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Disruptions of occludin and claudin-5 in brain endothelial cells in vitro and in brains of mice with acute liver failure
Chen, Florence
Ohashi, Norifumi
Li, Wensheng
Eckman, Christopher
Nguyen, Justin H.
Hepatology (Baltimore, Md.)
Brain edema in acute liver failure (ALF) remains lethal. The role of vasogenic mechanisms of brain edema has not been explored. We previously demonstrated that matrix metalloproteinase-9 (MMP-9) contributes to the pathogenesis of brain edema. Here, we show that MMP-9 mediates disruptions in tight junction proteins in vitro and in brains of mice with ALF. We transfected murine brain endothelial cells with MMP-9 cDNA using pc DNA3.1 (+)/Myc-His A expression vector. Tissue inhibitor of matrix metalloproteinases (TIMP-1) cDNA transfection or GM6001 was used to inhibit MMP-9. ALF was induced in mice with azoxymethane. Endogenous overexpression of MMP-9 in brain endothelial cells resulted in significant degradation of tight junction proteins occludin and claudin-5. The alterations in tight junction proteins correlated with increased permeability to FITC-dextran molecules. The degradation of tight junction proteins and the increased permeability were reversed by TIMP-1 and GM6001. Similar results were found when MMP-9 was exogenously added to brain EC. We also found that tight junction proteins degradation was reversed with GM6001 in brains of mice with ALF.
Tight junction proteins are significantly perturbed in brains of mice with ALF. These data corroborate the important role of MMP-9 in the vasogenic mechanism of brain edema in ALF.
acute liver failure; blood brain barrier permeability
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Neisseria meningitidis Induces Brain Microvascular Endothelial Cell Detachment from the Matrix and Cleavage of Occludin: A Role for MMP-8
Schubert-Unkmeir, Alexandra
Konrad, Christian
Slanina, Heiko
Czapek, Florian
Hebling, Sabrina
Frosch, Matthias
PLoS Pathogens
Disruption of the blood-brain barrier (BBB) is a hallmark event in the pathophysiology of bacterial meningitis. Several inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), nitric oxide and matrix metalloproteinases (MMPs), contribute to this disruption. Here we show that infection of human brain microvascular endothelial cells (HBMEC) with Neisseria meningitidis induced an increase of permeability at prolonged time of infection. This was paralleled by an increase in MMP-8 activity in supernatants collected from infected cells. A detailed analysis revealed that MMP-8 was involved in the proteolytic cleavage of the tight junction protein occludin, resulting in its disappearance from the cell periphery and cleavage to a lower-sized 50-kDa protein in infected HBMEC. Abrogation of MMP-8 activity by specific inhibitors as well as transfection with MMP-8 siRNA abolished production of the cleavage fragment and occludin remained attached to the cell periphery. In addition, MMP-8 affected cell adherence to the underlying matrix. A similar temporal relationship was observed for MMP activity and cell detachment. Injury of the HBMEC monolayer suggested the requirement of direct cell contact because no detachment was observed when bacteria were placed above a transwell membrane or when bacterial supernatant was directly added to cells. Inhibition of MMP-8 partially prevented detachment of infected HBMEC and restored BBB permeability. Together, we established that MMP-8 activity plays a crucial role in disassembly of cell junction components and cell adhesion during meningococcal infection.
Author Summary
A crucial step in the pathogenesis of bacterial meningitis is the disturbance of cerebral microvascular endothelial function, resulting in blood-brain barrier (BBB) breakdown. Matrix metalloproteinases (MMPs) have been implicated in BBB damage in bacterial meningitis in several studies. MMPs are a family of zinc-dependent endopeptidases that catalyze the proteolysis of extracellular matrix proteins, but can also cleave a range of other molecules, including cell adhesion molecules. In this study we showed that brain endothelial cells produced MMPs—in particular MMP-8—upon infection with Neisseria meningitidis, a bacterium that causes meningitis and septic shock. We found that MMP-8 was then involved in disruption of the tight junction protein occludin. In addition to the effect of MMP-8 on the tight junction component, MMP-8 activity also accounted for brain endothelial cell detachment that occurred during prolonged time of infection with N. meningitidis. When we inhibited MMP-8 activity, occludin disruption was completely abolished and cell detachment could be partially prevented, which resulted in restored BBB permeability. Our data reveal a molecular mechanism of cellular dysfunction during meningococcal meningitis that enhances our understanding how MMPs affect cerebral endothelial function and that can aid in our understanding and prevention of this disease.
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Bexarotene Reduces Blood-Brain Barrier Permeability in Cerebral Ischemia-Reperfusion Injured Rats
Xu, Lu
Cao, Fang
Xu, Feng
He, Baicheng
Dong, Zhi
PLoS ONE
Matrix metalloproteinase-9 (MMP-9) over-expression disrupts the blood-brain barrier (BBB) in the ischemic brain. The retinoid X receptor agonist bexarotene suppresses MMP-9 expression in endothelial cells and displays neuroprotective effects. Therefore, we hypothesized that bexarotene may have a beneficial effect on I/R-induced BBB dysfunction.
A total of 180 rats were randomized into three groups (n = 60 each): (i) a sham-operation group, (ii) a cerebral ischemia-reperfusion (I/R) group, and (iii) an I/R+bexarotene group. Brain water content was measured by the dry wet weight method. BBB permeability was analyzed by Evans Blue staining and the magnetic resonance imaging contrast agent Omniscan. MMP-9 mRNA expression, protein expression, and activity were assessed by reverse transcription polymerase chain reaction, Western blotting, and gelatin zymography, respectively. Apolipoprotein E (apoE), claudin-5, and occludin expression were analyzed by Western blotting.
After 24 h, 48 h, and 72 h post-I/R, several effects were observed with bexarotene administration: (i) brain water content and BBB permeability were significantly reduced; (ii) MMP-9 mRNA and protein expression as well as activity were significantly decreased; (iii) claudin-5 and occludin expression were significantly increased; and (iv) apoE expression was significantly increased.
Bexarotene decreases BBB permeability in rats with cerebral I/R injury. This effect may be due in part to bexarotene's upregulation of apoE expression, which has been previously shown to reduce BBB permeability through suppressing MMP-9-mediated degradation of the tight junction proteins claudin-5 and occludin. This work offers insight to aid future development of therapeutic agents for cerebral I/R injury in human patients.
Related Articles
Estrogen Decrease in Tight Junctional Resistance Involves Matrix-Metalloproteinase-7-Mediated Remodeling of Occludin
Gorodeski, George I.
Estrogen modulates tight junctional resistance through estrogen receptor-α-mediated remodeling of occludin. The objective of the study was to understand the mechanisms involved. Experiments using human normal vaginal-cervical epithelial cells showed that human normal vaginal-cervical epithelial cells secrete constitutively matrix-metalloproteinase-7 (MMP-7) into the luminal solution and that MMP-7 is necessary and sufficient to produce estrogen decrease of tight junctional resistance and remodeling of occludin. Treatment with estrogen stimulated activation of the pro-MMP-7 intracellularly and augmented secretion of the activated MMP-7 form. Steady-state levels of MMP-7 mRNA and protein were not affected by estrogen. Estrogen modulated phosphorylation of the MMP-7, but the changes were most likely secondary to changes in cellular MMP-7 mass. Estrogen increased coimmunoreactivity of MMP-7 with the Golgi protein GPP130. Tunicamycin and brefeldin-A had no effect on cellular MMP-7 but monensin (inhibitor of Golgi traffic) blocked estrogen effects, suggesting estrogen site of action is at the Golgi system. Estrogen increased generalized secretory activity, including of luminal exocytosis of polycarbohydrates. However, estrogen increased coimmunoreactivity of MMP-7 with synaptosomal-associated protein of 25 kDa in apical membranes, suggesting soluble N-ethylmaleimide sensitive fusion factor attachment protein receptor-facilitated exocytosis of MMP-7. Treatment with the vesicular-ATPase inhibitor bafilomycin A1 inhibited activation of MMP-7. These data suggest that estrogen up-regulates activation of the MMP-7 intracellularly, at the level of Golgi, and augments secretion of activated MMP-7 through soluble N-ethylmaleimide sensitive fusion factor attachment protein receptor-dependent exocytosis. On the other hand, estrogen acidification of the luminal solution would tend to alkalinize exocytotic vesicles and may lead to decreased activation of the MMP-7. These mechanisms acting in concert could be important for regulation and control of estrogen modulation of paracellular permeability in vivo.
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Matrix Metalloproteinase-2 and -9 Secreted by Leukemic Cells Increase the Permeability of Blood-Brain Barrier by Disrupting Tight Junction Proteins
Feng, Saran
Cen, Jiannong
Huang, Yihong
Shen, Hongjie
Yao, Li
Wang, Yuanyuan
Chen, Zixing
PLoS ONE
Central nervous system (CNS) involvement remains an important cause of morbidity and mortality in acute leukemia, the mechanisms of leukemic cell infiltration into the CNS have not yet been elucidated. The blood-brain barrier (BBB) makes CNS become a refugee to leukemic cells and serves as a resource of cells that seed extraneural sites. How can the leukemic cells disrupt this barrier and invasive the CNS, even if many of the currently available chemotherapies can not cross the BBB? Tight junction in endothelial cells occupies a central role in the function of the BBB. Except the well known role of degrading extracellular matrix in metastasis of cancer cells, here we show matrix metalloproteinase (MMP)-2 and -9, secreted by leukemic cells, mediate the BBB opening by disrupting tight junction proteins in the CNS leukemia. We demonstrated that leukemic cells impaired tight junction proteins ZO-1, claudin-5 and occludin resulting in increased permeability of the BBB. However, these alterations reduced when MMP-2 and -9 activities were inhibited by RNA interference strategy or by MMP inhibitor GM6001 in an in vitro BBB model. We also found that the disruption of the BBB in company with the down-regulation of ZO-1, claudin-5 and occludin and the up-regulation of MMP-2 and -9 in mouse brain tissues with leukemic cell infiltration by confocal imaging and the assay of in situ gelatin zymography. Besides, GM6001 protected all mice against CNS leukemia. Our findings suggest that the degradation of tight junction proteins ZO-1, claudin-5 and occludin by MMP-2 and -9 secreted by leukemic cells constitutes an important mechanism in the BBB breakdown which contributes to the invasion of leukemic cells to the CNS in acute leukemia.
