Browsing by Author "Das Ghatak, Piya"
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Item Disposable Patterned Electroceutical Dressing (PED-10) Is Safe for Treatment of Open Clinical Chronic Wounds(Mary Ann Liebert, 2019-04-01) Roy, Sashwati; Prakash, Shaurya; Mathew-Steiner, Shomita S.; Das Ghatak, Piya; Lochab, Varun; Jones, Travis H.; Mohana Sundaram, Prashanth; Gordillo, Gayle M.; Subramaniam, Vish V.; Sen, Chandan K.; Surgery, School of MedicineObjective: To evaluate if patterned electroceutical dressing (PED) is safe for human chronic wounds treatment as reported by wound care providers. Approach: This work reports a pilot feasibility study with the primary objective to determine physically observable effects of PED application on host tissue response from a safety evaluation point of view. For this pilot study, patients receiving a lower extremity amputation with at least one open wound on the part to be amputated were enrolled. Patients were identified through the Ohio State University Wexner Medical Center (OSUWMC) based on inclusion and exclusion criteria through prescreening through the Comprehensive Wound Center's (CWC) Limb Preservation Program and wound physicians and/or providers at OSUWMC. Wounds were treated with the PED before amputation surgery. Results: The intent of the study was to identify if PED was safe for clinical application based on visual observations of adverse or lack of adverse events on skin and wound tissue. The pilot testing performed on a small cohort (N = 8) of patients showed that with engineered voltage regulation of current flow to the open wound, the PED can be used with little to no visually observable adverse effects on chronic human skin wounds. Innovation: The PED was developed as a second-generation tunable electroceutical wound care dressing, which could potentially be used to treat wounds with deeper infections compared with current state of the art that treats wounds with treatment zone limited to the surface near topical application. Conclusion: Technology advances in design and fabrication of electroceutical dressings were leveraged to develop a tunable laboratory prototype that could be used as a disposable low-cost electroceutical wound care dressing on chronic wounds. Design revisions of PED-1 (1 kΩ ballast resistor) circumvented previously observed adverse effects on the skin in the vicinity of an open wound. PED-10 (including a 10 kΩ ballast resistor) was well tolerated in the small cohort of patients (N = 8) on whom it was tested, and the observations reported here warrant a larger study to determine the clinical impact on human wound healing and infection control.Item Identification of a Biofilm-Inducible Wound Macrophage Subset Characterized by Unique MARCO Expression: Significance in Chronic Wound Infection(2023-08) Das Ghatak, Piya; Roy, Sashwati; Gordillo, Gayle M.; Khanna, Savita; Xuan, YiStaphylococcus aureus (SA) biofilm infection, a common occurrence in human chronic wounds, exhibits sustained inflammation resulting in wound chronicity. Current studies claim that SA biofilm transforms host macrophages (m) towards an alternatively activated phenotype that suppresses the inflammatory response. Given a key role of m in wound healing, we investigate the phenotype and function of wound m (wm) in SA biofilm infection. To address the biofilm component of SA pathogenicity, two isogenic mutant strains of wild-type SA (USA300) were used that possess varying degrees of biofilm-forming ability. The strains, USA300::sarA (ΔsarA) and USA300::rexB (ΔrexB), have hypo- and hyper biofilm-forming ability, respectively. Mechanistic studies were performed on peripheral blood monocytes subjected to conditioned media (CM) derived from ΔsarA and ΔrexB biofilm cultures. scRNAseq and bulk RNAseq studies revealed presence of a unique m subtype that displays a distinct gene expression signature when exposed to CM derived from ΔrexB mutant as compared to CM from ΔsarA mutant. We identified macrophage receptors with collagenous structure (MARCO) as one of the highly abundant uniquely expressed biofilm-responsive genes in wm. MARCO, a member of the scavenger receptor, belongs to a larger group of pattern recognition receptors primarily involved in immunosurveillance and plays important roles in host defense and apoptotic cell clearance activities. scRNAseq and immunohistochemistry from human chronic biofilm-infected wounds demonstrated abundance of MARCO expression on wm. Significant upregulation of MARCO was observed in ΔrexB CM treated m in a dose and time-dependent manner as compared to ΔsarA CM treated m. Knockdown of biofilm-induced MARCO in m resulted in dysregulated m functions and inflammatory responses, suggesting its critical significance in biofilm infection. Immunohistochemistry studies in SA biofilm-infected porcine wounds validated the in vitro findings. In summary, this study identifies a SA biofilm inducible m receptor that has a significant role in wound chronicity.Item Inhibition of TFEB activation promotes Coxiella burnetii growth(2021-05) Das Ghatak, Piya; Gilk, Stacey D.; Bauer, Margaret E.; Robinson, Christopher M.Coxiella burnetii is the etiologic agent of Q fever, a zoonotic disease characterized by flu-like sickness in acute cases; endocarditis may occur and turn deadly if not treated correctly in chronic patients. Coxiella, an obligate intracellular bacterium, requires establishment of a replicative niche in the host cell. After being phagocytosed by the eukaryotic cell, the bacterium resides in a tight-fitting nascent phagosome which matures through the host canonical endocytic pathway, acquiring endosomal/lysosomal markers as well as acidic pH. Initial acidification of the Coxiella containing vacuole (CCV) is central to the bacterium’s pathogenesis because translocation of bacterial effector