The growth factor progranulin attenuates neuronal injury induced by cerebral ischemia-reperfusion through the suppression of neutrophil recruitment.

BACKGROUND: To improve the clinical outcome of patients who suffered ischemic stroke, cerebral ischemia-reperfusion (I/R) injury is one of the major concerns that should be conquered. Inflammatory reactions are considered a major contributor to brain injury following cerebral ischemia, and I/R exacerbates these reactions. The aim of this study was to investigate the possible ameliorative effects of progranulin (PGRN) against I/R injury in mice. METHODS: In vivo I/R was induced in four-week-old male ddY mice by 2 h of MCAO (middle cerebral artery occlusion) followed by 22 h of reperfusion. We evaluate expression of PGRN in I/R brain, efficacy of recombinant-PGRN (r-PGRN) treatment and its therapeutic time-window on I/R injury. Two hours after MCAO, 1.0 ng of r-PRGN or PBS was administered via intracerebroventricular. We assess neutrophil infiltration, expression of tumor necrosis factor (TNF)-α, matrix metalloproteinase-9 (MMP-9) and phosphorylation of nuclear factor-κB (NF-κB) by immunofluorescense staining and Western blotting. We also investigate neutrophil chemotaxis and intercellular adhesion molecule-1 (ICAM-1) expression in vitro inflammation models using isolated neutrophils and endothelial cells. RESULTS: We found that expression of PGRN was decreased in the I/R mouse brain. r-PGRN treatment at 2 h after MCAO resulted in a reduction in the infarct volume and decreased brain swelling; this led to an improvement in neurological scores and to a reduction of mortality rate at 24 h and 7 d after MCAO, respectively. Immunohistochemistry, Western blotting, and gelatin zymography also confirmed that r-PGRN treatment suppressed neutrophil recruitment into the I/R brain, and this led to a reduction of NF-κB and MMP-9 activation. In the in vitro inflammation models, PGRN suppressed both the neutrophil chemotaxis and ICAM-1 expression caused by TNF-α in endothelial cells. CONCLUSIONS: PGRN exerted ameliorative effects against I/R-induced inflammation, and these effects may be due to the inhibition of neutrophil recruitment into the I/R brain.

P2Y11 purinoceptor mediates the ATP-enhanced chemotactic response of rat neutrophils.

ATP and hydrolysis products of ATP like adenosine regulate the chemotaxis of neutrophils by activating purinoceptors and adenosine receptors. The present study was designed to examine exogenous ATP, activation of purinoceptors, and activation of A(3) adenosine receptor as key steps in the signal cascades that control cell orientation and migration of rat neutrophils. One or more of those steps might be potential therapeutic targets for treatment of inflammatory diseases. The chemotaxis of rat neutrophils was stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and measured with an EZ-TAXIScan apparatus. The effects of apyrase, exogenous ATP, suramin (P2X and P2Y blocker), PPADS (a P2X blocker), TNP-ATP (P2X(1) and P2X(3) antagonist), and Reactive Blue 2 (a P2Y blocker) on the chemotactic response were also investigated. Rat neutrophil chemotaxis was significantly suppressed by apyrase. fMLP induced rat neutrophil chemotaxis was potentiated by ATP, blocked by suramin, not affected by PPADS or TNP-ATP, and significantly inhibited by RB-2. Western blotting showed that A(3), P2Y(2), and P2Y(11) were expressed in rat neutrophils. The chemotactic response of rat neutrophils to fMLP stimulation is potentiated by ATP via P2Y(11) purinoceptors but not P2X purinoceptors or A(3) adenosine receptor, and that the response plays a critical role in host defense and pathogenicity.

Effects of changes in vertical occlusal dimension on heart rate fluctuations in guinea pigs.

BACKGROUND: We have previously reported that the decrease of the vertical occlusal dimension (VOD) led to heart failure and abnormalities in creatine phosphokinase (CPK) in guinea pigs. In the present study, we investigated the autonomic activity and the origin of the abnormality in CPK under different occlusal conditions. MATERIALS AND METHODS: Guinea pigs were separated into the following five groups: untreated control, reduced VOD, slit, restored VOD and increased VOD groups and compared for their electrocardiogram and heart rate fluctuations for two weeks using Fluclet, computer software. RESULTS: The control group revealed no changes in heart rate fluctuations or posture. The reduced VOD group exhibited a two-phase wave of heart rate fluctuations, with the first peak 0-2 days after teeth grinding, and the second peak starting from 4 days after teeth grinding until sudden death (usually 12th day), accompanied by head drop. The slit and the restored VOD groups exhibited only the first peak. The increased VOD group, with approximately 3 mm-thick acrylic pellets bonded to the posterior teeth, showed no heart rate fluctuations. Body weight loss was most prominent in the reduced VOD group, and became much milder in the order of increased VOD, restored and slit groups. The reduced VOD group exhibited transient elevation of skeletal muscle type of CPK isozyme activity within two days after treatment. CONCLUSION: The present study suggests that the first peak of heart rate fluctuations is caused by pulpal stimulation, and the second peak by excessive contraction (excessive excitation of the motor output system and the autonomic nervous system) of the masticatory muscles. On the other hand, increased VOD did not influence either the motor or the autonomic nervous system.

