Browsing by Author "Ghosh, Nandini"
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Item A Prospective, Randomized, Controlled Study to Evaluate the Effectiveness of a Fabric-Based Wireless Electroceutical Dressing Compared to Standard-of-Care Treatment Against Acute Trauma and Burn Wound Biofilm Infection(Mary Ann Liebert, 2024) Chan, Rodney K.; Nuutila, Kristo; Mathew-Steiner, Shomita S.; Diaz, Victoria; Anselmo, Kristin; Batchinsky, Maria; Carlsson, Anders; Ghosh, Nandini; Sen, Chandan K.; Roy, Sashwati; Surgery, School of MedicineObjective: Despite advances in the use of topical and parenteral antimicrobial therapy and the practice of early tangential burn wound excision to manage bacterial load, 60% of the mortality from burns is attributed to bacterial biofilm infection. A low electric field (∼1 V) generated by the novel FDA-cleared wireless electroceutical dressing (WED) was previously shown to significantly prevent and disrupt burn biofilm infection in preclinical studies. Based on this observation, the purpose of this clinical trial was to evaluate the efficacy of the WED dressing powered by a silver–zinc electrocouple in the prevention and disruption of biofilm infection. Approach: A prospective, randomized, controlled, single-center clinical trial was performed to evaluate the efficacy of the WED compared with standard-of-care (SoC) dressing to treat biofilms. Burn wounds were randomized to receive either SoC or WED. Biopsies were collected on days 0 and 7 for histology, scanning electron microscopy (SEM) examination of biofilm, and for quantitative bacteriological analyses. Results: In total, 38 subjects were enrolled in the study. In 52% of the WED-treated wounds, little to no biofilm could be detected by SEM. WED significantly lowered or prevented increase of biofilm in all wounds compared with the pair-matched SoC-treated wounds. Innovation: WED is a simple, easy, and rapid method to protect the wound while also inhibiting infection. It is activated by a moist environment and the electrical field induces transient and micromolar amounts of superoxide anion radicals that will prevent bacterial growth. Conclusion: WED decreased biofilm infection better compared with SoC. The study was registered in clinicaltrials.gov as NCT04079998.Item Bacterial Pyocyanin Inducible KRT6A Accelerates Closure of Epithelial Defect Under Conditions of Mitochondrial Dysfunction(Elsevier, 2023) Ghatak, Subhadip; Hemann, Craig; Boslett, James; Singh, Kanhaiya; Sharma, Anu; El Masry, Mohamed S.; Abouhashem, Ahmed Safwat; Ghosh, Nandini; Mathew-Steiner, Shomita S.; Roy, Sashwati; Zweier, Jay L.; Sen, Chandan K.; Surgery, School of MedicineRepair of epithelial defect is complicated by infection and related metabolites. Pyocyanin is one such metabolite which is secreted during Pseudomonas aeruginosa infection. Keratinocyte migration is required for the closure of skin epithelial defects. The current work sought to understand pyocyanin-keratinocyte interaction and its significance in tissue repair. SILAC proteomics identified mitochondrial dysfunction as the top pathway responsive to pyocyanin exposure in human keratinocytes. Consistently, functional studies demonstrated mitochondrial stress, depletion of reducing equivalents, and ATP. Strikingly, despite all the above, pyocyanin markedly accelerated keratinocyte migration. Investigation of underlying mechanisms revealed a new function of KRT6A in keratinocytes. KRT6A was pyocyanin inducible and accelerated closure of epithelial defect. Acceleration of closure was associated with poor quality healing including compromised expression of apical junction proteins. This work recognizes KRT6A for its role of enhancing keratinocyte migration under conditions of threat posed by pyocyanin. Qualitatively deficient junctional proteins under conditions of defensive acceleration of keratinocyte migration explains why an infected wound close with deficient skin barrier function as previously reported.Item Driving adult tissue repair via re-engagement of a pathway required for fetal healing(Elsevier, 2023) Ghatak, Subhadip; Khanna, Savita; Roy, Sashwati; Thirunavukkarasu, Mahesh; Pradeep, Seetur R.; Wulff, Brian C.; El Masry, Mohamed S.; Sharma, Anu; Palakurti, Ravichand; Ghosh, Nandini; Xuan, Yi; Wilgus, Traci A.; Maulik, Nilanjana; Yoder, Mervin C.; Sen, Chandan K.; Surgery, School of MedicineFetal cutaneous wound closure and repair differ from that in adulthood. In this work, we identify an oxidant stress sensor protein, nonselenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), that is abundantly expressed in normal fetal epidermis (and required for fetal wound closure), though not in adult epidermis, but is variably re-induced upon adult tissue wounding. NPGPx is a direct target of the miR-29 family. Following