collagen fibers. The appearance of these lipids was similar to that of lipids in lysosomes of foam cells.     

Quantitative Analysis of Mycobacterial and Propionibacterial DNA in Lymph Nodes of Japanese and European Patients with Sarcoidosis

The cause(s) of sarcoidosis is unknown. Mycobacterium spp. are suspected in Europe and Propionibacterium spp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes of Propionibacterium acnes, Propionibacterium granulosum, Mycobacterium tuberculosis, Mycobacterium avium subsp. paratuberculosis, and Escherichia coli (as the control) were counted by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was found in no sarcoid sample. M. tuberculosis was found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum were far more than those of M. tuberculosis. P. acnes or P. granulosum was found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in such samples were less than those in sarcoid samples. Propionibacterium spp. are more likely than Mycobacteria spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.