scholarly journals The US3 Protein of Pseudorabies Virus Drives Viral Passage across the Basement Membrane in Porcine Respiratory Mucosa Explants

2016 ◽  
Vol 90 (23) ◽  
pp. 10945-10950 ◽  
Author(s):  
Jochen A. S. Lamote ◽  
Sarah Glorieux ◽  
Hans J. Nauwynck ◽  
Herman W. Favoreel
Keyword(s):  
Epithelial Cells ◽  
Basement Membrane ◽  
Pseudorabies Virus ◽  
Rho Gtpase ◽  
Ex Vivo ◽  
Respiratory Mucosa ◽  
First Report ◽  
Rhoa Signaling ◽  
Porcine Respiratory ◽  
Cell Spread

ABSTRACT Passage of the basement membrane (BM), which forms a barrier between the epithelium and the underlying lamina propria, represents an important step in the early pathogenesis of different alphaherpesviruses. Rho GTPase signaling plays an important role in transmigration of cells across the BM during physiological and pathological processes. We reported earlier that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) interferes with Rho GTPase signaling and causes a reorganization of the host cell cytoskeleton, which as a consequence, enhances viral cell-to-cell spread in epithelial cell cultures. Here, using an ex vivo system of porcine nasal respiratory mucosa explants that allows to study PRV invasion through the BM, we found that a PRV strain that lacks US3 expression (ΔUS3 PRV) showed a reduced spread in mucosal epithelium and was virtually unable to breach the BM, in contrast to isogenic wild-type (WT) or US3 rescue PRV strains. Interestingly, addition of IPA3, an inhibitor of p21-activated kinases that blocks the effects of US3 on the cytoskeleton, suppressed the ability of WT PRV to spread across the BM. In addition, artificial suppression of RhoA signaling using CPC3 (cell-permeable C3 transferase) to mimic the effects of US3 on Rho GTPase signaling, significantly increased passage of ΔUS3 PRV through the BM, whereas it did not significantly affect BM passage of WT or US3 rescue PRV. In conclusion, these data indicate that US3 plays an important role in PRV mucosal invasion across the BM, which involves its interference with Rho GTPase signaling. This is the first report describing an alphaherpesvirus protein that drives viral BM passage. IMPORTANCE Many viruses, including alphaherpesviruses, primarily replicate in epithelial cells of surface mucosae, such as the respiratory mucosa. Some of these viruses breach the basement membrane underlying these epithelial cells to reach underlying connective tissue and blood vessels and invade the host. Hence, epithelial spread and basement membrane passage represent crucial but still poorly understood early steps in (alphaherpes)virus pathogenesis. Here, using ex vivo porcine respiratory mucosa explants, we show that the conserved US3 protein of the porcine alphaherpesvirus pseudorabies virus (PRV) is critical for passage of PRV across the basement membrane and contributes to efficient viral epithelial spread. In addition, we show that US3-mediated viral epithelial spread and passage across the basement membrane depend at least in part on the ability of this viral protein to modulate cellular Rho GTPase signaling. This is the first report that identifies an alphaherpesvirus protein that drives viral basement membrane passage.

scholarly journals P2Y2nucleotide receptor activation enhances the aggregation and self-organization of dispersed salivary epithelial cells

2014 ◽  
Vol 307 (1) ◽  
pp. C83-C96 ◽  
Author(s):  
Farid G. El-Sayed ◽  
Jean M. Camden ◽  
Lucas T. Woods ◽  
Mahmoud G. Khalafalla ◽  
Michael J. Petris ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Epithelial Cells ◽  
Rho Gtpase ◽  
Ex Vivo ◽  
Growth Factor Receptor ◽  
Cell Aggregation ◽  
Receptor Activation ◽  
Self Organization ◽  
Salivary Epithelial Cells