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Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover
Phillips, P A
McCarroll, J A
Park, S
Wu, M-J
Pirola, R
Korsten, M
Wilson, J S
Apte, M V
Background: Pancreatic fibrosis is a characteristic feature of chronic pancreatic injury and is thought to result from a change in the balance between synthesis and degradation of extracellular matrix (ECM) proteins. Recent studies suggest that activated pancreatic stellate cells (PSCs) play a central role in pancreatic fibrogenesis via increased synthesis of ECM proteins. However, the role of these cells in ECM protein degradation has not been fully elucidated.
Aims: To determine: (i) whether PSCs secrete matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) and, if so (ii) whether MMP and TIMP secretion by PSCs is altered in response to known PSC activating factors such as tumour necrosis factor α (TNF-α), transforming growth factor β1 (TGF-β1), interleukin 6 (IL-6), ethanol, and acetaldehyde.
Methods: Cultured rat PSCs (n=3–5 separate cell preparations) were incubated at 37°C for 24 hours with serum free culture medium containing TNF-α (5–25 U/ml), TGF-β1 (0.5–1 ng/ml), IL-6 (0.001–10 ng/ml), ethanol (10–50 mM), or acetaldehyde (150–200 μM), or no additions (controls). Medium from control cells was examined for the presence of MMPs by zymography using a 10% polyacrylamide-0.1% gelatin gel. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine gene expression of MMP9 and the tissue inhibitors of metalloproteinases TIMP1 and TIMP2. Western blotting was used to identify a specific MMP, MMP2 (a gelatinase that digests basement membrane collagen and the dominant MMP observed on zymography) and a specific TIMP, TIMP2. Reverse zymography was used to examine functional TIMPs in PSC secretions. The effect of TNF-α, TGF-β1, and IL-6 on MMP2 secretion was assessed by densitometry of western blots. The effect of ethanol and acetaldehyde on MMP2 and TIMP2 secretion was also assessed by this method.
Results: Zymography revealed that PSCs secrete a number of MMPs including proteinases with molecular weights consistent with MMP2, MMP9, and MMP13. RT-PCR demonstrated the presence of mRNA for metalloproteinase inhibitors TIMP1 and TIMP2 in PSCs while reverse zymography revealed the presence of functional TIMP2 in PSC secretions. MMP2 secretion by PSCs was significantly increased by TGF-β1 and IL-6, but was not affected by TNF-α. Ethanol and acetaldehyde induced secretion of both MMP2 and TIMP2 by PSCs.
Conclusions: Pancreatic stellate cells have the capacity to synthesise a number of matrix metalloproteinases, including MMP2, MMP9, and MMP13 and their inhibitors TIMP1 and TIMP2. MMP2 secretion by PSCs is significantly increased on exposure to the proinflammatory cytokines TGF-β1 and IL-6. Both ethanol and its metabolite acetaldehyde increase MMP2 as well as TIMP2 secretion by PSCs.
Implication: The role of pancreatic stellate cells in extracellular matrix formation and fibrogenesis may be related to their capacity to regulate the degradation as well as the synthesis of extracellular matrix proteins.
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Matrix Metalloproteinase-9 Leads to Claudin-5 Degradation via the NF-κB Pathway in BALB/c Mice with Eosinophilic Meningoencephalitis Caused by Angiostrongylus cantonensis
Chiu, Ping-Sung
Lai, Shih-Chan
PLoS ONE
The epithelial barrier regulates the movement of ions, macromolecules, immune cells and pathogens. The objective of this study was to investigate the role of the matrix metalloproteinase (MMP)-9 in the degradation of tight junction protein during infection with rat nematode lungworm Angiostrongylus cantonensis. The results showed that phosphorylation of IκB and NF-κB was increased in mice with eosinophilic meningoencephalitis. Treatment with MG132 reduced the phosphorylation of NF-κB and the activity of MMP-9, indicating upregulation of MMP-9 through the NF-κB signaling pathway. Claudin-5 was reduced in the brain but elevated in the cerebrospinal fluid (CSF), implying that A. cantonensis infection caused tight junction breakdown and led to claudin-5 release into the CSF. Degradation of claudin-5 coincided with alteration of the blood-CSF barrier permeability and treatment with the MMP inhibitor GM6001 attenuated the degradation of claudin-5. These results suggested that degradation of claudin-5 was caused by MMP-9 in angiostrongyliasis meningoencephalitis. Claudin-5 could be used for the pathophysiologic evaluation of the blood-CSF barrier breakdown and tight junction disruption after infection with A. cantonensis.
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Matrix metalloproteinase-2-mediated occludin degradation and caveolin-1-mediated claudin-5 redistribution contribute to blood brain barrier damage in early ischemic stroke stage
Liu, Jie
Jin, Xinchun
Liu, Ke J.
Liu, Wenlan
The Journal of Neuroscience
Blood brain barrier (BBB) disruption occurs early enough to be within the thrombolytic time window, and this early ischemic BBB damage is closely associated with hemorrhagic transformation and thus emerging as a promising target for reducing the hemorrhagic complications of thrombolytic stroke therapy. However, the mechanisms underlying early ischemic BBB damage remain poorly understood. Here we investigated the early molecular events of ischemic BBB damage using in vitro oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery occlusion (MCAO) models. Exposure of bEND3 monolayer to OGD for 2 h significantly increased its permeability to FITC-labeled dextran, and promoted the secretion of metalloproteinase-2 and 9 (MMP-2/9) and cytosolic translocation of caveolin-1 (Cav-1). This same OGD treatment also led to rapid degradation of tight junction protein occludin and dissociation of claudin-5 from the cytoskeleton, which contributed to OGD-induced endothelial barrier disruption. Using selective MMP-2/9 inhibitor SB-3CT or their neutralizing antibodies or Cav-1 siRNA, we found that MMP-2 was the major enzyme mediating OGD-induced occludin degradation, while Cav-1 was responsible for claudin-5 redistribution. The interaction between Cav-1 and claudin-5 was further confirmed by coimmunoprecipitation. Consistent with these in vitro findings, we observed fluorescence tracer extravasation, increased gelatinolytic activity and elevated interstitial MMP-2 levels in ischemic subcortical tissue after 2-h MCAO. Moreover, occludin protein loss and claudin-5 redistribution were detected in ischemic cerebromicrovessels. These data indicate that cerebral ischemia initiates two rapid parallel processes, MMP-2-mediated occludin degradation and Cav-1-mediated claudin-5 redistribution, to cause BBB disruption at early stroke stages relevant to acute thrombolysis.
Cerebral ischemia; oxygen-glucose deprivation; blood brain barrier; matrix metalloproteinase; caveolin-1; tight junction proteins
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Exposure to vehicle emissions results in altered blood brain barrier permeability and expression of matrix metalloproteinases and tight junction proteins in mice
Oppenheim, Hannah A
Lucero, JoAnn
Guyot, Anne-Cécile
Herbert, Lindsay M
McDonald, Jacob D
Mabondzo, Aloïse
Lund, Amie K
Particle and Fibre Toxicology
Traffic-generated air pollution-exposure is associated with adverse effects in the central nervous system (CNS) in both human exposures and animal models, including neuroinflammation and neurodegeneration. While alterations in the blood brain barrier (BBB) have been implicated as a potential mechanism of air pollution-induced CNS pathologies, pathways involved have not been elucidated.
To determine whether inhalation exposure to mixed vehicle exhaust (MVE) mediates alterations in BBB permeability, activation of matrix metalloproteinases (MMP) -2 and −9, and altered tight junction (TJ) protein expression.
Apolipoprotein (Apo) E−/− and C57Bl6 mice were exposed to either MVE (100 μg/m3 PM) or filtered air (FA) for 6 hr/day for 30 days and resulting BBB permeability, expression of ROS, TJ proteins, markers of neuroinflammation, and MMP activity were assessed. Serum from study mice was applied to an in vitro BBB co-culture model and resulting alterations in transport and permeability were quantified.
MVE-exposed Apo E−/− mice showed increased BBB permeability, elevated ROS and increased MMP-2 and −9 activity, compared to FA controls. Additionally, cerebral vessels from MVE-exposed mice expressed decreased levels of TJ proteins, occludin and claudin-5, and increased levels of inducible nitric oxide synthase (iNOS) and interleukin (IL)-1β in the parenchyma. Serum from MVE-exposed animals also resulted in increased in vitro BBB permeability and altered P-glycoprotein transport activity.
These data indicate that inhalation exposure to traffic-generated air pollutants promotes increased MMP activity and degradation of TJ proteins in the cerebral vasculature, resulting in altered BBB permeability and expression of neuroinflammatory markers.
Air pollution; Blood brain barrier; Tight junction proteins; Matrix metalloproteinase; Neuroinflammation
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Neutrophils Compromise Retinal Pigment Epithelial Barrier Integrity
Zhou, Jiehao
He, Shikun
Zhang, Ning
Spee, Christine
Zhou, Peng
Ryan, Stephen J.
Kannan, Ram
Hinton, David R.
Journal of Biomedicine and Biotechnology
We hypothesized that neutrophils and their secreted factors mediate breakdown of the integrity of the outer blood-retina-barrier by degrading the apical tight junctions of the retinal pigment epithelium (RPE). The effect of activated neutrophils or neutrophil cell lysate on apparent permeability of bovine RPE-Choroid explants was evaluated by measuring [3H] mannitol flux in a modified Ussing chamber. The expression of matrix metalloproteinase- (MMP-) 9 in murine peritoneal neutrophils, and the effects of neutrophils on RPE tight-junction protein expression were assessed by confocal microscopy and western blot. Our results revealed that basolateral incubation of explants with neutrophils decreased occludin and ZO-1 expression at 1 and 3 hours and increased the permeability of bovine RPE-Choroid explants by >3-fold (P < .05). Similarly, basolateral incubation of explants with neutrophil lysate decreased ZO-1 expression at 1 and 3 hours (P < .05) and increased permeability of explants by 75%. Further, we found that neutrophils prominently express MMP-9 and that incubation of explants with neutrophils in the presence of anti-MMP-9 antibody inhibited the increase in permeability. These data suggest that neutrophil-derived MMP-9 may play an important role in disrupting the integrity of the outer blood-retina barrier.
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Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis
Goldbach-Mansky, Raphaela
Lee, Jennifer M
Hoxworth, Joseph M
Smith II, David
Duray, Paul
Schumacher, Ralph H
Yarboro, Cheryl H
Klippel, John
Kleiner, David
El-Gabalawy, Hani S
Arthritis Research
Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion.
In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.
To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.
Patients and method:
A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.
Of the 66 patients studied, 45 fulfilled American College of Rheumatology criteria for rheumatoid arthritis (RA), with 32 (71%) being rheumatoid factor positive. Of the 21 non-RA patients, seven had a spondylarthropathy and 14 had undifferentiated arthritis. Radiographically, 12 of the RA patients had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed erosive disease of this extent. In the tissue, latent MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining layer and stroma, whereas MMP-9 was expressed more sparsely and focally. MMP-14, TIMP-2, and MMP-2 were all detected in similar areas of the lining layer on consecutive histologic sections. Tissue expression of MMP-14, the activator for pro-MMP-2, was significantly higher in RA than in non-RA patients (8.4 ± 5 versus 3.7 ± 4 cells/high-power field; P = 0.009). In contrast, the expression of TIMP-2, an inhibitor of MMP-2, was lower in the RA than in the non-RA samples (25 ± 12 versus 39 ± 9 cells/high-power field; P = 0.01). Synovial tissue expressions of MMP-2, MMP-14, and TIMP-2 were virtually undetectable in normal synovial tissue samples. The synovial tissue samples of patients with erosive disease had significantly higher levels of active MMP-2 than did those of patients without erosions (Fig. 1). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.
With the exception of serum MMP-2, which was not elevated over normal, serum levels of all of the other MMPs and TIMPs were elevated to varying degrees, and were not predictive of erosive disease. Interestingly, MMP-1 and C-reactive protein, both of which were associated with the presence of erosions, were positively correlated with each other (r = 0.42; P < 0.001).
MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.
In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.
early synovitis; erosion; metalloproteinase; matrix metalloproteinase-2; rheumatoid arthritis
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Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation
Wiechmann, Allan F.
Ceresa, Brian P.
Howard, Eric W.
PLoS ONE
Background and Objectives
The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs) are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium.
Methodology/Principal Findings
Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2), membrane type 1-MMP (MT1-MMP) and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime.
MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface cell desquamation and renewal may be orchestrated by nocturnal circadian signals.
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Matrix Metalloproteinase-9 Expression Is Enhanced in Renal Parietal Epithelial Cells of Zucker Diabetic Fatty Rats and Is Induced by Albumin in In Vitro Primary Parietal Cell Culture
Zhang, Yuanyuan
George, Jasmine
Li, Yun
Olufade, Rebecca
Zhao, Xueying
PLoS ONE
As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy. Albumin overload may induce MMP-9 expression and secretion by PECs via the activation of p44/42 MAPK pathway.
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Dexamethasone Ameliorates H2S-Induced Acute Lung Injury by Alleviating Matrix Metalloproteinase-2 and -9 Expression
Wang, Jun
Zhang, Huazhong
Su, Chenglei
Chen, Junjie
Zhu, Baoli
Zhang, Hengdong
Xiao, Hang
Zhang, Jinsong
PLoS ONE
Acute lung injury (ALI) is one of the fatal outcomes after exposure to high levels of hydrogen sulfide (H2S), and the matrix metalloproteinases (MMPs) especially MMP-2 and MMP-9 are believed to be involved in the development of ALI by degrading the extracellular matrix (ECM) of blood-air barrier. However, the roles of MMP-2 and MMP-9 in H2S-induced ALI and the mechanisms of dexamethasone (DXM) in treating ALI in clinical practice are still largely unknown. The present work was aimed to investigate the roles of MMP-2 and MMP-9 in H2S-induced ALI and the protective effects of DXM. In our study, SD rats were exposed to H2S to establish the ALI model and in parallel, A549 cells were incubated with NaHS (a H2S donor) to establish cell model. The lung HE staining, immunohistochemisty, electron microscope assay and wet/dry ratio were used to identify the ALI induced by H2S, then the MMP-2 and MMP-9 expression in both rats and A549 cells were detected. Our results revealed that MMP-2 and MMP-9 were obviously increased in both mRNA and protein level after H2S exposure, and they could be inhibited by MMP inhibitor doxycycline (DOX) in rat model. Moreover, DXM significantly ameliorated the symptoms of H2S-induced ALI including alveolar edema, infiltration of inflammatory cells and the protein leakage in BAFL via up-regulating glucocorticoid receptor(GR) to mediate the suppression of MMP-2 and MMP-9. Furthermore, the protective effects of DXM in vivo and vitro study could be partially blocked by co-treated with GR antagonist mifepristone (MIF). Our results, taken together, demonstrated that MMP-2 and MMP-9 were involved in the development of H2S-induced ALI and DXM exerted protective effects by alleviating the expression of MMP-2 and MMP-9. Therefore, MMP-2 and MMP-9 might represent novel pharmacological targets for the treatment of H2S and other hazard gases induced ALI.
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The Mechanisms of Cerebral Vascular Dysfunction and Neuroinflammation by MMP-mediated Degradation of VEGFR-2 in Alcohol Ingestion
Abdul Muneer, P. M.
Alikunju, Saleena
Szlachetka, Adam M.
Haorah, James
Arteriosclerosis, thrombosis, and vascular biology
Blood-brain barrier (BBB) dysfunction caused by activation of matrix metalloproteinases (MMPs) is a pathological feature in vascular/neurological disease. Here, we describe the mechanisms of BBB dysfunction and neuroinflammation as a result of MMP-3/9 activation and disruption of VEGF-A/VEGFR-2 interaction, impairing effective angiogenesis.
Methods and Results
We investigate the hypothesis in human brain endothelial cells and animal model of chronic alcohol ingestion. Proteome array analysis, zymography, immunofluorescence and Western blotting techniques detected the activation, expression and levels of MMP-3 and MMP-9. We found that degradation of VEGFR-2 and BBB proteins viz, occludin, claudin-5, and ZO-1 by MMP-3/9 causes rupture of capillary endothelium and BBB leakiness. Impairment of BBB integrity was demonstrated by increased permeability of dye tracers and Fluo-3/calcein-AM labeled monocytes adhesion or infiltration, and decrease in trans-endothelial electrical resistance. Alcohol-induced degradation of endothelial VEGFR-2 by MMP-3/9 led to a subsequent elevation of cellular/serum VEGF-A level. The decrease in VEGFR-2 with subsequent increase in VEGF-A level led to apoptosis and neuroinflammation via the activation of caspase-1 and IL-1β release. The use of MMPs, VEGFR-2 and caspase-1 inhibitors helped to dissect the underlying mechanisms.
Alcohol-induced MMPs activation is a key mechanism for dysfunction of BBB via degradation of VEGFR-2 protein and activation of caspase-1 or IL-1β release. Targeting VEGF-induced MMP-3/9 activation can be a novel preventive approach to vascular inflammatory disease in alcohol abuse.
Alcohol; blood-brain barrier; vascular inflammation; VEGF/VEGFR-2; matrix metalloproteinase
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Activation of Protein Tyrosine Kinases and Matrix Metalloproteinases Causes Blood-Brain Barrier Injury: Novel Mechanism for Neurodegeneration Associated with Alcohol Abuse
Haorah, James
Schall, Kathy
Ramirez, Servio H.
Persidsky, Yuri
Blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC) regulates the passage of molecules and leukocytes in and out of the brain. Activation of matrix metalloproteinases (MMPs) and alteration of basement membrane (BM) associated with BBB injury were documented in stroke patients. While chronic alcoholism is a risk factor for developing stroke, underlying mechanisms are not well understood. We hypothesized that ethanol (EtOH)-induced protein tyrosine kinase (PTK) signaling resulted a loss of BBB integrity via MMPs activation and degradation of BM component, collagen IV. Treatment of BMVEC with EtOH or acetaldehyde (AA) for 2–48 hr increased MMP-1, -2 and -9 activities or decreased the levels of tissue inhibitors of MMPs (TIMP-1, -2) in a PTK-dependent manner without affecting protein tyrosine phosphatase activity. Enhanced PTK activity after EtOH exposure correlated with increased phosphorylated proteins of selective receptor and non-receptor PTKs. Up-regulation of MMPs activities and protein contents paralleled a decrease in collagen IV content, and inhibitors of EtOH metabolism, MMP-2 and 9, or PTK reversed all these effects. Using human BMVEC assembled into BBB models, we found that EtOH/AA diminished barrier tightness, augmented permeability, and monocyte migration across the BBB via activation of PTKs and MMPs. These findings suggest that alcohol associated BBB injury could be mediated by MMPs via BM protein degradation and could serve as a co-morbidity factor for neurological disorders like stroke or neuroinflammation. Furthermore, our preliminary experiments indicated that human astrocytes secreted high levels of MMP-1 and 9 following exposure to EtOH, suggesting the role of BM protein degradation and BBB compromise as a result of glial activation by ethanol. These results provide better understanding of multifaceted effects of alcohol on the brain and could help develop new therapeutic interventions.
Blood-brain barrier; brain endothelial cell; ethanol metabolism; matrix metalloproteinases; protein tyrosine kinase; protein tyrosine phosphatase
Related Articles
The effects of 1α,25-dihydroxyvitamin D3 on matrix metalloproteinase and prostaglandin E2 production by cells of the rheumatoid lesion
Tetlow, Lynne C
Woolley, David E
Arthritis Research
The biologically active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], acts through vitamin D receptors, which were found in rheumatoid tissues in the present study. IL-1β-activated rheumatoid synovial fibroblasts and human articular chondrocytes were shown to respond differently to exposure to 1α,25(OH)2D3, which has different effects on the regulatory pathways of specific matrix metalloproteinases and prostaglandin E2.
1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the biologically active metabolite of vitamin D3, acts through an intracellular vitamin D receptor (VDR) and has several immunostimulatory effects. Animal studies have shown that production of some matrix metalloproteinases (MMPs) may be upregulated in rat chondrocytes by administration of 1α,25(OH)2D3; and cell cultures have suggested that 1α,25(OH)2D3 may affect chondrocytic function. Discoordinate regulation by vitamin D of MMP-1 and MMP-9 in human mononuclear phagocytes has also been reported. These data suggest that vitamin D may regulate MMP expression in tissues where VDRs are expressed. Production of 1α,25(OH)2D3 within synovial fluids of arthritic joints has been shown and VDRs have been found in rheumatoid synovial tissues and at sites of cartilage erosion. The physiological function of 1α,25(OH)2D3 at these sites remains obscure. MMPs play a major role in cartilage breakdown in the rheumatoid joint and are produced locally by several cell types under strict control by regulatory factors. As 1α,25(OH)2D3 modulates the production of specific MMPs and is produced within the rheumatoid joint, the present study investigates its effects on MMP and prostaglandin E2 (PGE2) production in two cell types known to express chondrolytic enzymes.