proteins into the host cell by the type 4B secretion system (T4BSS) initiates only after it senses the acidic environment. The effector proteins are required for subverting different host cell functions in favor of Coxiella growth, CCV maturation and are crucial for bacterial virulence. Contrary to the belief that since CCV matures through the host endocytic pathway, CCV is as acidic as lysosome, we found that CCV is significantly less acidic (pH~5.2) than lysosomes (pH~4.8) and inducing further CCV acidification causes Coxiella lysis. Furthermore, increasing lysosomal biogenesis in the host cell is detrimental for Coxiella growth. So, we hypothesized that Coxiella blocks lysosomal biogenesis in host cells to maintain the CCV pH just optimal for its growth. Lysosomal biogenesis is regulated by the master transcription factor EB (TFEB). Its ability to act as a transcription factor depends on its subcellular localization, which relies on its phosphorylation state. TFEB, when phosphorylated is cytosolic and inactive, whereas dephosphorylated TFEB translocates to the nucleus and is active, binding to promoter regions of lysosomal genes of the CLEAR network, thus controlling lysosome biogenesis. Therefore, we hypothesized that Coxiella blocks TFEB translocation to the nucleus, thus inhibiting lysosome biogenesis. We determined that Coxiella grows significantly better in TFEB-KO cells than they do in parentals. Also, using a torin-induced TFEB translocation model, we observed remarkably decreased TFEB activation in the Coxiella infected cells as was evident by less TFEB translocation to nucleus. Overall, data obtained from this work suggest that Coxiella inhibits lysosome biogenesis by blocking TFEB nuclear translocation.Item Nanoscopic and Functional Characterization of Keratinocyte-Originating Exosomes in the Wound Fluid of Non-Diabetic and Diabetic Chronic Wound Patients(Elsevier, 2023) Guda, Poornachander R.; Sharma, Anu; Anthony, Adam J.; ElMasry, Mohamed S.; Couse, Andrew D.; Das Ghatak, Piya; Das, Amitava; Timsina, Lava; Trinidad, Jonathan C.; Roy, Sashwati; Clemmer, David E.; Sen, Chandan K.; Ghatak, Subhadip; Surgery, School of MedicineExosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk in vivo. However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes (hExoκ) from human chronic wound fluid. Furthermore, we studied the significance of hExoκ in diabetic wounds. LC-MS-MS detection of KRT14 in hExoκ and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of hExoκ. dSTORM nanoimaging identified KRT14+ extracellular vesicles (EVκ) in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated hExoκ from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated hExoκ (Ø75–150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed hExoκ Ø as 80–145nm. The abundance of hExoκ was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The hExoκ isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic hExoκ Uptake of hExoκ by monocyte-derived macrophages (MDM) was low for diabetics versus non-diabetics. Unlike hExoκ from non-diabetics, the addition of diabetic hExoκ to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of hExoκ from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity.Item Stabilized collagen matrix dressing improves wound macrophage function and epithelialization(Federation of American Society of Experimental Biology, 2019-02) El Masry, Mohamed S.; Chaffee, Scott; Das Ghatak, Piya; Mathew-Steiner, Shomita S.; Das, Amitava; Higuita-Castro, Natalia; Roy, Sashwati; Anani, Raafat A.; Sen, Chandan K.; Surgery, School of MedicineDecellularized matrices of biologic tissue have performed well as wound care dressings. Extracellular matrix–based dressings are subject to rapid degradation by excessive protease activity at the wound environment. Stabilized, acellular, equine pericardial collagen matrix (sPCM) wound care dressing is flexible cross-linked proteolytic enzyme degradation resistant. sPCM was structurally characterized utilizing scanning electron and atomic force microscopy. In murine excisional wounds, sPCM was effective in mounting an acute inflammatory response. Postwound inflammation resolved rapidly, as indicated by elevated levels of IL-10, arginase-1, and VEGF, and lowering of IL-1β and TNF-α. sPCM induced antimicrobial proteins S100A9 and β-defensin-1 in keratinocytes. Adherence of Pseudomonas aeruginosa and Staphylococcus aureus on sPCM pre-exposed to host immune cells in vivo was inhibited. Excisional wounds dressed with sPCM showed complete closure at d 14, while control wounds remained open. sPCM accelerated wound re-epithelialization. sPCM not only accelerated wound closure but also improved the quality of healing by increased collagen deposition and maturation. Thus, sPCM is capable of presenting scaffold functionality during the course of wound healing. In addition to inducing endogenous antimicrobial defense systems, the dressing itself has properties that minimize biofilm formation. It mounts robust inflammation, a process that rapidly resolves, making way for wound healing to advance.—El Masry, M. S., Chaffee, S., Das Ghatak, P., Mathew-Steiner, S. S., Das, A., Higuita-Castro, N., Roy, S., Anani, R. A., Sen, C. K. Stabilized collagen matrix dressing improves wound macrophage function and epithelialization.Item Staphylococcus aureus Biofilm Infection Compromises Wound Healing by Causing Deficiencies in Granulation Tissue Collagen(Wolters Kluwer, 2020-06) Roy, Sashwati; Santra, Suman; Das, Amitava; Dixith, Sriteja; Sinha, Mithun; Ghatak, Subhadip; Ghosh, Nandini; Banerjee, Pradipta; Khanna, Savita; Mathew-Steiner, Shomita; Das Ghatak, Piya; Blackstone, Britani N.; Powell, Heather M.; Bergdall, Valerie K.; Wozniak, Daniel J; Sen, Chandan K.; Surgery, School of MedicineObjective: 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.