A Modified Collagen Dressing Induces Transition of Inflammatory to Reparative Phenotype of Wound Macrophages

Collagen containing wound-care dressings are extensively used. However, the mechanism of action of these dressings remain unclear. Earlier studies utilizing a modified collagen gel (MCG) dressing demonstrated improved vascularization of ischemic wounds and better healing outcomes. Wound macrophages are pivotal in facilitating wound angiogenesis and timely healing. The current study was designed to investigate the effect of MCG on wound macrophage phenotype and function. MCG augmented recruitment of macrophage at the wound-site, attenuated pro-inflammatory and promoted anti-inflammatory macrophage polarization. Additionally, MCG increased anti-inflammatory IL-10, IL-4 and pro-angiogenic VEGF production, indicating a direct role of MCG in resolving wound inflammation and improving angiogenesis. At the wound-site, impairment in clearance of apoptotic cell bioburden enables chronic inflammation. Engulfment of apoptotic cells by macrophages (efferocytosis) resolves inflammation via a miR-21-PDCD4-IL-10 pathway. MCG-treated wound macrophages exhibited a significantly bolstered efferocytosis index. Such favorable outcome significantly induced miR-21 expression. MCG-mediated IL-10 production was dampened under conditions of miR-21 knockdown pointing towards miR-21 as a causative factor. Pharmacological inhibition of JNK attenuated IL-10 production by MCG, implicating miR-21-JNK pathway in MCG-mediated IL-10 production by macrophages. This work provides direct evidence demonstrating that a collagen-based wound-care dressing may influence wound macrophage function and therefore modify wound inflammation outcomes

Structural Characterization of Tick Cement Cones Collected From \u3ci\u3ein vivo\u3c/i\u3e and Artifical Membrane Blood-Fed Lone Star Ticks (\u3ci\u3eAmbylomma americanum\u3c/i\u3e)

The Lone Star tick, Amblyomma americanum, is endemic to the southeastern United States and capable of transmitting pathogenic diseases and causing non-pathogenic conditions. To remain firmly attached to the host, the tick secretes a proteinaceous matrix termed the cement cone which hardens around the tick’s mouthparts to assist in the attachment of the tick as well as to protect the mouthparts from the host immune system. Cement cones collected from ticks on a host are commonly contaminated with host skin and hair making analysis of the cone difficult. To reduce the contamination found in the cement cone, we have adapted an artificial membrane feeding system used to feed long mouthpart ticks. Cones collected from in vivo and membrane fed ticks are analyzed to determine changes in the cone morphology. Comparisons of the cement cones using light microscopy shows similar structures and color however using scanning electron microscopy the cones have drastically different structures. The in vivo cones contain fibrils, sheets, and are heavily textured whereas cones from membrane fed ticks are remarkably smooth with no distinct structures. Analysis of the secondary protein structures using FTIR-ATR show both in vivo and membrane fed cement cones contain β sheets but only in vivo cement cones contain helical protein structures. Additionally, proteomic analysis using LC–MS/MS identifies many proteins including glycine rich proteins, metalloproteases, and protease inhibitors. Proteomic analysis of the cones identified both secreted and non-secreted tick proteins. Artificial membrane feeding is a suitable model for increased collection of cement cones for proteomic analysis however, structurally there are significant differences

Chromogranin A regulates gut permeability via the antagonistic actions of its proteolytic peptides

AIM: A 'leaky' gut barrier has been implicated in the initiation and progression of a multitude of diseases, e.g., inflammatory bowel disease (IBD), irritable bowel syndrome, and celiac disease. Here we show how pro-hormone Chromogranin A (CgA), produced by the enteroendocrine cells, and Catestatin (CST: hCgA352-372 ), the most abundant CgA-derived proteolytic peptide, affect the gut barrier. METHODS: Colon tissues from region-specific CST-knockout (CST-KO) mice, CgA-knockout (CgA-KO) and WT mice were analyzed by immunohistochemistry, Western blot, ultrastructural and flowcytometry studies. FITC-dextran assays were used to measure intestinal barrier function. Mice were supplemented with CST or CgA fragment pancreastatin (PST: CgA250-301 ). The microbial composition of cecum was determined. CgA and CST levels were measured in blood of IBD patients. RESULTS: Plasma levels of CST were elevated in IBD patients. CST-KO mice displayed (i) elongated tight, adherens junctions and desmosomes similar to IBD patients, (ii) elevated expression of Claudin 2, and (iii) gut inflammation. Plasma FITC-dextran measurements showed increased intestinal paracellular permeability in the CST-knockout mice. This correlated with a higher ratio of Firmicutes to Bacteroidetes, a dysbiotic pattern commonly encountered in various diseases. Supplementation of CST-knockout mice with recombinant CST restored paracellular permeability and reversed inflammation, whereas CgA-knockout mice supplementation with CST and/or PST in CgA-KO mice showed that intestinal paracellular permeability is regulated by the antagonistic roles of these two peptides: CST reduces and PST increases permeability. CONCLUSION: The pro-hormone CgA regulates the intestinal paracellular permeability. CST is both necessary and sufficient to reduce permeability and primarily acts by antagonizing PST