To investigate VDR expression in rheumatoid tissues and to examine the effects of 1α,25-dihydroxyvitamin D3 on cultured rheumatoid synovial fibroblasts (RSFs) and human articular chondrocytes (HACs) with respect to MMP and PGE2 production.
Rheumatoid synovial tissues were obtained from arthroplasty procedures on patients with late-stage rheumatoid arthritis; normal articular cartilage was obtained from lower limb amputations. Samples were embedded in paraffin, and examined for presence of VDRs by immunolocalisation using a biotinylated antibody and alkaline-phosphatase-conjugated avidin-biotin complex system. Cultured synovial fibroblasts and chondrocytes were treated with either 1α,25(OH)2D3, or interleukin (IL)-1β or both. Conditioned medium was assayed for MMP and PGE2 by enzyme-linked immunosorbent assay (ELISA), and the results were normalised relative to control values.
The rheumatoid synovial tissue specimens (n = 18) immunostained for VDRs showed positive staining but at variable distributions and in no observable pattern. VDR-positive cells were also observed in association with some cartilage-pannus junctions (the rheumatoid lesion). MMP production by RSFs in monolayer culture was not affected by treatment with 1α,25(OH)2D3 alone, but when added simultaneously with IL-1β the stimulation by IL-1β was reduced from expected levels by up to 50%. In contrast, 1α,25(OH)2D3 had a slight stimulatory effect on basal production of MMPs 1 and 3 by monolayer cultures of HACs, but stimulation of MMP-1 by IL-1β was not affected by the simultaneous addition of 1α,25(OH)2D3 whilst MMP-3 production was enhanced (Table 1). The production of PGE2 by RSFs was unaffected by 1α,25(OH)2D3 addition, but when added concomitantly with IL-1β the expected IL-1 β-stimulated increase was reduced to almost basal levels. In contrast, IL-1β stimulation of PGE2 in HACs was not affected by the simultaneous addition of 1α,25(OH)2D3 (Table 2). Pretreatment of RSFs with 1α,25(OH)2D3 for 1 h made no significant difference to IL-1β-induced stimulation of PGE2, but incubation for 16 h suppressed the expected increase in PGE2 to control values. This effect was also noted when 1α,25(OH)2D3 was removed after the 16h and the IL-1 added alone. Thus it appears that 1α,25(OH)2D3 does not interfere with the IL-1β receptor, but reduces the capacity of RSFs to elaborate PGE2 after IL-1β induction.
Cells within the rheumatoid lesion which expressed VDR were fibroblasts, macrophages, lymphocytes and endothelial cells. These cells are thought to be involved in the degradative processes associated with rheumatoid arthritis (RA), thus providing evidence of a functional role of 1α,25(OH)2D3 in RA. MMPs may play important roles in the chondrolytic processes of the rheumatoid lesion and are known to be produced by both fibroblasts and chondrocytes. The 1α,25(OH)2D3 had little effect on basal MMP production by RSFs, although more pronounced differences were noted when IL-1β-stimulated cells were treated with 1α,25(OH)2D3, with the RSF and HAC showing quite disparate responses. These opposite effects may be relevant to the processes of joint destruction, especially cartilage loss, as the ability of 1α,25(OH)2D3 to potentiate MMP-1 and MMP-3 expression by 'activated' chondrocytes might facilitate intrinsic cartilage chondrolysis in vivo. By contrast, the MMP-suppressive effects observed for 1α,25(OH)2D3 treatment of 'activated' synovial fibroblasts might reduce extrinsic chondrolysis and also matrix degradation within the synovial tissue. Prostaglandins have a role in the immune response and inflammatory processes associated with RA. The 1α,25(OH)2D3 had little effect on basal PGE2 production by RSF, but the enhanced PGE2 production observed following IL-1β stimulation of these cells was markedly suppressed by the concomitant addition of 1α,25(OH)2D3. As with MMP production, there are disparate effects of 1α,25(OH)2D3 on IL-1β stimulated PGE2 production by the two cell types; 1α,25(OH)2D3 added concomitantly with IL-1β had no effect on PGE2 production by HACs. In summary, the presence of VDRs in the rheumatoid lesion demonstrates that 1α,25(OH)2D3 may have a functional role in the joint disease process. 1α,25(OH)2D3 does not appear to directly affect MMP or PGE2 production but does modulate cytokine-induced production.
Comparative effects of 1 α,25-dihydroxyvitamin D3 (1 α,25D3) on interleukin (IL)-1-stimulated matrix metalloproteinase (MMP)-1 and MMP-3 production by rheumatoid synovial fibroblasts and human articular chondrocytes in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
Comparative effects of 1α,25-dihydroxyvitamin D3 (1α,25D3) on Interleukin (IL)-1-stimulated prostaglandin E2 production by rheumatoid synovial fibroblasts and human articular chondrocyte in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
1α,25-dihydroxyvitamin D3; matrix metalloproteinase; prostaglandin E2; rheumatoid arthritis
Related Articles
Normobaric hyperoxia attenuates early blood-brain barrier disruption by inhibiting MMP-9-mediated occludin degradation in focal cerebral ischemia
Liu, Wenlan
Hendren, Jill
Qin, Xu-Jun
Shen, Jiangang
Liu, Ke Jian
Journal of neurochemistry
Early blood-brain barrier (BBB) disruption, resulting from excessive neurovascular proteolysis by matrix metalloproteinases (MMPs), is closely associated with hemorrhagic transformation events in ischemic stroke. We have shown that normobaric hyperoxia (NBO) treatment reduces MMP-9 increase in the ischemic brain. The aim of this study was to determine whether NBO could attenuate MMP-9-mediated early BBB disruption following ischemic stroke. Rats were exposed to NBO (95% O2) or normoxia (30% O2) during 90-min middle cerebral artery occlusion, followed by 3-hr reperfusion. NBO-treated rats showed significant reduction in Evan's blue extravasation in the ischemic hemisphere compared with normoxic rats. Topographically, Evan's blue leakage was mainly seen in the subcortical regions including striatum, which was accompanied by increased gelatinolytic activity and reduced immunostaining for tight junction protein occludin. Increased gelatinolytic activities and occludin protein loss were also observed in isolated ischemic microvessels. Gel gelatin zymography identified that MMP-9 was the main enzymatic source in the cerebral microvessels. Incubation of brain slices or isolated microvessels with purified MMP-9 revealed specific degradation of occludin. Inhibition of MMP-9 by NBO or MMP-inhibitor BB1101 significantly reduced occludin protein loss in ischemic microvessels. These results suggest that NBO attenuates early BBB disruption, and inhibition of MMP-9-mediated occludin degradation is an important mechanism for this protection.
blood-brain barrier; matrix metalloproteinases; oxygen; stroke
Related Articles
Clinicopathologic significance of claudin-6, occludin, and matrix metalloproteinases −2 expression in ovarian carcinoma
Wang, Liping
Jin, Xiangshu
Lin, Dongjing
Liu, Zhijing
Zhang, Xiaowei
Lu, Yan
Liu, Yuanyuan
Wang, Min
Yang, Minlan
Li, Jiuxia
Quan, Chengshi
Diagnostic Pathology
Tight junctions (TJs) are mainly composed of claudins, occludin, and tight junction adhesion molecules (JAM). The invasive and metastatic phenotype of highly invasive cancer cells has been related to abnormal structure and function of TJs, and with expression of activated matrix metalloproteinases (MMPs). The relevance of these mechanisms responsible for the invasion and metastasis of ovarian carcinoma is unclear. Similarly, it is not known if the expression of claudin-6, occludin and MMP2 is related with the clinical properties of these tumors.
Expression of claudin-6, occludin, and MMP2 was detected in samples of human ovarian cancer tissues by immunohistochemistry and correlated with the clinical properties of the tumors.
The positive expression rates of claudin-6 and MMP-2 were higher in ovarian papillary serous carcinomas than n ovarian serous adenomas (P < 0.05). There were no differences in the expression of occludin (P > 0.05). The expression of claudin-6 and occludin in ovarian cancer was not correlated with patient age, pathological grade, clinical stage, and metastasis (P > 0.05). MMP-2 expression was enhanced with increased clinical stage and metastasis (P < 0.05), but was unrelated to patient age or tumor grade (P > 0.05). There were no apparent correlations between expression of claudin-6, occludin and MMP-2 in ovarian cancer tissue (P > 0.05).
Our data suggest, for the first time, that the claudin-6 and MMP-2 are up-regulated in ovarian papillary serous carcinomas, MMP-2 expression was enhanced with increased clinical stage and metastasis. Claudin-6 and MMP-2 may play a positive role in the invasion and metastasis of ovarian cancer.
Virtual slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1775628454106511.
Tight junctions; Ovarian cancer; Claudin-6; Occludin; MMP-2
Related Articles
Early inhibition of MMP activity in ischemic rat brain promotes expression of tight junction proteins and angiogenesis during recovery
Yang, Yi
Thompson, Jeffrey F
Taheri, Saeid
Salayandia, Victor M
McAvoy, Thera A
Hill, Jeff W
Yang, Yirong
Estrada, Eduardo Y
Rosenberg, Gary A
Journal of Cerebral Blood Flow & Metabolism
In cerebral ischemia, matrix metalloproteinases (MMPs) have a dual role by acutely disrupting tight junction proteins (TJPs) in the blood–brain barrier (BBB) and chronically promoting angiogenesis. Since TJP remodeling of the neurovascular unit (NVU) is important in recovery and early inhibition of MMPs is neuroprotective, we hypothesized that short-term MMP inhibition would reduce infarct size and promote angiogenesis after ischemia. Adult spontaneously hypertensive rats had a transient middle cerebral artery occlusion with reperfusion. At the onset of ischemia, they received a single dose of the MMP inhibitor, GM6001. They were studied at multiple times up to 4 weeks with immunohistochemistry, biochemistry, and magnetic resonance imaging (MRI). We observed newly formed vessels in peri-infarct regions at 3 weeks after reperfusion. Dynamic contrast-enhanced MRI showed BBB opening in new vessels. Along with the new vessels, pericytes expressed zonula occludens-1 (ZO-1) and MMP-3, astrocytes expressed ZO-1, occludin, and MMP-2, while endothelial cells expressed claudin-5. The GM6001, which reduced tissue loss at 3 to 4 weeks, significantly increased new vessel formation with expression of TJPs and MMPs. Our results show that pericytes and astrocytes act spatiotemporally, contributing to extraendothelial TJP formation, and that MMPs are involved in BBB restoration during recovery. Early MMP inhibition benefits neurovascular remodeling after stroke.
