The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor

Antimicrobial peptides produced by epithelial cells and neutrophils represent essential elements of innate immunity, and include the defensin and cathelicidin family of antimicrobial polypeptides. The human cathelicidin cationic antimicrobial protein-18 is an antimicrobial peptide precursor predominantly expressed in neutrophils, and its active peptide LL-37 is released from the precursor through the action of neutrophil serine proteinases. LL-37 has been shown to display antimicrobial activity against a broad spectrum of microorganisms, to neutralize LPS bioactivity, and to chemoattract neutrophils, monocytes, mast cells, and T cells. In this study we show that LL-37 activates airway epithelial cells as demonstrated by activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and increased release of IL-8. Epithelial cell activation was inhibited by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, by blocking anti-EGFR and anti-EGFR-ligand Abs, and by the metalloproteinase inhibitor GM6001. These data suggest that LL-37 transactivates the EGFR via metalloproteinase-mediated cleavage of membrane-anchored EGFR-ligands. LL-37 may thus constitute one of the mediators by which neutrophils regulate epithelial cell activity in the lung.     

A standard tissue as a control for histochemical and immunohistochemical staining

The variable quality of histochemical and immunohistochemical staining of tissues may be attributed to pre-analytical and analytical variables. Both categories of variables frequently are undefined or inadequately controlled during specimen collection and preparation. Pre-analytical variables may alter the molecular composition of tissues, which results in variable staining; such variations may cause problems when different tissues are used as staining controls. We developed a standard tissue for use as a staining control. Our standard tissue contains five components: 1) nine combined human cell lines mixed with stroma from human spleen; 2) a squamous cancer cell line, A431; 3) fungus; 4) transverse sections of the mosquitofish and 5) normal human spleen. The first three components were embedded in HistoGel^? and all components were processed to paraffin and used to construct a single standard paraffin block. The muscles of mosquitofish and arteries of the spleen are positive controls for eosin staining, while other tissues are useful for assessing hematoxylin staining. The mosquitofish tissues also are excellent controls for the Masson trichrome stain and all mucin-related histochemical stains that we tested. The goblet cells of the intestine and skin stained strongly with Alcian blue, pH 2.5 (AB-2.5), mucicarmine, colloidal iron, periodic acid Schiff (PAS) or PAS-hematoxylin (PASH) and combination stains such as colloidal iron-PASH. Cell lines were not useful for evaluating histochemical stains except for PASH. The splenic stroma was a useful control for AB-2.5; however, eosin and mucin stains stained cell lines poorly, probably due to their rapid growth and associated loss of some differentiated characteristics such as production of mucins. Nevertheless, the cell lines were a critical control for immunohistochemical stains. Immunostaining of specific cell lines was consistent with the presence of markers, e.g., EGFr in DU145 cells. The cell lines expressed a wide range of markers, so they were useful controls for immunohistochemical staining including EGFr, HER2, E-cadherin, cytokeratins, Ki67, PCNA, estrogen receptor, progesterone receptor, CD3, CD20 and CD45, activated (cleaved) caspase 3 and Bcl-2. The cell lines also were a control for the TUNEL stain.     

Interspecific variation of body shape and sexual dimorphism in three coexisting species of the genus <Emphasis Type="Italic">Petrotilapia</Emphasis> (Teleostei: Cichlidae) from Lake Malawi

Differences in color patterns have been the most used feature in describing cichlid species belonging to genus Petrotilapia from Lake Malawi. In this study, we quantified morphological variation in body shape within and among three coexisting Petrotilapia species using landmark-based geometric morphometric methods. Statistic analyses revealed significant body shape differences among species but not between sexes. Post hoc multiple comparisons based on Mahalanobis distances revealed that P. nigra was significantly different from P. genalutea and Petrotilapia sp., whereas the latter two were not significantly different. The splines generated showed that the most pronounced variation was in the head region, in which P. nigra had a relatively longer and deeper head than the other two. The most clear-cut distinction was in gape length; P. genalutea had the longest gape, followed by Petrotilapia sp., whereas P. nigra had the shortest gape. Body depth was shallower in P. nigra than the others. When comparing sexes by their centroid size, ANOVA revealed that males were bigger than females. Therefore, we conclude that color is not the only feature that can distinguish these congeners. We discuss the observed sexual dimorphism in terms of sexual selection and relate morphological variation among species to feeding behavior, which may help explain their coexistence in nature.     

IL-4 and IL-13 exposure during mucociliary differentiation of bronchial epithelial cells increases antimicrobial activity and expression of antimicrobial peptides

The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity.PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity.IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.     

Trehalose Increases Freeze-Thaw Damage in Liposomes Containing Chloroplast Glycolipids

Chloroplast thylakoids contain three classes of glycolipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG). We have investigated the stability of large unilamellar vesicles made from egg phosphatidylcholine (EPC) and different chloroplast glycolipids during freezing to -18 degreesC, as a function of the presence of three sugars: glucose, sucrose, or trehalose. Contrary to the situation in thylakoids, where cryoprotection increases from glucose < sucrose < trehalose, liposomes containing 50% DGDG showed the opposite behavior. In fact, carboxyfluorescein leakage increased over the control values (freezing in the absence of sugar) in the presence of trehalose. This effect was not seen in vesicles made from pure EPC, or a mixture of EPC and MGDG, or EPC and SQDG. Liposomes made from mixtures of all three glycolipids, however, showed even more leakage in the presence of trehalose than liposomes containing only DGDG and EPC. Copyright 1998 Academic Press.     