Hyposalivation resulting from salivary gland dysfunction leads to poor oral health and greatly reduces the quality of life of patients. Current treatments for hyposalivation are limited. However, regenerative medicine to replace dysfunctional salivary glands represents a revolutionary approach. The ability of dispersed salivary epithelial cells or salivary gland-derived progenitor cells to self-organize into acinar-like spheres or branching structures that mimic the native tissue holds promise for cell-based reconstitution of a functional salivary gland. However, the mechanisms involved in salivary epithelial cell aggregation and tissue reconstitution are not fully understood. This study investigated the role of the P2Y2nucleotide receptor (P2Y2R), a G protein-coupled receptor that is upregulated following salivary gland damage and disease, in salivary gland reconstitution. In vitro results with the rat parotid acinar Par-C10 cell line indicate that P2Y2R activation with the selective agonist UTP enhances the self-organization of dispersed salivary epithelial cells into acinar-like spheres. Other results indicate that the P2Y2R-mediated response is dependent on epidermal growth factor receptor activation via the metalloproteases ADAM10/ADAM17 or the α5β1integrin/Cdc42 signaling pathway, which leads to activation of the MAPKs JNK and ERK1/2. Ex vivo data using primary submandibular gland cells from wild-type and P2Y2R−/−mice confirmed that UTP-induced migratory responses required for acinar cell self-organization are mediated by the P2Y2R. Overall, this study suggests that the P2Y2R is a promising target for salivary gland reconstitution and identifies the involvement of two novel components of the P2Y2R signaling cascade in salivary epithelial cells, the α5β1integrin and the Rho GTPase Cdc42.

scholarly journals A trypsin-like serine protease is involved in pseudorabies virus invasion through the basement membrane barrier of porcine nasal respiratory mucosa

2011 ◽  
Vol 42 (1) ◽  
pp. 58 ◽  
Author(s):  
Sarah Glorieux ◽  
Herman W Favoreel ◽  
Lennert Steukers ◽  
Annelies P Vandekerckhove ◽  
Hans J Nauwynck
Keyword(s):  
Basement Membrane ◽  
Serine Protease ◽  
Pseudorabies Virus ◽  
Respiratory Mucosa ◽  
Membrane Barrier

scholarly journals Thymic Cystic Degeneration, Pseudoepitheliomatous Hyperplasia, and Hemorrhage in a Dog with Brodifacoum Toxicosis

2009 ◽  
Vol 46 (3) ◽  
pp. 449-452 ◽  
Author(s):  
B. H. Rickman ◽  
N. Gurfield
Keyword(s):  
Adipose Tissue ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Cystic Degeneration ◽  
First Report ◽  
Pseudoepitheliomatous Hyperplasia ◽  
Corticomedullary Differentiation ◽  
Thymic Cysts ◽  
Nonhuman Species

Thymic cysts with pseudoepitheliomatous hyperplasia are described in a 7-month-old female American Eskimo Dog that died of complications from brodifacoum poisoning. Grossly, there was hemothorax with marked cranial mediastinal hemorrhage. Histologically, thymic lobules were expanded and distorted by irregular cysts, lined by single to multiple layers of plump to slightly attenuated polygonal squamous epithelial cells supported by a basement membrane (pseudoepitheliomatous hyperplasia). The thymus had a paucity of lymphocytes and lacked corticomedullary differentiation. Extensive hemorrhage within the cysts and thymic parenchyma extended into the adjacent adipose tissue. To the authors' knowledge, this is the first report of cystic thymic degeneration with pseudoepitheliomatous hyperplasia in a nonhuman species.

scholarly journals Lung macrophages drive mucus production and steroid-resistant inflammation in chronic bronchitis

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Kristina Andelid ◽  
Karolina Öst ◽  
Anders Andersson ◽  
Esha Mohamed ◽  
Zala Jevnikar ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Chronic Bronchitis ◽  
Cross Talk ◽  
Alveolar Macrophages ◽  
Ex Vivo ◽  
Bronchial Epithelial Cells ◽  
Steroid Resistance ◽  
Bronchial Epithelial ◽  
Steroid Resistant ◽  
Copd Patients