astrocytes; blood–brain barrier; brain recovery; focal ischemia; matrix proteinases; pericytes angiogenesis
Related Articles
D'Aversa, Teresa G.
Eugenin, Eliseo A.
Lopez, Lillie
Berman, Joan W.
Neuropathology and applied neurobiology
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), characterized by demyelination of white matter, loss of myelin forming oligodendrocytes, changes in the blood-brain-barrier (BBB), and leukocyte infiltration. Myelin basic protein (MBP) is a component of the myelin sheath. Degradation of myelin is believed to be an important step that leads to MS pathology. Transmigration of leukocytes across the vasculature, and a compromised BBB participate in the neuroinflammation of MS. We examined the expression and regulation of the chemokine CCL2 and the cytokine IL-6 in human endothelial cells (EC), a component of the BBB, after treatment with MBP.
EC were treated with full length MBP. CCL2 and IL-6 protein were determined by ELISA. Western blot analysis was used to determine signaling pathways. A BBB model was treated with MBP and permeability was assayed using albumin conjugated to Evan's blue dye. The levels of the tight junction proteins occludin and claudin-1, and matrix metalloprotease (MMP)-2 were assayed by Western blot.
MBP significantly induced CCL2 and IL-6 protein from EC. This induction was partially mediated by the p38 MAPK pathway as there was phosphorylation after MBP treatment. MBP treatment of a BBB model caused an increase in permeability that correlated with a decrease in occludin and claudin-1, and an induction of MMP-2.
These data demonstrate that MBP induces chemotactic and inflammatory mediators. MBP also alters BBB permeability and tight junction expression, indicating additional factors that may contribute to the BBB breakdown characteristic of MS.
MBP; MMP2; IL-6; CCL2; EAE; blood-brain-barrier; MS
Related Articles
Protective Role of Matrix Metalloproteinase-9 in Ozone-Induced Airway Inflammation
Yoon, Hyoung-Kyu
Cho, Hye-Youn
Kleeberger, Steven R.
Environmental Health Perspectives
Exposure to ozone causes airway inflammation, hyperreactivity, lung hyper-permeability, and epithelial cell injury. An early inflammatory response induced by inhaled O3 is characterized primarily by release of inflammatory mediators such as cytokines, chemokines, and airway neutrophil accumulation. Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of oxidative lung disorders including acute lung injury, asthma, and chronic obstructive pulmonary disease.
We hypothesized that MMPs have an important role in the pathogenesis of O3-induced airway inflammation.
We compared the lung injury responses in either Mmp7- (Mmp7−/−) or Mmp9-deficient (Mmp9−/−) mice and their wild-type controls (Mmp7+/+, Mmp9+/+) after exposure to 0.3 ppm O3 or filtered air.
Relative to air-exposed controls, MMP-9 activity in bronchoalveolar lavage fluid (BALF) was significantly increased by O3 exposure in Mmp9+/+ mice. O3-induced increases in the concentration of total protein (a marker of lung permeability) and the numbers of neutrophils and epithelial cells in BALF were significantly greater in Mmp9−/− mice compared with Mmp9+/+ mice. Keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 levels in BALF were also significantly higher in Mmp9−/− mice than in Mmp9+/+ mice after O3 exposure, although no differences in mRNA expression for these chemokines were found between genotypes. Mean BALF protein concentration and numbers of inflammatory cells were not significantly different between Mmp7+/+ and Mmp7−/− mice after O3 exposure.
Results demonstrated a protective role of MMP-9 but not of MMP-7, in O3-induced lung neutrophilic inflammation and hyperpermeability. The mechanism through which Mmp9 limits O3-induced airway injury is not known but may be via posttranscriptional effects on proinflammatory CXC chemokines including KC and MIP-2.
chemokine; knockout mice; lung; MMP-9; O3; oxidant
Related Articles
West Nile virus-induced disruption of the blood–brain barrier in mice is characterized by the degradation of the junctional complex proteins and increase in multiple matrix metalloproteinases
Roe, Kelsey
Kumar, Mukesh
Lum, Stephanie
Orillo, Beverly
Nerurkar, Vivek R.
Verma, Saguna
The Journal of General Virology
2012;93(Pt 6):1193-1203.
West Nile virus (WNV) encephalitis is characterized by neuroinflammation, neuronal loss and blood–brain barrier (BBB) disruption. However, the mechanisms associated with the BBB disruption are unclear. Complex interactions between the tight junction proteins (TJP) and the adherens junction proteins (AJP) of the brain microvascular endothelial cells are responsible for maintaining the BBB integrity. Herein, we characterized the relationship between the BBB disruption and expression kinetics of key TJP, AJP and matrix metalloproteinases (MMPs) in the mice brain. A dramatic increase in the BBB permeability and extravasation of IgG was observed at later time points of the central nervous system (CNS) infection and did not precede virus–CNS entry. WNV-infected mice exhibited significant reduction in the protein levels of the TJP ZO-1, claudin-1, occludin and JAM-A, and AJP β-catenin and vascular endothelial cadherin, which correlated with increased levels of MMP-1, -3 and -9 and infiltrated leukocytes in the brain. Further, intracranial inoculation of WNV also demonstrated increased extravasation of IgG in the brain, suggesting the role of virus replication in the CNS in BBB disruption. These data suggest that altered expression of junction proteins is a pathological event associated with WNV infection and may explain the molecular basis of BBB disruption. We propose that WNV initially enters CNS without altering the BBB integrity and later virus replication in the brain initiates BBB disruption, allowing enhanced infiltration of immune cells and contribute to virus neuroinvasion via the 'Trojan-horse' route. These data further implicate roles of multiple MMPs in the BBB disruption and strategies to interrupt this process may influence the WNV disease outcome.
Related Articles
Matrix Metalloproteinase-9 in Homocysteine-Induced Intestinal Microvascular Endothelial Paracellular and Transcellular Permeability
Munjal, Charu
Tyagi, Neetu
Lominadze, David
Tyagi, Suresh C.
Journal of Cellular Biochemistry
Although elevated levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with inflammatory bowel disease, the mechanism of Hcy action is unclear. In the present study we tested the hypothesis that HHcy activates matrix metalloproteinase-9 (MMP-9), which in turn enhances permeability of human intestinal microvascular endothelial cell (HIMEC) layer by decreasing expression of endothelial junction proteins and increasing caveolae formation. HIMECs were grown in Transwells and treated with 500 μM Hcy in the presence or absence of MMP-9 activity inhibitor. Hcy-induced permeability to FITC-conjugated bovine serum albumin (FITC-BSA) was assessed by measuring fluorescence intensity of solutes in the Transwells' lower chambers. The cell-cell interaction and cell barrier function was estimated by measuring trans-endothelial electrical impedance. Confocal microscopy and flow cytometry were used to study cell junction protein expressions. Hcy-induced changes in transcellular transport of HIMECs were estimated by observing formation of functional caveolae defined as caveolae labeled by cholera toxin and antibody against caveolin-1 and one that have taken up FITC-BSA. Hcy instigated HIMEC monolayer permeability through activation of MMP-9. The increased paracellular permeability was associated with degradation of vascular endothelial cadherin and zona occludin-1 and transcellular permeability through increased caveolae formation in HIMECs. Elevation of Hcy content increases permeability of HIMEC layer affecting both paracellular and transcellular transport pathways, and this increased permeability was alleviated by inhibition of MMP-9 activity. These findings contribute to clarification of mechanisms of inflammatory bowel disease development.
Inflammatory bowel disease; microvascular remodeling; paracellular transport; transcellular transport
Related Articles
Ischemic Postconditioning Decreases Cerebral Edema and Brain Blood Barrier Disruption Caused by Relief of Carotid Stenosis in a Rat Model of Cerebral Hypoperfusion
Yang, Fuwei
Zhang, Xiaojie
Sun, Ying
Wang, Boyu
Zhou, Chuibing
Luo, Yinan
Ge, Pengfei
PLoS ONE
Background and Purpose
Complications due to brain edema and breakdown of blood brain barrier are an important factor affecting the treatment effects of patients with severe carotid stenosis. In this study, we investigated the protective effects of ischemic postconditioning on brain edema and disruption of blood brain barrier via establishing rat model of hypoperfusion due to severe carotid stenosis.
Wistar rat model of hypoperfusion due to severe carotid stenosis was established by binding a stainless microtube to both carotid arteries. Ischemic postconditioning procedure consisted of three cycles of 30 seconds ischemia and 30 seconds reperfusion. Brain edema was evaluated by measuring cerebral water content, and blood brain barrier permeability was assayed by examining cerebral concentration of Evans' Blue (EB) and fluorescein sodium (NaF). ELISA was used to analyze the expression of MMP-9, claudin-5 and occludin. The activity and location of MMP-9 was analyzed by gelatin zymography and in situ zymography, respectively. The distribution of tight junction proteins claudin-5 and occludin was observed by immunohistochemistry.
The increased brain water content and cerebral concentration of EB and NaF were suppressed by administration of ischemic postconditioning prior to relief of carotid stenosis. Zymographic studies showed that MMP-9 was mainly located in the cortex and its activity was significantly improved by relief of carotid stenosis and, but the elevated MMP-9 activity was inhibited markedly by ischemic postconditioning. Immunohistochemistry revealed that ischemic postconditioning improved the discontinuous distribution of claudin-5 and occludin. ELISA detected that the expression of up-regulated MMP-9 and down-regulated claudin-5 and occludin caused by carotid relief were all attenuated by ischemic postconditioning.
Ischemic postconditioning is an effective method to prevent brain edema and improve BBB permeability and could be used during relief of severe carotid stenosis.
Related Articles
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text:
Take a look at this!
The owners name is Jonny.
At the heart of it is a Yamaha developed 3s-ge beams engine ran on a megasquirt ecu. think of Toyota's version of a k20 and your sort of close.
The engine in standard form makes 190bhp, this one makes slightly more, plus a redline of 9k must be fun.
The rest of the car is just mad, with a wide arch Corsa kit moulded on to accommodate the increased track front and back.
Id love to get a shot ragging this with the individual throttle bodies poking through the firewall.
It must sound insane!