injury, abundance of miR-29 is lowered, permitting a prompt increase in NPGPx transcripts and protein expression in adult wound-edge tissue. NPGPx expression was required to mediate increased keratinocyte migration induced by miR-29 inhibition in vitro and in vivo. Increased NPGPx expression induced increased SOX2 expression and β-catenin nuclear localization in keratinocytes. Augmenting physiologic NPGPx expression via experimentally induced miR-29 suppression, using cutaneous tissue nanotransfection or targeted lipid nanoparticle delivery of anti-sense oligonucleotides, proved to be sufficient to overcome the deleterious effects of diabetes on this specific pathway to enhance tissue repair.Item The eIF2 kinase GCN2 directs keratinocyte collective cell migration during wound healing via coordination of reactive oxygen species and amino acids(American Society for Biochemistry and Molecular Biology, 2021-11) Miles, Rebecca R.; Amin, Parth H.; Diaz, Miguel Barriera; Misra, Jagannath; Aukerman, Erica; Das, Amitava; Ghosh, Nandini; Guith, Tanner; Knierman, Michael D.; Roy, Sashwati; Spandau, Dan F.; Wek, Ronald C.; Biochemistry and Molecular Biology, School of MedicineHealing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds.Item Filamentous bacteriophage delays healing of Pseudomonas-infected wounds(Elsevier, 2022) Bach, Michelle S.; de Vries, Christiaan R.; Khosravi, Arya; Sweere, Johanna M.; Popescu, Medeea C.; Chen, Qingquan; Demirdjian, Sally; Hargil, Aviv; Van Belleghem, Jonas D.; Kaber, Gernot; Hajfathalian, Maryam; Burgener, Elizabeth B.; Liu, Dan; Tran, Quynh-Lam; Dharmaraj, Tejas; Birukova, Maria; Sunkari, Vivekananda; Balaji, Swathi; Ghosh, Nandini; Mathew-Steiner, Shomita S.; El Masry, Mohamed S.; Keswani, Sundeep G.; Banaei, Niaz; Nedelec, Laurence; Sen, Chandan K.; Chandra, Venita; Secor, Patrick R.; Suh, Gina A.; Bollyky, Paul L.; Surgery, School of MedicineChronic wounds infected by Pseudomonas aeruginosa (Pa) are characterized by disease progression and increased mortality. We reveal Pf, a bacteriophage produced by Pa that delays healing of chronically infected wounds in human subjects and animal models of disease. Interestingly, impairment of wound closure by Pf is independent of its effects on Pa pathogenesis. Rather, Pf impedes keratinocyte migration, which is essential for wound healing, through direct inhibition of CXCL1 signaling. In support of these findings, a prospective cohort study of 36 human patients with chronic Pa wound infections reveals that wounds infected with Pf-positive strains of Pa are more likely to progress in size compared with wounds infected with Pf-negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian cells. These findings suggest Pf may have potential as a biomarker and therapeutic target in chronic wounds.Item Identification of a physiologic vasculogenic fibroblast state to achieve tissue repair(Springer Nature, 2023-02-28) Pal, Durba; Ghatak, Subhadip; Singh, Kanhaiya; Abouhashem, Ahmed Safwat; Kumar, Manishekhar; El Masry, Mohamed S.; Mohanty, Sujit K.; Palakurti, Ravichand; Rustagi, Yashika; Tabasum, Saba; Khona, Dolly K.; Khanna, Savita; Kacar, Sedat; Srivastava, Rajneesh; Bhasme, Pramod; Verma, Sumit S.; Hernandez, Edward; Sharma, Anu; Reese, Diamond; Verma, Priyanka; Ghosh, Nandini; Gorain, Mahadeo; Wan, Jun; Liu, Sheng; Liu, Yunlong; Castro, Natalia Higuita; Gnyawali, Surya C.; Lawrence, William; Moore, Jordan; Perez, Daniel Gallego; Roy, Sashwati; Yoder, Mervin C.; Sen, Chandan K.; Surgery, School of MedicineTissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.Item A Modified Collagen Dressing Induces Transition of Inflammatory to Reparative Phenotype of Wound Macrophages(Nature Research, 2019-10-04) Das, Amitava; Abas, Motaz; Biswas, Nirupam; Banerjee, Pradipta; Ghosh, Nandini; Rawat, Atul; Khanna, Savita; Roy, Sashwati; Sen, Chandan K.; Surgery, School of MedicineCollagen 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.Item Myo-Inositol in Fermented Sugar Matrix Improves Human Macrophage Function(Wiley, 2022) Ghosh, Nandini; Das, Amitava; Biswas, Nirupam; Mahajan, Sanskruti P.; Madeshiya, Amit K.; Khanna, Savita; Sen, Chandan K.; Roy, Sashwati; Surgery, School of MedicineScope Reactive oxygen species production by innate immune cells plays a central role in host defense against invading pathogens at wound-site. A weakened hos-defense results in persistent infection leading to wound chronicity. Fermented Papaya Preparation (FPP), a complex sugar matrix, bolstered respiratory burst activity and improved wound healing outcomes in chronic wound patients. The objective of the current study was to identify underlying molecular factor/s responsible for augmenting macrophage host defense mechanisms following FPP supplementation. Methods and results In depth LC-MS/MS analysis of cells