Abstract Background Patients with chronic obstructive pulmonary disease (COPD) frequently suffer from chronic bronchitis (CB) and display steroid-resistant inflammation with increased sputum neutrophils and macrophages. Recently, a causal link between mucus hyper-concentration and disease progression of CB has been suggested. Methods In this study, we have evaluated the steroid sensitivity of purified, patient-derived sputum and alveolar macrophages and used a novel mechanistic cross-talk assay to examine how macrophages and bronchial epithelial cells cross-talk to regulate MUC5B production. Results We demonstrate that sputum plug macrophages isolated from COPD patients with chronic bronchitis (COPD/CB) are chronically activated and only partially respond to ex vivo corticosteroid treatment compared to alveolar macrophages isolated from lung resections. Further, we show that pseudo-stratified bronchial epithelial cells grown in air–liquid-interface are inert to direct bacterial lipopolysaccharide stimulation and that macrophages are able to relay this signal and activate the CREB/AP-1 transcription factor complex and subsequent MUC5B expression in epithelial cells through a soluble mediator. Using recombinant protein and neutralizing antibodies, we identified a key role for TNFα in this cross-talk. Conclusions For the first time, we describe ex vivo pharmacology in purified human sputum macrophages isolated from chronic bronchitis COPD patients and identify a possible basis for the steroid resistance frequently seen in this population. Our data pinpoint a critical role for chronically activated sputum macrophages in perpetuating TNFα-dependent signals driving mucus hyper-production. Targeting the chronically activated mucus plug macrophage phenotype and interfering with aberrant macrophage-epithelial cross-talk may provide a novel strategy to resolve chronic inflammatory lung disease.

scholarly journals Die Kaliumhydrogenphosphat-induzierte Nephropathie des Hundes. I. Pathogenese der Tubulusatrophiee [Potassium Hydrogen Phosphate Induced Nephropathy in the Dog. I. Pathogenesis of Tubular Atrophy—author's trans.]

1980 ◽  
Vol 17 (6) ◽  
pp. 699-719 ◽  
Author(s):  
P. Schneider ◽  
G. Pappritz ◽  
R. Müller-Peddinghaus ◽  
M. Bauer ◽  
H. Lehmann ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Basement Membranes ◽  
Tubular Epithelial Cells ◽  
Beagle Dogs ◽  
Lysosomal Degradation ◽  
Tubular Basement Membrane ◽  
Tubular Atrophy ◽  
Study Results

A nephropathy with severe tubular atrophy was observed in Beagle dogs after oral administration of K2HPO4 for 14 or 38 weeks. We describe the complete lysosomal degradation of atrophying tubular epithelial cells. During two experiments of 14 and 38 weeks duration, respectively, a total of 15 Beagle dogs received 0.8 g K2HPO4/kg body weight daily with their food. All dogs were examined clinically at regular intervals. Renal biopsies were taken in the fourth week from beagles of the 14-week study. Results were compared with those of control dogs. At the end of the experiments the animals were killed and necropsies done. Different stains and histochemical reactions were applied to paraffin sections of the kidneys. Acid phosphatase and β-glucuronidase were found on cryostat sections. Kidneys fixed by perfusion of five Beagles from the 38-week study and three Beagles of the 14-week study, and from five control dogs, were examined electron microscopically. Ultrahistochemically, acid phosphatase was demonstrated. Clinically, the dogs in both experiments vomited, were cachectic, and had elevated creatinine and blood urea nitrogen. Morphologically, qualitatively identical changes were seen, but the renal damage was most marked at 38 weeks. There were disseminated tubular atrophy (usually of the proximal tubules), focal scar tissue and nephrocalcinosis. The following pathogenesis was established for the lesions of the proximal tubule: Tubular atrophy begins with loss of differentiation of epithelial cells. Enzyme histochemistry, ultrahistochemistry and electron microscopy show an increase in autophagic vacuoles and autophagolysosomes. The lysosomal bodies showing fusion enclose large parts of the cytoplasm as the process continues. Complete lysosomal degradation of epithelial cells and extrusion of large lysosomes into the tubular lumen follow. After complete enzymatic digestion of the intratubular detritus, the residue is empty, convoluted and collapsed tubular basement membrane. Atrophic tubular epithelial cells have many organelle-free zones at their base, which contain fine filamentous material resembling that of the basement membrane. The degradation processes described here may explain why clinically the urinary sediment contains few cylinders and epithelial cells and why proteinuria decreases significantly toward the end of the experiment. So far, it is not clear whether the tubular basement membrane is synthesized by the tubular cells, by fibroblasts or by both cell types. The presence of basement membrane-like material in tubular epithelial cells and in parietal epithelial cells of the glomerulus favors the view that epithelial cells produce the basement membranes and that increased production of basement membrane-like material is a sign of loss of differentiation.

