KL-6, a Human MUC1 Mucin,as a prognostic marker for diffuse alveolar hemorrhage syndrome

Background

Diffuse alveolar hemorrhage syndrome is a life threatening condition with diverse etiologies. Sensitive prognostic markers for diffuse alveolar hemorrhage have not been well investigated. Serum KL-6 is a biomarker for various interstitial lung disease associated with disease activity and prognosis. The purpose of the present study was to evaluate the clinical utility of serum KL-6 level as a prognostic marker for diffuse alveolar hemorrhage.

Methods

We retrospectively collected 41 consecutive patients clinically diagnosed as having diffuse alveolar hemorrhage who were admitted to the Intensive Care Unit of Hiroshima University Hospital between 2004 and 2011. Correlation between prognosis and age, sex, laboratory findings including serum KL-6, radiological findings, ventilatory modes or therapeutic regimens were evaluated.

Results

Baseline and peak serum KL-6 levels were significantly higher in non-survivors compared with survivors. An increase in KL-6 levels during the initial week was associated with a subsequent deterioration of the oxygenation index. Higher baseline KL-6 levels and higher peak KL-6 levels were strongly correlated with death. With a cut-off level of 700 U/mL for peak KL-6, the sensitivity, specificity and accuracy for non-survival were 75%, 85% and 78%, respectively. In the multivariate analysis, only the peak KL-6 level ≥700 U/ml was an independent poor prognostic factor for diffuse alveolar hemorrhage.

Conclusions

Peak serum KL-6 level ≥700 U/ml may become a clinically useful marker of poor prognosis for diffuse alveolar hemorrhage.

     

Prostaglandin production in cultured gastric mucosal cells: role of cAMP on its modulation

The effect of cAMP on prostaglandin production may depend on cell types. To clarify the relationship between PG and cAMP, we examined arachidonate's effects on PG synthesis and intracellular cAMP accumulation in monolayers of rat gastric mucosal cells. These cells produced PGE2, PGI2 and thromboxaneA2 (TXA2) in amounts of 316 +/- 18, 100 +/- 7 and 30 +/- 5 pg per 10(5) cells in 10 min, respectively, in response to 10 microM arachidonic acid (AA). The production of these PG, however, leveled off subsequently. Cells initially exposed to AA responded poorly to a subsequent stimulation by AA. AA simultaneously stimulated intracellular cAMP accumulation; this stimulatory effect on cAMP production was abolished by the pretreatment with indomethacin. Nevertheless, the pretreatments with dibutyryl cAMP (0.1-5 mM) did not alter the amount of subsequent AA-induced PGE2 production. Furthermore, the preincubation with 1mM isobutyl methyl xanthine also failed to affect PGE2 synthesis, while it increased intracellular cAMP accumulation. Our studies suggest AA stimulates intracellular cAMP formation in cultured gastric mucosal cells, linked with conversion of AA to cyclooxygenase metabolites, AA-induced PG production is limited in these cells, and it seems, however, unlikely that intracellular cAMP modulates AA metabolism to PG.     

Peripheral injection of lipopolysaccharide enhances expression of inflammatory cytokines in murine locus coeruleus: possible role of increased norepinephrine turnover

Cytokines and catecholamines are known to constitute a significant portion of the regulatory neuroimmune networks involved in maintaining homeostasis in the central nervous system (CNS). Although we have already reported an increase in norepinephrine (NE) turnover within the locus coeruleus (LC) at 2 and 4 h after the intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), the implication of this increase remains unclear. In view of evidence that norepinephrine (NE) acts in an anti-inflammatory manner by way of negatively regulating pro-inflammatory cytokine expression, we examined the inflammatory cytokine expression levels in the LC of C3H/HeN mice (male, 8 weeks old) after an i.p. LPS injection. The mRNA expression levels of the genes encoding IL-1beta and TNF-alpha within the LC increased during the first 2 h, and showed two peaks, the first at 4 h and the second lesser one at 15 h after the LPS injection. Microglia, which are one of the major cell types that produce pro-inflammatory cytokines in the CNS, were isolated from mouse neonate brains in order to clarify more precisely the relationship between the changes in NE content and the up-regulation of inflammatory cytokines in the LC. Simultaneous incubation of microglia with LPS and NE enhanced the expression of IL-1beta at both mRNA and protein levels, but reduced the mRNA and protein levels of TNF-alpha. These data support the hypothesis that NE negatively regulates the expression of pro-inflammatory cytokine expression, at least in the case of TNF-alpha, which action could contribute to the observed anti-inflammatory properties of NE. This report, based on the results of both in vivo and in vitro experiments, is the first to suggest a relationship between NE content and cytokine expression levels in the CNS.     