Arterivirus and nairovirus ovarian tumor domain-containing Deubiquitinases target activated RIG-I to control innate immune signaling

The innate immune response constitutes the first line of defense against viral infection and is extensively regulated through ubiquitination. The removal of ubiquitin from innate immunity signaling factors by deubiquitinating enzymes (DUBs) therefore provides a potential opportunity for viruses to evade this host defense system. It was previously found that specific proteases encoded by the unrelated arteri- and nairoviruses resemble the ovarian tumor domain-containing (OTU) family of DUBs. In arteriviruses, this domain has been characterized before as a papain-like protease (PLP2) that is also involved in replicase polyprotein processing. In nairoviruses, the DUB resides in the polymerase protein but is not essential for RNA replication. Using both in vitro and cell-based assays, we now show that PLP2 DUB activity is conserved in all members of the arterivirus family and that both arteri- and nairovirus DUBs inhibit RIG-I-mediated innate immune signaling when overexpressed. The potential relevance of RIG-I-like receptor (RLR) signaling for the innate immune response against arterivirus infection is supported by our finding that in mouse embryonic fibroblasts, the production of beta interferon primarily depends on the recognition of arterivirus RNA by the pattern-recognition receptor MDA5. Interestingly, we also found that both arteri- and nairovirus DUBs inhibit RIG-I ubiquitination upon overexpression, suggesting that both MDA5 and RIG-I have a role in countering infection by arteriviruses. Taken together, our results support the hypothesis that arteri- and nairoviruses employ their deubiquitinating potential to inactivate cellular proteins involved in RLR-mediated innate immune signaling, as exemplified by the deubiquitination of RIG-I.     

Lumbar muscle electromyographic dynamic topography during flexion-extension

The objective of this study is to introduce dynamic topography of surface electromyography (SEMG) to visualize lumbar muscle myoelectric activity and provides a new view to analyze muscle activity in vivo. A total of 20 healthy male subjects and 15 males LBP were enrolled. An electrode-array was applied to the lumbar region to collect SEMG. The root mean square (RMS) value was calculated for each channel, and then a 160×120 matrix was constructed using a linear cubic spline interpolation of each scan to create a 2-D color topographic image. Along a definite interval of action, a series of RMS topography matrices was concatenated as a function of position and time, to form a dynamic topographical video of lumbar muscle activity. Relative area (RA), relative width (RW), relative height (RH) and Width-to-Height Ratio (W/H) were chosen as the four quantitative parameters in measuring topographic features. Normal RMS dynamic topography was found to have a consistent, symmetric pattern with a high intensity area in the paraspinal area. LBP patients had a different RMS dynamic topography, with an asymmetric, broad, or disorganized distribution. Quantitative SEMG features were found significantly different between normal control and LBP. After physiotherapy rehabilitation, the dynamic topography images of LBP tended towards the normal pattern.There are obvious differences in lumbar muscle coordination between healthy subjects and LBP patients. The dynamic topography allows the continuous visualization of the distribution of surface EMG signals and the coordination of muscular contractions.     

Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PL^pro) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PL^pro was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PL^pro domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PL^pro, we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PL^pro to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PL^pro DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PL^pro domain was found to suppress IFN-β promoter activation, PL^pro variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PL^pro, and not its proteolytic activity per se, in the inhibition of IFN-β promoter activity. The ability to decouple the DUB activity of PL^pro from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PL^pro as a viral DUB during MERS-CoV infection.     

Cytokinins in pathogenesis and disease resistance of Pyrenophora teres-barley and Dreschslera maydis-maize interactions during early stages of infection

Infection of Hordeum vulgare L. by Pyrenophora teresand of Zea mays by Dreschslera maydis were characterized by green island formation, higher cytokinin levels and accumulation of metabolites in the infected areas. Higher cytokinin concentrations of the order 6-Y,Y-dimethylallylaminopurine > zeatinriboside > zeatin >dihydrozeatinriboside were detected at infection sites of susceptible hosts. By virtue of these cytokinins, infection sites may be acting as metabolic sinks helping proliferation of the pathogen. Existence of translocatory sinks at infection zones was confirmed from autoradiographic studies,where, accumulation of labeled metabolites was prominent at infection sites of susceptible hosts. Upon infection the lower cytokinin levels of resistant hosts decreased further with progress of infection. In the infected resistant hosts the concentrations of zeatin/zeatinriboside were the maximum among the four identified cytokinins. The pathogen is also capable of secreting cytokinins as evident from quantification of cytokinins in culture filtrate extracts using HPLC. Since detached leaves were used in the experiments the increase/decrease of various cytokinin levels may be attributed to pathogen influence. The increase in cytokinin levels in the susceptible host may be aiding the growth of the pathogen on one hand, while the decrease in the infected resistant host may signal the host to activate defenses against a potential pathogen at the early stage of infection.This revised version was published online in October 2005 with corrections to the Cover Date.