Staphylococcus aureus Biofilm Infection Compromises Wound Healing by Causing Deficiencies in Granulation Tissue Collagen

Objective: The objective of this work was to causatively link biofilm properties of bacterial infection to specific pathogenic mechanisms in wound healing. Background: Staphylococcus aureus is one of the four most prevalent bacterial species identified in chronic wounds. Causatively linking wound pathology to biofilm properties of bacterial infection is challenging. Thus, isogenic mutant stains of S. aureus with varying degree of biofilm formation ability was studied in an established preclinical porcine model of wound biofilm infection. Methods: Isogenic mutant strains of S. aureus with varying degree (ΔrexB > USA300 > ΔsarA) of biofilm-forming ability were used to infect full-thickness porcine cutaneous wounds. Results: Compared with that of ΔsarA infection, wound biofilm burden was significantly higher in response to ΔrexB or USA300 infection. Biofilm infection caused degradation of cutaneous collagen, specifically collagen 1 (Col1), with ΔrexB being most pathogenic in that regard. Biofilm infection of the wound repressed wound-edge miR-143 causing upregulation of its downstream target gene matrix metalloproteinase-2. Pathogenic rise of collagenolytic matrix metalloproteinase-2 in biofilm-infected wound-edge tissue sharply decreased collagen 1/collagen 3 ratio compromising the biomechanical properties of the repaired skin. Tensile strength of the biofilm infected skin was compromised supporting the notion that healed wounds with a history of biofilm infection are likely to recur. Conclusion: This study provides maiden evidence that chronic S. aureus biofilm infection in wounds results in impaired granulation tissue collagen leading to compromised wound tissue biomechanics. Clinically, such compromise in tissue repair is likely to increase wound recidivism

Bound to unbound state route and propagation of dark solitons showing acoustical memory

The dark and bright solitons in different systems are already known. If the intrinsic field is only considered, then the modal dynamics for small oscillations could be characterized by the bound state in a limited range of frequency, revealed via associated Legendre polynomial. The pairing and interplay between the dark and bright solitons occur. The disappearance of the bound state after a critical frequency gives rise to dark solitons in the unbound states that propagate through the domains. Above the upper boundary of the bound states, the estimated frequencies of dark solitons match with those experimentally found for ‘acoustical memory’

Removal of Non-Specifically Bound Proteins Using Rayleigh Waves Generated on ST-Quartz Substrates

Label-free biosensors are plagued by the issue of non-specific protein binding which negatively affects sensing parameters such as sensitivity, selectivity, and limit-of-detection. In the current work, we explore the possibility of using the Rayleigh waves in ST-Quartz devices to efficiently remove non-specifically bound proteins via acoustic streaming. A coupled-field finite element (FE) fluid structure interaction (FSI) model of a surface acoustic wave (SAW) device based on ST-Quartz substrate in contact with a liquid loading was first used to predict trends in forces related to SAW-induced acoustic streaming. Based on model predictions, it is found that the computed SAW body force is sufficient to overcome adhesive forces between particles and a surface while lift and drag forces prevent reattachment for a range of SAW frequencies. We further performed experiments to validate the model predictions and observe that the excitation of Rayleigh SAWs removed non-specifically bound (NSB) antigens and antibodies from sensing and non-sensing regions, while rinsing and blocking agents were ineffective. An amplified RF signal applied to the device input disrupted the specific interactions between antigens and their capture antibody as well. ST-quartz allows propagation of Rayleigh and leaky SH-SAW waves in orthogonal directions. Thus, the results reported here could allow integration of three important biosensor functions on a single chip, i.e., removal of non-specific binding, mixing, and sensing in the liquid phase

Strain-Specific Propagation by an Amyloid-Beta Dodecamer

Soluble oligomers of the amyloid-β (Aβ) peptide have emerged as the primary neurotoxic agents in Alzheimer disease (AD). Increasing evidence implicates aggregates of Aβ to propagate towards fibrils in a strain-specific manner. Morphological differences observed within fibrils are known to arise due to micro-structural differences among the smaller aggregates that form the fibril building blocks. Furthermore, polymorphic fibrils correlate with the clinically observed phenotypes in AD pathology, cementing the idea that conformational strains of oligomers may have significance in phenotypic outcomes. Given the existing diversity among Aβ oligomers, it becomes imperative to determine the ability of various oligomeric strains to faithfully propagate their structure. However, dearth in a molecular understanding of Aβ oligomers has confounded the insights into such a mechanism. We have previously established that a distinct dodecamer of Aβ42, called large fatty acid-derived oligomers (LFAOs), are formed as an off-pathway species under interfacial conditions. Here we demonstrate that at high concentrations, LFAOs seed Aβ towards fibrils containing distinct morphological features different from those generated in the absence of the seed. More importantly, the fibril structure seems to contain units of parent LFAO seeds, indicating a biophysically faithful propagation of the seed. The observations reported here may have profound significance in deciphering the emerging roles of Aβ oligomer phenotypes in prion-like propagation and dissemination of toxicity in AD