IL‐17 is Involved in Helicobacter pylori‐Induced Gastric Inflammatory Responses in a Mouse Model

Background: Helicobacter pylori (H. pylori) is the major cause of chronic active gastritis and peptic ulcer disease. Recent studies have shown that H. pylori produces various cytokines that are related to neutrophil or mononuclear cell accumulation. Interleukin‐17 (IL‐17) is the founding member of an emerging family of inflammatory cytokines whose biological activities remain incompletely defined. In this study, the contributions of IL‐17 to the induction of gastric inflammation and to the protection from H. pylori infection were investigated using IL‐17 gene‐knockout (IL‐17^?/–) mice. Materials and Methods: IL‐17^?/–and wild‐type C57BL/6 mice were challenged with H. pylori CPY2052 (2 × 10⁸ CFU/mL) and the histological and microbiological evaluation were carried out at specified times. IL‐17 and myeloperoxidase (MPO) protein levels in tissues were assayed in duplicate using ELISA kits. Results: In wild‐type mice, IL‐17 was undetected at baseline; however, the protein expression of IL‐17 was induced after infection with H. pylori. A severe infiltration of neutrophils appeared in the submucosa and the lamina propria in wild‐type mice. In contrast, the degree of neutrophil infiltration in IL‐17^?/– mice was significantly lower than that in wild‐type mice. Although wild‐type mice infected with H. pylori showed drastically higher MPO activity compared with uninfected wild‐type mice, any significant increase in the enzyme activity was not revealed in infected IL‐17^?/– mice. The number of H. pylori colonized in the stomach of IL‐17^?/– mice was significantly lower than that of wild‐type mice from 1 to 6 months after infection. Conclusions: These results suggest that IL‐17 may play an important role in the inflammatory response to the H. pylori infection and ultimately influence the outcome of the H. pylori‐associated disease.     

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells

Background

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined.

Methods

Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR.

Results

The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition.

Conclusions

Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.     

Novel modular domain PB1 recognizes PC motif to mediate functional protein-protein interactions

Modular domains mediating specific protein-protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67(phox), which activates the microbicidal phagocyte NADPH oxidase. Each of these specifically recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40(phox) (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67(phox), which abolishes its interaction with Cdc24p, confers on cells temperature- sensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif-containing region of p40(phox), which restores the interaction with the altered Bem1p. This domain-swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules.     

Fluctuation of Endogenous Cytokinin Contents in Rice during Its Life Cycle—Quantification of Cytokinins by Selected Ion Monitoring Using Deuterium-labelled Internal Standards

The change in the levels of endogenous cytokinins in the shoot and grain of rice (Oryza sativa L. japonica cultivar Nihonbare) during its growth and development was followed by selected ion monitoring using deuterium-labelled internal standards. Five cytokinins, zeatin (Z), cis-Z, ribosyl-zeatin (RZ), cis-RZ and 6-(3-methyl-but-2-enylamino)purine riboside were detected in the shoot, root and ear of rice. The maximum contents of RZ and Z in the shoot and root were found at the maximum tillering stage. In the ear, the levels of Z and RZ changed markedly. It is therefore suspected that RZ and Z play some roles in the tillering and seed growth in rice.     

Gene rearrangements in snake mitochondrial genomes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster

Mitochondrial DNA (mtDNA) regions corresponding to two major tRNA gene clusters were amplified and sequenced for the Japanese pit viper, himehabu. In one of these clusters, which in most vertebrates characterized to date contains three tightly connected genes for tRNA(Ile), and tRNA(Gln), and tRNA(Met), a sequence of approximately 1.3 kb was found to be inserted between the genes for tRNA(Ile) and tRNA(Gln). The insert consists of a control-region-like sequence possessing some conserved sequence blocks, and short flanking sequences which may be folded into tRNA(Pro), tRNA(Phe), and tRNA(Leu) genes. Several other snakes belonging to different families were also found to possess a control-region-like sequence and tRNA(Leu) gene between the tRNA(Ile)and tRNA(Gln) genes. We also sequenced a region surrounded by genes for cytochrome b and 12S rRNA, where the control region and genes for tRNA(Pro) and tRNA(Phe) are normally located in the mtDNAs of most vertebrates. In this region of three examined snakes, a control-region- like sequence exists that is almost completely identical to the one found between the tRNA(Ile) and tRNA(Gln) genes. The mtDNAs of these snakes thus possess two nearly identical control-region-like sequences which are otherwise divergent to a large extent between the species. These results suggest that the duplicate state of the control-region- like sequences has long persisted in snake mtDNAs, possibly since the original insertion of the control-region-like sequence and tRNA(Leu) gene into the tRNA gene cluster, which occurred in the early stage of the divergence of snakes. It is also suggested that the duplicated control-region-like sequences at two distant locations of mtDNA have evolved concertedly by a mechanism such as frequent gene conversion. The secondary structures of the determined tRNA genes point to the operation of simplification pressure on the T psi C arm of snake mitochondrial tRNAs.      

Immunohistochemical localization of sodium-potassium ATPase in human normal stomach and gastric adenocarcinomas

We studied the expression pattern of Na, K-ATPase beta 1 subunit in human normal stomachs and in gastric adenocarcinomas by using anti-Na, K-ATPase beta 1 subunit-specific monoclonal antibody. Tissue samples were processed in formalin solution or in a cold acetic acid-ethanol solution, routinely processed, embedded in paraffin, and an immunoperoxidase method for Na, K-ATPase beta 1 subunit was performed. After antigen retrieval using a steamer in citrate buffer (pH 6.0), tissue sections initially fixed in cold acetic acid-ethanol showed intense immunoreactivity with the antibody at the lateral or basolateral cytoplasmic membrane of normal gastric epithelial cells, at the cytoplasmic membrane of gastric carcinoma cells according to the level of differentiation, and at the cytoplasmic membrane and in the cytoplasm of Schwann cells and neurons in the mesenteric plexus of the gastric wall. Acetic acid-ethanol and paraffin embedding is a useful method for the investigation of the immunohistochemical localization of Na, K-ATPase in normal and diseased tissues.