subset: commoncrawl


text:
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Join 20,774 other Security Guard professionals receiving free weekly job listings to their inbox every week.
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text:
Eh, while I like the game, I'd say after how much effort B/W/B2/W2 put into the story, a lot of this game seems very weak in that regard. The rivals are all annoying (stop interrupting my adventure to drag me to some extra thing or give me some speech on friendship or dancing). Team Flare is kind of all over the place in their motives, and they're really just kind of... stupid, overall. There really didn't feel like there was enough of a build-up to the final confrontation with them, AZ and the Pokemon on the cover seem shoehorned in. I know there's foreshadowing to AZ, but his story didn't really make me feel anything. And the gym leaders. Besides Korrina, and a pre-gym scene with electric kid and Wulfric each, they seem to just be there for five minutes before you trounce them. They're so unmemorable that I keep mixing up the fairy and psychic leaders in my head. Gyms themselves were cool though.
I was also a bit "wait, who?" on the champion in this game, but X/Y has one of my favorite Elite 4s in all the games.
I still really like the game, but after the last few installments this one just feels like the main selling point was graphics and everything else took a hit for it.


text:
Marketing Major
Marketing is an organizational function and a set of processes for creating, communicating, and delivering value to customers and for managing customer relationships in ways that benefit the organization and its stakeholders. Marketing plays an important role in society by making sure products and services are available in the form and price that is acceptable to consumers. Marketing is also important to business since marketing directly contributes to achieve business objectives of growth and profits.
Student Learning/Engagement/Teaching
Marketing courses are designed to both ground students in the current theories and give them active practice in applying the concepts.
All Marketing majors gain valuable first-hand experience often working with clients as they design marketing campaigns in many of the marketing classes.
This integration of theory and practice prepares students for the career opportunities ahead and provides them with an engaging experience as they work closely with marketing faculty.
Typical Careers for Marketing Majors
More than 30% of the entire civilian workforce performs marketing activities. Marketing offers great career opportunities in such areas as professional selling, marketing research, advertising, retail buying, distribution management, product management, product development and wholesaling. More CEOs such as Lee Iacocca (Chrysler) and John Akers (IBM) come from sales and marketing backgrounds than from any other field.
As the global marketplace becomes more challenging, companies all over the world and of all sizes have to become better marketers. Thus marketing jobs are plentiful and offer the opportunity to earn a rewarding income.
Where our students go
Drury University marketing graduates have begun their careers at companies such as Clorox, Shoppers Advantage, Edward Jones International, Prime Trucking, Farmers Insurance, and Cerner International. Some marketing graduates decide to pursue graduate studies at top schools such as Washington University, University of West Virginia, St. Louis University, and University of South Carolina.
Course Requirements
Students pursuing the bachelor of business administration degree (BBA) with a major in marketing are required to complete the following course requirements:
|CORE Requirements
39-43 hours*
|BBA Tool Courses
25 hours
|BBA Core Courses
21 hours
|Marketing Major Courses
16 hours
19-23 hours
Total BBA hours:
124-128 hours
*Drury CORE hours have been reduced from 45-59 to 39-43 as credit hours (6-7) are included in the BBA Tool Courses hours.
BBA Tool Courses:
All students pursuing a BBA degree with a major in marketing are required to complete the following tool courses:
ACCT 209: Principles of Accounting
ACCT 210: Accounting for Management
ECON 201: Basic Economic Theory
MATH 227: Introduction to Statistics
MGMT 103: Business Foundations
MGMT 170: Computer Proficiency Exam
MGMT 204: Organizational Behavior
MGMT 228: Analytical Methods
MGMT 250: Management Information Systems
BBA Core Courses:
All students pursuing a BBA degree with a major in marketing are required to complete the following core courses:
FINC 331: Corporate Finance
MGMT 205-208: Study Abroad
MGMT 301: Leadership and Organizations
MGMT 319: Business Law & Ethics
MGMT 424: Business Simulation Workshop
MGMT 446: Strategic Management
MGMT 480: Professional Business Experience
MKTG 337: Marketing
Marketing Major Courses:
Students earning a BBA degree with a major in marketing also must complete the following courses for the major:
ECON 311: Price Theory
MKTG 348: Marketing Research
Choose three of the following:
MKTG 338: Professional Selling and Presentation
MKTG 339: Principles of Advertising
MKTG 341: Product Development and Brand Strategy
MKTG 344: Consumer Behavior
The faculty of the Breech School reserves the right to modify the above program and degree requirements at any time.
Admission Policy
Official admission to the Breech School of Business Administration is required* to enroll in any upper-division course (numbered 300/400) with prefixes of ACCT, ECON, FINC, MGMT and MKTG.
To be admitted to the Breech School of Business Administration, a student must:
- Be officially admitted to Drury University.
- Complete an official declaration of major form indicating the major, minor or special program that the student intends to pursue. At this time, students will be required to read and sign the Breech Student Honor Code.
- Have completed at least 42 hours of college level credit, including a minimum of 12 semester credit hours at Drury University.
- Have completed all of the following preparatory courses (or their transfer equivalents) with a minimum grade of C- in each of the following courses:
- ACCT 209: Principles of Accounting
- ECON 201: Basic Economic Theory
- MATH 227: Introduction to Statistics
- MGMT 170: Computer Proficiency Exam (must achieve grade of 'S')
- MGMT 250: Management Information Systems
Choose one of the following:
- MATH 230: Business Calculus
- MATH 231: Calculus I
- Have a cumulative GPA of 2.75 on all Drury University coursework.
Conditional Admission. A student not achieving the 2.75 overall GPA, or not achieving the required grades of C- in the preparatory courses listed above, or not completing at least 42 hours of college-level credit, including a minimum of 12 semester credit hours at Drury University, may be granted up to two (2) semesters of conditional admission to the Breech School. Conditional admission cannot be granted to a student not achieving both the required GPA and a minimum grade of C- in the specified preparatory courses.
During the semester(s) of conditional admission, the student will be expected to meet all requirements for full admission. The student, subject to advisor approval, may enroll in a total of 13 hours of upper-division Breech School coursework during the terms of conditional admission. Prior to registration, students are required to complete the Permission to Enroll in Upper-Division Breech School Courses form with their academic advisor.
Students Majoring in Non-Breech School Majors/Minors
Students who must enroll in a Breech School 300 or 400 level offering to fulfill requirements of a program NOT offered through the Breech School of Business Administration may be exempted from this admission policy, provided they have completed all other specific prerequisites for the course. Prior to registration, students are required to complete the Permission to Enroll in Upper-Division Breech School Courses form with their academic advisor.


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The Life And Times Of The World's Richest Women
Susanne Klatten, an heiress to the BMW fortune, may be the 68th richest person in the world, but her life hasn't been so peachy lately. The German mama of three had a two-month affair with a creep named Helg Sgarbi, who secretly videotaped many of their encounters. Then he and several buddies blackmailed her, demanding… More »


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For 101 years, the Albany YMCA was closed on Sundays, for its 102nd year they decided to give something new a try and opened on Sundays.
On the one year anniversary of its new schedule the YMCA was giving out ice cream sundaes, to show their appreciation to members for making the schedule a success.
Having the YMCA stay open all weekend is something members like Louise Little appreciate, "I can come Sunday afternoon, because I'm so busy during the week, I can't get here all the time during the week,and (now) I can come on Sunday afternoon."
Officials from the YMCA said they decided to open on Sunday's after customers started requesting it.