Mesenchymal miR-21 regulates branching morphogenesis in murine submandibular gland in vitro.

Branching morphogenesis in murine submandibular glands (SMG) is regulated by growth factors, extracellular matrix (ECM) and many biological processes through interactions between the epithelium and the mesenchyme. MicroRNAs (miRNAs) are a set of small, non-protein-coding RNAs that regulate gene expression at the post-transcriptional level. We hypothesized that branching morphogenesis is partly regulated by miRNAs. Forty-four miRNAs and novel miRNA candidates were detected in SMG at embryonic day 13 by a cloning method combined with Argonaute-2 immunoprecipitation. MicroRNA21 (miR-21) expression in the mesenchyme was up-regulated and accelerated by epidermal growth factor, which is known to enhance branching morphogenesis in vitro. Down-regulation of miR-21 in the mesenchyme by locked nucleic acids was associated with a decrease in the number of epithelial buds. Relative quantification of candidates for target genes of miR-21 indicated that two messenger RNAs (for Reck and Pdcd4) were down-regulated in the mesenchyme, where miR-21 expression levels were up-regulated. These results suggest that branching morphogenesis is regulated by miR-21 through gene expression related to ECM degradation in the mesenchyme.

Phospholipase D-dependent and -independent p38MAPK activation pathways are required for superoxide production and chemotactic induction, respectively, in rat neutrophils stimulated by fMLP.

Mitogen-activated protein kinase (MAPK)-mediated signal transduction pathways convert signals by extracellular stimulation into a variety of cellular functions. However, the roles of MAPKs in neutrophils are not well understood. To elucidate the temporal roles of p38MAPK during rat neutrophil activation stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), we examined the kinetics of this enzyme and the role of p38MAPK related to neutrophil functions (superoxide production and chemotaxis). SB203580, a potent and specific inhibitor of p38MAPK, significantly depressed both superoxide production and chemotaxis. Ethanol and 1-butanol, inhibitors of phospholipase D (PLD), suppressed p38MAPK activation in neutrophils under conditions (1 microM fMLP for 5 min) that stimulated superoxide production; and they significantly depressed superoxide production in rat neutrophils stimulated by fMLP. However, neither inhibitor had any effect on the activation of p38MAPK under the conditions (10 nM fMLP for 60 min) that gave optimal chemotaxis. These results indicate that multiple signaling pathways were involved in stimulating p38MAPK and that p38MAPK played different roles in regulating neutrophil function depending on the conditions for stimulation with fMLP. In addition, the activation of p38MAPK occurred dependent on or independent of PLD activation in neutrophils stimulated with fMLP.

[Evaluation of bronchial arterial infusion (BAI) for lung cancer with brain metastasis].

MATERIALS AND METHODS: Seven high risk patients of lung cancer with brain metastasis to which BAI was done were examined. The standard systemic chemotherapy was not indicated in these cases due to the patients' systemic condition. BAI was performed using CDDP (40-80 mg/m2)+CPT-11 (40-60 mg/m2). The therapeutic effects, side effects, quality of life (QOL) and prognosis were examined. RESULTS: All cases revealed a stable disease according to the rule of the RECIST. Side effects over grade 2 were not recognized. QOL was improved because clinical effects were recognized in all seven cases with respiratory symptoms (cough in 6, dyspnea at movement in 5 and hemosputum in 1). Six patients died three-thirty months after the treatment of brain metastasis, and the median survival was twelve months. The causes of death were recurrence of brain tumor in 3, increase of primary lesion in 1, recurrence of brain tumor and increase of primary lesion in 2. The prognosis of the patient who had progressing T and N factors was bad. COMMENT: BAI was effective in keeping the quality of life for a lung cancer patient with brain metastasis. It was thought that BAI should be performed positively in the high risk patient who did not have progressing T and N factors.