scholarly journals Eviscerated Corneas as Tissue Source for Ex Vivo Expansion of Limbal Epithelial Cells on Platelet-Rich Plasma Gels

2016 ◽  
Vol 41 (12) ◽  
pp. 1543-1547 ◽  
Author(s):  
Leonard G. Heydenrych ◽  
Donald F. du Toit ◽  
Colleen M. Aldous
Keyword(s):  
Epithelial Cells ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Ex Vivo Expansion ◽  
Tissue Source

Abstract 5369: Mesenchymal Stem Cells Overexpressing CXCR4 (CXCR4 + -MSCs) Attenuate Remodeling of Post-Myocardial Infarction by Releasing Anti-Fibrotic Enzymes

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Tao Wang ◽  
Yigang Wang ◽  
Dongsheng Zhang ◽  
Tiemin Zhao ◽  
Atif Ashraf ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Ventricular Remodeling ◽  
Interstitial Fibrosis ◽  
Ex Vivo ◽  
Genetically Engineered ◽  
Control Group ◽  
Hypoxic Conditions ◽  
Antibody Staining ◽  
Adenoviral Transduction

We hypothesize that CXCR4 + -MSCs penetrate and proliferate in infracted heart by releasing collagen degrading enzymes. We genetically engineered male mouse MSCs using ex vivo adenoviral transduction for over-expression of CXCR4/GFP or GFP alone. MSCs (G-I) or CXCR4 + -MSCs (G-II) or CXCR4 + -MSCs treated with epigallocarechin gallate (EGCG, 50μg/ml), a MT1-matrix metalloproteinases (MMPs) inhibitor (G-III) or CXCR4 + -MSCs with AMD3100 (5 μg/mL), a CXCR4-selective antagonist (G-IV). A Trans-Matrigel Chemoinvasion Assay was used to evaluate the ability of MSCs to cross the basement membrane. MMPs were analyzed by Western blot and MMP antibody staining. Sex mismatched MSCs were infused into female mice via a tail vein injection 3 days after MI. Mice in G-III were treated with EGCG (100 mg/kg, oral gavage, daily for 2 weeks) to inhibit MMPs and G-IV was treated with AMD3100 (1 mg/kg, i.p. given continually for 6 days after MI). LV fibrosis was detected by Picrosirius red staining. Echocardiography was performed at 4 weeks after MI and hearts were harvested for histological analysis. In vitro, cell migration was significantly higher in G-II in the presence of SDF-1α as compared with other groups, ( p <0.01). EGCG or AMD3100 markedly prevented this response. MMP-9 and MT1-MMP were upregulated significantly only in G-II (p<0.01) exposed to hypoxia. Infiltration of GFP and Y chromosome positive cells in the peri- or infarct area was increased significantly in G-II. CXCR4 + -MSCs penetrated more effectively into the infarcted region and survived in the ischemic environment as compared to control group. These effects were reduced with EGCG or AMD3100. The ventricular remodeling and interstitial fibrosis were also reduced in G-II but not in other groups. G-II also had less LV dilation (diastolic dimension 4.9±0.2 vs. 6.2±0.3 mm, p<0.05), EF (62±3 vs. 44±4%, p<0.05). Infarct size (31±3.8 vs 43±4.7% of LV, p<0.05) and collagen area fraction (16±2 vs. 28±4 %, p<0.05) were significantly reduced in G-2 compared to G-I. Under hypoxic conditions MMPs were upregulated in CXCR4 + -MSCs which crossed the basement membrane by releasing enzymes leading to breakdown or reduction of scar formation thus facilitating cell homing and proliferation.

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