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Ulteo Partners with Microsoft to Further Integrate the Open Virtual Desktop into Windows
Open source startup selected for Initiative for the Economic Development of the Software publishers and Start-up by MS France.
Ulteo announced a partnership with Microsoft France to further integrate the open source Open Virtual Desktop into Windows environments. The OVD allows end users to publish Linux or Windows business applications from virtual environment, rather than install the applications (and therefore the operating system) onto a local machine.
Microsoft has given Ulteo access to proprietary products such as Sharepoint, Microsoft HyperV, App-V and Azure. Ulteo is using this access to create more remote access options for IT departments to build around. In addition to customer servers and data centers, remote access via the cloud will soon be a viable option for the OVD.
Version 2.0 of the Open Virtual Desktop is available now with 2.5 in beta. More information can be found at ulteo.com.
Issue 192/2016
Buy this issue as a PDF
Nextcloud Announces Raspberry Pi-Powered Home Cloud Box
Innovative system adds a hard drive and Ubuntu Core to the RPi for an IoT hub.
Linus Torvalds Confirms the Date of the First Linux Release
Linux is two weeks younger than we thought!
End of OpenOffice?
The Apache Software Foundation considers retiring OpenOffice
Adobe Gives New Life to NPAPI Plugin for Linux
Adobe won't kill the plugin in 2017
LinuxCon North America Convenes in Toronto, Canada
Linux Foundation's big event celebrates the 25th anniversary of Linux
Linux Turns 25
Linux has evolved from "won't be a professional" project to one of the most professional software projects in the history of computers.
Mirantis Joins Hands with SUSE To Offer RHEL Support; Red Hat Not Happy
Competitors get in the game with RHEL without Red Hat
Linux on Windows 10 Poses a Security Risk
Security researchers have already notified Microsoft; some fixes are available
Mirantis to Refactor OpenStack Fuel for Kubernetes
The company is collaborating with Google and Intel to use Kubernetes as an engine for Fuel
Canonical Joins Document Foundation Advisory Board
The upcoming release of LibreOffice will be available as a snap package


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Tippy toe walker....still!!
Comments (6)
I don't think the tip toe thing is a big deal, I've seen some people talk about it as part of a sensory disorder but I don't think that's always the case.
Mommy to Little Miss Adriana 12/23/2009 and Sir Nicholas 11/29/2012
Mine does it all the time as well. I think it's just a quirk and something he enjoys doing. I know it can be associated with autism and learning delays but I don't see any other symptoms that concern me.
My daughter is also a toe walker. We are always trying to correct her. There are developmental risks involved with toe walking. It can shorten the calf muscles, which can require physio, braces and possibly surgery. I am hoping as my daughter starts skiing and skating more this year she will toe walk less.
6 year old girl
2 year old boy
I know my brain goes to CP when I think of toe walkers, but if your son can walk normally, it is less likely to be that. I agree with PP that long term it can cause issues though. I would think as he gets older he will be more prone to listen to you more and want to walk like his friends.
DD December 2009
DS September 2014
Learn more
Look how far your child has come.
Review your child's growth milestones.
Read about 6 years 8 months : Week 2.
Your growing child
Your 6 1/2-year-old: Is it ADHD?
Your 6 3/4-year-old: The power of positivity
Your 6 3/4-year-old: Is your child gifted?
Your 6 3/4-year-old: Potty mouth
Your 6 3/4-year-old: Dealing with death
Learn more
What's next for your growing child?
Get a glimpse of what's to come.
Read about 6 years 10 months.


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iPhone App
This project received 67 bids from talented freelancers with an average bid price of $1489 USD.Get free quotes for a project like this
Skills Required
Project Budget$50 - $3000 USD
Total Bids67
Project Description
I'm needing a simple app created. It just needs to be able to simply have the ability to copy and paste a link, compose a brief message with a character limit and send it to a contact in your phone via email. I've got a web app already created, just need a mobile app.
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- Set your budget and the timeframe
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- Times Online


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An end of innocence after death
The first question Joe DeCecco asked police after finding out the killers who buried a hatchet in the skull of a 78-year-old woman were allegedly two 13-year-olds, including her great-grandson, was a simple and incredulous one:
"Did these kids grow up here?" asked the veteran Sheboygan County district attorney. "Did they grow up here in Sheboygan?"
The last murder in idyllic little Sheboygan Falls was in 1996. There are maybe one or two first-degree homicides per year on average in all of Sheboygan County.
This isn't a big metropolitan area where ingrained poverty, drugs, gangs and family dysfunction have helped foster horrific crime by young teenagers. This is small-town Wisconsin, and these kids, Antonio Barbeau and Nathan Paape, look like they haven't even reached puberty. I have a 13-year-old myself. Kids that age have consciences and an innate innocence that lingers.
Something different may have grown in Sheboygan.
According to the criminal complaint: The boys asked Paape's mother to give them a ride from Sheboygan, where they live, to Barbeau's great-grandmother's house in Sheboygan Falls seven miles away. That's a meandering, pleasant drive, especially when the leaves are deep red beside the sun-dappled waters of the Sheboygan River. You pass baseball diamonds and parks and the stables of Kohler. It's the sort of drive you'd take with a picnic basket on your lap. These boys, say police, secretly carried a hammer and a hatchet.
"These two boys were cold, callous and calculating," said Sheboygan Falls Police Chief Steven Riffel.
Barbeau's great-grandmother's house in the quiet Westwynde Bluffs subdivision has a welcome sign affixed to a plant stand in which she helped things grow. Barbara Olson is said to have been like that - welcoming, nurturing.
After Paape's mother, oblivious, dropped them off, they walked in, struck the old woman several times with the blunt end of the hatchet, then turned it around while she lay groaning on the floor, according to the criminal complaint.
"Nathan stated he hit her twice with the hammer and then Antonio, using the blade end of the hatchet, struck her in the head and the hatchet stuck there and that both of them had to pull the hatchet loose," according to the complaint.
When they were done with her, it is alleged, they went out for pizza. Yes, pizza.
DeCecco is a prosecutor, not a minister. But I can't get over the fact these kids are 13 and from Sheboygan. I asked him if the killing gives him pause about human nature.
"No, no, this is an exception. I have been a prosecutor for about 23 years. Of all the people I have prosecuted, there are only two that gave me pause like this case does." Those two had a "dead affect in describing the crime: no emotion, no apparent remorse."
He thinks there are a "limited number of people in the world" who aren't just immoral but are "amoral," people who "have no moral compass at all."
This case has a long way to go, so while Barbeau and Paape have also appeared remorseless to DeCecco, he is resisting definitively lumping these kids in with what the professors would call sociopaths. He did say, however, that some people "don't see the world like we all see it . . . We just have a sense of conscience, and if we do something wrong, most of us are regretful . . . But there are some people in the world who never have those feelings."
These boys did indeed grow up in the Sheboygan area. But if they did this, I don't think that tells us what we want to know - where they really come from.
That's a place unknown to the rest of us and one where, we can only hope, there aren't many more of them.
Mike Nichols is an author and a senior fellow with the Wisconsin Policy Research Institute, a nonpartisan conservative think tank. This column reflects his personal perspective. Email firstname.lastname@example.com.
Mike Nichols Archive
- May 23, 2014 | WPRI chief responds to critic
- Jun 28, 2013 | Hundreds of blooms over the past 15 years
- Jun 21, 2013 | Productive advice: stay productive
- Jun 14, 2013 | Appleton bars scanning their patrons
- Jun 8, 2013 | Racial disparity in pot busts troubling
- May 31, 2013 | A snake with long legs
- May 24, 2013 | Success after high school unpredictable
- May 17, 2013 | Regulate, yes, but don't ban sales
- May 10, 2013 | Learning from a neighbor's success
- May 3, 2013 | Junk food on the taxpayer's dime
- Apr 26, 2013 | All the news - about him
- Apr 19, 2013 | Nihilism unleashed in Boston
- Apr 12, 2013 | English still a necessity for most
- Apr 5, 2013 | Property rights that really aren't rights at all
- Mar 30, 2013 | Off our plate and into shrubs
- Mar 22, 2013 | Still safely in control at airports
Across the Board
The Journal Sentinel Editorial Board's blog on the day's happenings as they happen.


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HARRISBURG - Bring on the posturing and political maneuvering.
It is June in Harrisburg, when the state budget takes center stage and legislators plot ways to squeeze in priorities - and stomp on others.
And on Monday, the House began debate on a $28.3 billion spending plan that found Republicans advocating for proposals that closely resemble what Gov. Corbett outlined in his budget address in February, and Democrats arguing for more money for public schools and the expansion of Medicaid in Pennsylvania.
The spending blueprint being considered by the House was crafted by Republicans who control the chamber, and would send $100 million more to public schools and restore funding for certain health-related items, such as diabetes and epilepsy support programs and poison-control centers.


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Plumber game HD pro
By junjingtong
Open iTunes to buy and download apps.
Plumber game
Plumber Full is a puzzle game.
Rotate the pipes around and set them up
so they connect from one to the end pipe
before time runs out.
What's New in Version 1.9
Add new game scene (100 levels)
Fix xmas scene's bug
iPad Screenshots
View in iTunes
- Category: Games
- Updated: 11 November 2013
- Version: 1.9
- Size: 39.4 MB
- Languages: English, Japanese, Simplified Chinese, Traditional Chinese
- Developer: xin guan
- © 2014 Easymobi
Compatibility: Requires iOS 4.3 or later. Compatible with iPad.