supplemented with FPP led to identification of myo-inositol as a key determinant of FPP activity towards improving macrophage function. Myo-inositol, in quantities that is present in FPP, significantly improved macrophage respiratory burst and phagocytosis via de novo synthesis pathway of ISYNA1. Additionally, myo-inositol transporters, HMIT and SMIT1, played a significant role in such activity. Blocking these pathways using siRNA attenuated FPP-induced improved macrophage host defense activities. FPP supplementation emerges as a novel approach to increase intracellular myo-inositol levels. Such supplementation also modified wound microenvironment in chronic wound patients to augment myo-inositol levels in wound fluid. Conclusion These observations indicate that myo-inositol in FPP influences multiple aspects of macrophage function critical for host defense against invading pathogens.Item Oncostatin M Improves Cutaneous Wound Re-Epithelialization and Is Deficient under Diabetic Conditions(Elsevier, 2022) Das, Amitava; Madeshiya, Amit K.; Biswas, Nirupam; Ghosh, Nandini; Gorain, Mahadeo; Rawat, Atul; Mahajan, Sanskruti P.; Khanna, Savita; Sen, Chandan K.; Roy, Sashwati; Surgery, School of MedicineImpaired re-epithelialization characterized by hyperkeratotic non-migratory wound epithelium is a hallmark of non-healing diabetic wounds. In chronic wounds, copious release of oncostatin M (OSM) from wound macrophages is evident. OSM is a potent keratinocyte activator. This work sought to understand the signal transduction pathway responsible for wound-re-epithelialization, the primary mechanism underlying wound closure. Daily topical treatment of full-thickness excisional wounds of C57bl/6 mice with recombinant murine OSM improved wound re-epithelialization and accelerated wound closure by bolstering keratinocyte proliferation and migration. OSM activated the JAK-STAT pathway as manifested by STAT3 phosphorylation. Such signal transduction in the human keratinocyte induced TP63, the master regulator of keratinocyte function. Elevated TP63 induced integrin beta 1, a known effector of keratinocyte migration. In diabetic wounds, OSM was more abundant compared to the level in non-diabetic wounds. However, in diabetic wounds OSM activity was compromised by glycation. Aminoguanidine, a deglycation agent, rescued compromised keratinocyte migration caused by glycated OSM. Finally, topical application of recombinant OSM improved keratinocyte migration and accelerated wound closure in db/db mice. This work recognizes that despite its abundance at the wound-site, OSM is inactivated by glycation and topical delivery of exogenous OSM is likely to be productive in accelerating diabetic wound closure.Item Pseudomonas Aeruginosa Theft Biofilm Require Host Lipids of Cutaneous Wound(Wolters Kluwer, 2023) Sinha, Mithun; Ghosh, Nandini; Wijesinghe, Dayanjan S.; Mathew-Steiner, Shomita S.; Das, Amitava; Singh, Kanhaiya; El Masry, Mohamed; Khanna, Savita; Inoue, Hiroyuki; Yamazaki, Katsuhisa; Kawada, Manabu; Gordillo, Gayle M.; Roy, Sashwati; Sen, Chandan K.; Surgery, School of MedicineObjective: This work addressing complexities in wound infection, seeks to test the reliance of bacterial pathogen Pseudomonas aeruginosa (PA) on host skin lipids to form biofilm with pathological consequences. Background: PA biofilm causes wound chronicity. Both CDC as well as NIH recognizes biofilm infection as a threat leading to wound chronicity. Chronic wounds on lower extremities often lead to surgical limb amputation. Methods: An established preclinical porcine chronic wound biofilm model, infected with PA or Pseudomonas aeruginosa ceramidase mutant (PA ∆Cer ), was used. Results: We observed that bacteria drew resource from host lipids to induce PA ceramidase expression by three orders of magnitude. PA utilized product of host ceramide catabolism to augment transcription of PA ceramidase. Biofilm formation was more robust in PA compared to PA ∆Cer . Downstream products of such metabolism such as sphingosine and sphingosine-1-phosphate were both directly implicated in the induction of ceramidase and inhibition of peroxisome proliferator-activated receptor (PPAR)δ, respectively. PA biofilm, in a ceram-idastin-sensitive manner, also silenced PPARδ via induction of miR-106b. Low PPARδ limited ABCA12 expression resulting in disruption of skin lipid homeostasis. Barrier function of the wound-site was thus compromised. Conclusions: This work demonstrates that microbial pathogens must co-opt host skin lipids to unleash biofilm pathogenicity. Anti-biofilm strategies must not necessarily always target the microbe and targeting host lipids at risk of infection could be productive. This work may be viewed as a first step, laying fundamental mechanistic groundwork, toward a paradigm change in biofilm management.