Retinal microvasculature and vasoreactivity changes in hypertension using optical coherence tomography-angiography

Graefe's Archive for Clinical and Experimental Ophthalmology - To evaluate the retinal vasculature and vasoreactivity of patients with hypertension (HTN) using spectral domain optical coherence...     

Structure-activity studies and therapeutic potential of host defense peptides of human thrombin

Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length- and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (>12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest.     

Morphometric computer‐assisted image analysis of oral epithelial cells in normal epithelium and leukoplakia

J Oral Pathol Med (2010) 39 149–154 Background: There are very few studies documenting morphometric parameters of normal oral mucosa and leukoplakia. The present study was undertaken to establish the morphometric parameters of the parabasal and spinous cells of normal oral epithelium. Analysis of changes occurring in these cells in leukoplakia was also done. Methods: This study was conducted on tissue sections of clinically normal oral mucosa and leukoplakia. Morphometric analysis was done for parabasal and spinous cells. Statistical analysis was done using one way ANOVA and Mann–Whitney test. Results: Morphometric parameters were greater in the spinous cells than in parabasal cells in normal oral mucosa. Leukoplakia showed greater cellular and nuclear parameters than normal mucosa. Conclusion: Normal oral epithelium showed site‐wise difference in cell and nuclear measurements. Nuclear parameters showed a statistically significant change than cellular parameters in dysplasia. These changes were expressed in the earliest stage of transformation to dysplasia.     

Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown that P. aeruginosa secretes outer-membrane vesicles that deliver a small RNA to human airway epithelial cells (AECs), in which it suppresses the innate immune response. Here, we demonstrate that interdomain communication through small RNA–containing membrane vesicles is bidirectional and that microRNAs (miRNAs) in extracellular vesicles (EVs) secreted by human AECs regulate protein expression, antibiotic sensitivity, and biofilm formation by P. aeruginosa. Specifically, human EVs deliver miRNA let-7b-5p to P. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1-3, and increases the ability of beta-lactam antibiotics to reduce biofilm formation by targeting the beta-lactamase AmpC. Let-7b-5p is bioinformatically predicted to target not only PpkA, ClpV1, and AmpC in P. aeruginosa but also the corresponding orthologs in Burkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let-7b-5p to reduce biofilm formation and increase beta-lactam sensitivity is not limited to P. aeruginosa. Here, we provide direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryote, resulting in subsequent phenotypic alterations in the prokaryote as a result of this interdomain communication. Since let-7–family miRNAs are in clinical trials to reduce inflammation and because chronic P. aeruginosa lung infections are associated with a hyperinflammatory state, treatment with let-7b-5p and a beta-lactam antibiotic in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are the leading cause of nosocomial infections worldwide primarily due to their multidrug resistance (1). P. aeruginosa contributes to 5 to 10% of the acute exacerbations in chronic obstructive pulmonary disease (COPD), which afflicts 10% of the world’s population and soon will be the third leading cause of death in the world (2–5). P. aeruginosa also chronically colonizes the lungs of ∼60% of adults with cystic fibrosis (CF), and its presence is strongly associated with reduced forced expiratory volume and a progressive loss of lung function (6–10). In addition, P. aeruginosa ventilator-associated pneumonia mortality rates can be as high as 30% (11). During chronic infection, P. aeruginosa establishes antibiotic-resistant biofilms, which are notoriously difficult to treat and are associated with serious adverse medical outcomes (12–14). Therefore, new strategies are needed to control recalcitrant chronic infections by P. aeruginosa and to prevent the formation of antibiotic-resistant biofilms.Intercellular communication in the lungs is essential to maintain homeostasis and to raise an appropriate immune response to pathogens (15–18). In addition to cytokines and chemokines secreted by the myriad cell types in the lungs, eukaryotic cells also communicate by secreting extracellular vesicles (EVs) that contain proteins, lipid mediators, and microRNAs (miRNAs), which are 21- to 25-nucleotide–long noncoding RNAs that regulate gene expression (15, 16, 19, 20). EVs containing miRNAs have been identified in numerous biological samples including blood, urine, exhaled-breath condensates, and bronchoalveolar-lavage fluid (16, 18, 19, 21–23). When EVs fuse with target cells, they deliver their contents, including miRNAs that alter target-cell gene expression and function (24–28).Cross-kingdom RNA interference (RNAi) mediated by EVs can play a crucial role in host–pathogen communication between plants and fungi (29–31). A few recent studies show that eukaryotic miRNAs affect prokaryotic organisms living in the gut: 1) Transfection of eukaryotic miRNAs into Fusobacterium nucleatum and Escherichia coli affects messenger RNA (mRNA) abundance and bacterial growth (32); 2) miR-30d given orally to mice increases the abundance of the commensal microbe Akkermansia muciniphila (33); and 3) Ginger-derived miRNAs are taken up by gut microbes, altering the microbiome (34). Although these studies reveal that eukaryotic miRNAs affect bacteria, to our knowledge, there are no published studies demonstrating that eukaryotic EVs deliver miRNAs to bacteria or elucidating the mechanism of action of eukaryotic miRNAs in bacteria. Recently, we demonstrated that P. aeruginosa secretes outer-membrane vesicles that diffuse through mucus and fuse with human airway epithelial cells (AECs), delivering short interfering RNAs (siRNAs) that down-regulate the host immune response without direct contact between the bacterium and the host cells (35). Since siRNAs secreted by P. aeruginosa target eukaryotic genes, we hypothesized that eukaryotic miRNAs can also target bacterial genes to alter gene expression and phenotype. Motivated by the clinical challenge presented by antibiotic-resistant P. aeruginosa biofilms, we designed experiments to test whether EVs secreted by human AECs affect biofilm formation and also potentiate the ability of antibiotics to reduce these biofilms.     

Thymic stromal lymphopoietin exerts antimicrobial activities

Thymic stromal lymphopoietin (TSLP) is an interleukin-7-like cytokine expressed by epithelial cells and reported to be involved in allergic diseases and atopic eczema. The presence of several predicted α-helical regions in TSPL, a structure characterizing many classical antimicrobial peptides (AMPs), prompted us to investigate whether TSLP exerts antimicrobial activities. Recombinant human TSLP exerted antimicrobial activity, particularly against Gram-negative bacteria. Using synthetic overlapping peptide 20-mers of TSLP, it was demonstrated that the antimicrobial effect is primarily mediated by the C-terminal region of the protein. MKK34 (MKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLK), a peptide spanning a C-terminal α-helical region in TSLP, showed potent antimicrobial activities, in physiological salt conditions and in the presence of human plasma. Fluorescent studies of peptide-treated bacteria, electron microscopy and liposome leakage models showed that MKK34 exerted membrane-disrupting effects comparable to those of the classical AMP LL-37. Moreover, TSLP was degraded into multiple fragments by staphylococcal V8 proteinase. One major antimicrobial degradation fragment was found to encompass the C-terminal antimicrobial region defined by the MKK34 peptide. We here describe a novel antimicrobial role for TSLP. The antimicrobial activity is primarily mediated by the C-terminal part of the protein. In combination with the previously known cytokine function of TSLP, our result indicates dual functions of the molecule and a previously unknown role in host defense.