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ZombiezMum (12) Bangkok, Thailand | January 2, 2016
very big place but good service. good food but beer is a bit too expensive.
punkybrewster (39) California | January 1, 2016| Updated January 2, 2016
Go for the low prices and food, but the beer is water and no real selection.
justinbkk (16) Tulsa, Oklahoma | December 23, 2015
Typical 3 German beers that are made for the masses. A few bottles of other Belgian and German beers, but nothing in the line of craft beer.
mikemmacd (15) Halifax, Nova Scotia | December 11, 2015
Typical German beer house. Move along.
evilempire (67) sheffield, England | December 4, 2014
big tourist trap... almost a parody of a german brauhaus. some bizarre entertainment, food decent but expensive, beer expensive and while better than the yellow bubbly water sold most places still below par
PaWa (3) | November 25, 2013
Quite cool big place, the brewhouse in the center is pretty nice and well taken care of, it was actually in use when i came by, nice to smell the good odor of boiling wort. Service is nothing to care about, since there is about more than 75 servers on the floor (no joke i count them...) and we are less than 10 custommers... Beer is ok, just dont mind the little off taste here and there, its just nice to have some craft beer in Thailand... Anyway from a month here i noticed they are not pretty good in fermentation sience... I say it worth the move im happy to be here! give it a try!
TickerTerryDJ (17) Redditch, England | May 2, 2013
Large,busy and lively.Usually serving 3 beers which are good by Thai standard and welcome change from all the bottled fizz.Food is good and there is entertainment too.Easy enough to get to and worth checking out.
bigball (15) , Singapore | May 23, 2012
fun fun fun.. the beers not all that great, but people come to have a good time with a packed atmosphere, smiles from the crew, good food, decent brews and theres even entertainment.
JapanTony (144) , Japan | February 3, 2012
Massive place that seats 500+. Lots of musical acts keep things lively. Attentive staff serving quickly. Bilingual menus. Food menu is pretty extensive, with some dishes better than others. Only 3 beers on tap the day I was there (sometimes they have a 4th). Pils and Weizen were not bad, Dunkel a little disappointing. Bit out of the way, but the BRT express busline stop is just outside. Then about 10 minutes north to get to Sathron/Chong Nonsi (BTS skytrain station)./
Deninbangkok (1) , Thailand | August 23, 2011
Please ignore the Pradit Janie am part of the address, the hall is on the corner of Rama 3 and Naradhiwas ratchanakharin roads. Show is different every night, pace picks up around 8pm. Food is excellent. Beer is best in Thailand, which might not say much, but the Weizen is fruity and the dunkel is good for a hot country( a truly thick dark beer in Thailand would be bad upon stepping out in tropical heat.


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Determining the best location, time of year, and care to successfully transplant your tree is essential. This guide is about transplanting a rose of sharon.
We always had bad luck replanting others free give-a-ways to us. My daughter gave us this Rose of Sharon tree and we talked and watered daily and guess what? Success and a beautiful plant from her yard to mine. Now, when I go and see my little flower garden, I see the tree of love and success again? Don't give up...
By Chris from Buffalo, NY
When is the best time to dig up and replant a Rose of Sharon tree? Thank you.
Hardiness Zone: 5a
By Bbgone from Chicago, IL
You can transplant them in early spring or fall.
I have tons of rose of Sharon bushes and you can replant them at any time,spring or summer. One word of advise, they do re seed themselves everywhere. It has become a major task to pull up the baby ones before they take root, and they do root quickly. I have planted pinks, purples and whites all together and when they bloom they are beautiful. I have started to cut the tops off so they do more of a bushing out than a tree. I have an entire border that's about six feet tall as a "fence" in my backyard. Good luck with them.


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Santa Fund/Holiday Adopt-a-Family Program
Registration for our 2016 Holiday Santa Fund Program will take place at The First Church at 1 Concord Street, Nashua on the following dates and times:
Friday, Oct. 21 9:00 am – Noon
Wednesday, Oct. 26 3:30 – 6:00 pm
Thursday, Nov. 3 3:30 – 6:00 pm
Monday, Nov. 7 3:00- 5:00 p.m.
Please note that no children will be allowed at registration; Also you may not apply if you are already receiving assistance from any other agency or organization. You must bring ALL documentation listed on the attached information sheet, available in both English and Spanish.
Thank you to all of those in the community who gave so generously to our Holiday Program in 2015. With your help, we were able to provide toys and warm clothing to 981 children in need. Special thanks to BAE, Right Network, Church of the Good Shepherd and many other individuals, organizations and businesses for your generosity!
Since our inception, we have offered a program to help ensure smiles to the children of our community on Christmas morning. Santa lives in each and every one of us, and through this program the true meaning of the Christmas spirit becomes apparent.
In 1994, the Front Door Agency joined with the Telegraph to ensure the Santa Fund resources are allocated to those most in need. Through this partnership we have provided gifts, food, toys and clothing to nearly 1,100 children each and every year.
Local businesses, civic groups, individuals and religious organizations all participate in adopting children for the holidays. It is estimated that through the generosity of the community we pass through over $100,000 in in-kind contributions.
Registrations for assistance are held in late October and early November.


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Youth not a problem for Iota in scrimmage
KAPLAN – It's no secret that the Iota Bulldogs will be a young squad this year.
Apparently no one told them that however.
The Bulldogs, which have 16 freshmen, 23 sophomores, eight juniors and four seniors, will lean heavily on the younger players throughout the year, and if Thursday night's scrimmage was any indicator, the younger Dogs are ready for their shot.
"I was very pleased with our effort," said Neal Legé, Iota's head coach. "We have a very young ball club and they did well today. It's all we can ask right now, we are taking baby steps and we just want to get better week to week."
The three-way scrimmage featured the Bulldogs and Eunice Bobcats traveling to Kaplan to take on the Pirates.
After two scrimmages between Iota, Kaplan and Eunice that showcased the younger sides of each squad, the first team offenses and defenses took the field in different pairings.
Iota's first team defense took on Kaplan's first team offense at first. The Bulldogs' defense started strong effectively forcing two three and outs but in the Pirates third series, Kaplan started to get the ball moving with the help of big plays.
Three double digit runs mixed helped the Pirates quickly move the scrimmage into the red zone and on the seventh play of the series, Kaplan found itself in the endzone.
Iota's defense would not take the field again until the final scrimmage of the evening, taking on a Eunice offense that scored three touchdowns in its scrimmage against Kaplan earlier.
In that scrimmage, Iota alternated a solid stop with a penalty for the first several plays. The two penalties, resulting in 20 yards coupled with Eunice's offensive efficiency led to another red zone visit for Iota's defense. This time however, the defense would stand, leading to Eunice traveling the football field just to come up without anything to show for it.
"We did well," said Legé of the Bulldogs' defense. "We have a lot of guys playing on both sides of the ball and they got tired, but they dealt with it and played hard."
In both scrimmages, Iota kept their opponents off of the score board.
Iota's first team offense went back-to-back scrimmages facing first Eunice's defense then Kaplan's.
When the Bulldogs and Bobcats squared off, Iota's new look offense had trouble finding the end zone. Twenty-seven combined rushing yards between junior Brandon Fantroy and sophomore Michael Thevis were quickly equalized by the Bobcats adjustments and just as quickly as that, Iota was forced to restart offensively.
In the second series, Iota struggled to break off big plays, except for a more than 40 yard rush by Fantroy and the series and scrimmage against Eunice's defense ended with an interception thrown by junior Thomas Murrell.
Fantroy finished the first scrimmage with five carries for 69 yards and Thevis tacked on seven more yards in five carries. Murrell's two attempted passes fell incomplete. He had one interception.
The second offensive scrimmage for Iota saw a bit more success, however.
When the Pirate's defense took on Iota's offense, three 3 and outs were forced before Iota's offense finally saw consistent ball movement. In that fourth series, Murrell would complete two lateral passes and one final touchdown pass to Ryan Chapman to end the series with three of four passes complete for 52 yards.
"After looking at the stats and everything, it's obvious we are a better team in the split-back veer," said Legé of Iota's offense. "But we got some guys that are out right now that will make us a better power-I team."
Iota will next be in action Thursday in Crowley at Gardner Memorial Stadium for the Vermilion/Acadia Jamboree. Kickoff for Iota's game against North Vermilion is slated for 6 p.m.


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Mazda has announced that the RX-8 'SPIRIT R' Special Edition is now available in New Zealand.
In advance of Mazda RX-8 production ending in June 2012, SPIRIT R will be the final special edition model, offering a striking commemoration for Mazda's iconic four door, four seater rotary engine sports car.
SPIRIT R is named after the final limited edition Mazda RX-7 and this pedigree is reflected in stylish features that include an exclusive Recaro seat design and custom 19-inch alloy wheels.
The Mazda RX-8 SPIRIT R has a 6-speed manual transmission with exclusive features such as Bilstein sports suspension, distinctive red front and rear brake callipers.
Exterior features include 225/40 R19 89W tyres and 19-inch alloy wheels finished in stunning bronze with black centre cap. Front, side and rear spoilers improve aerodynamics and add street appeal. Black bezels within front and rear combination lamps and front fog lamps bring the race car to the fore and a SPIRIT R badge on C-pillar seals it in the Mazda history books.
The SPIRIT R is a sports car that means business with custom designed interior fittings. Distinctive SPIRIT R Recaro bucket seats are trimmed with black and red leather for effect. In addition, contrast red stitching on the steering wheel, gear shift, parking brake and door trim, set this RX-8 apart from the rest.
The limited edition RX-8 is available in three exterior colour options, Aluminium Metallic, Sparkling Black Mica and Crystal White Pearl Mica.
This vehicle is available in very limited numbers and for the same price as the standard model at $56,695 plus on-road costs. Every new RX-8 Spirit R comes with mazdacare as standard, including 3-years/100,000km (whichever occurs first) Mazda Scheduled Servicing, 3-years unlimited kilometre Mazda Factory Warranty and Mazda On Call Roadside Assistance.


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Product Information
Product Information
Casio Watch
The Casual Sports watch from Casio(r) incorporates an array of functional features, including 50-meter water resistance, a stopwatch, electro-luminescent backlighting, a countdown timer, and more. The nylon band is soft and durable.
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It is amazing to me how "the majority" of people are dissatisfied with the direction our country is moving. However, they continue to go to primary elections and vote the same regime right back into office.
I am profoundly disappointed with the Republican primary election results. To think, when I'm out and about in this city, I am surrounded by people who like to gripe about everything under the sun, yet they do nothing to solve the problems about which they continually moan and groan.
This election, we the people missed another opportunity to unite and tell the government establishment that we are not satisfied with the direction our country is moving. Rather, a majority of our populace stood up and said please keep giving it to us just the same way you always have.
Unfortunately, those of us who are tired of the same ideas and the 'Republican In Name Only' elite must suffer another 700-plus days thinking maybe next time.
If we are going to restore our constitutional republic to its conservative values, we must not be apathetic. We should research the candidates, determine who aligns best with our world view and support them with our time, money and ultimately our vote. We can't just show up at the polls and cast our vote for the name that is most recognized or the candidate who looks the best in their campaign advertisements.
If voters aren't going to do their due diligence, please just stay home.
Colin McCubbin