Browsing by Author "Das, Amitava"
Now showing 1 - 10 of 10
Results Per Page
Sort Options
Item Electroceutical Management of Bacterial Biofilms and Surgical Infection(Liebert, 2020) Sen, Chandan K.; Mathew-Steiner, Shomita S.; Das, Amitava; Sundaresan, Vishnu Baba; Roy, Sashwati; Surgery, School of MedicineSignificance: In the host–microbe microenvironment, bioelectrical factors influence microbes and hosts as well as host–microbe interactions. This article discusses relevant mechanistic underpinnings of this novel paradigm. It also addresses how such knowledge may be leveraged to develop novel electroceutical solutions to manage biofilm infection. Recent Advances: Systematic review and meta-analysis of several hundred wound studies reported a 78.2% prevalence of biofilms in chronic wounds. Biofilm infection is a major cause of delayed wound healing. In the host–microbe microenvironment, bioelectrical factors influence interactions between microbes and hosts. Critical Issues: Rapid biological responses are driven by electrical signals generated by ion currents moving across cell membranes. Bacterial life, growth, and function rely on a bioelectrical milieu, which when perturbed impairs their ability to form a biofilm, a major threat to health care. Electrokinetic stability of several viral particles depend on electrostatic forces. Weak electrical field strength, otherwise safe for humans, can be anti-microbial in this context. In the host, the electric field enhanced keratinocyte migration, bolstered immune defenses, improved mitochondrial function, and demonstrated multiple other effects consistent with supporting wound healing. A deeper mechanistic understanding of bioelectrical principles will inform the design of next-generation electroceuticals. Future Directions: This is an opportune moment in time as there is a surge of interest in electroceuticals in medicine. Projected to reach $35.5 billion by 2025, electroceuticals are becoming a cynosure in the global market. The World Health Organization reports that more than 50% of surgical site infections can be antibiotic resistant. Electroceuticals offer a serious alternative.Item Electroceutical Treatment of Pseudomonas aeruginosa Biofilms(Springer Nature, 2019-02-14) Dusane, Devendra H.; Lochab, Varun; Jones, Travis; Peters, Casey W.; Sindeldecker, Devin; Das, Amitava; Roy, Sashwati; Sen, Chandan K.; Subramaniam, Vish V.; Wozniak, Daniel J.; Prakash, Shaurya; Stoodley, Paul; Surgery, School of MedicineElectroceutical wound dressings, especially those involving current flow with silver based electrodes, show promise for treating biofilm infections. However, their mechanism of action is poorly understood. We have developed an in vitro agar based model using a bioluminescent strain of Pseudomonas aeruginosa to measure loss of activity and killing when direct current was applied. Silver electrodes were overlaid with agar and lawn biofilms grown for 24 h. A 6 V battery with 1 kΩ ballast resistor was used to treat the biofilms for 1 h or 24 h. Loss of bioluminescence and a 4-log reduction in viable cells was achieved over the anode. Scanning electron microscopy showed damaged cells and disrupted biofilm architecture. The antimicrobial activity continued to spread from the anode for at least 2 days, even after turning off the current. Based on possible electrochemical ractions of silver electrodes in chlorine containing medium; pH measurements of the medium post treatment; the time delay between initiation of treatment and observed bactericidal effects; and the presence of chlorotyrosine in the cell lysates, hypochlorous acid is hypothesized to be the chemical agent responsible for the observed (destruction/killing/eradication) of these biofilm forming bacteria. Similar killing was obtained with gels containing only bovine synovial fluid or human serum. These results suggest that our in vitro model could serve as a platform for fundamental studies to explore the effects of electrochemical treatment on biofilms, complementing clinical studies with electroceutical dressings.Item Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing(ACS, 2020-09) Zhou, Xiaoju; Brown, Brooke A.; Siegel, Amanda P.; El Masry, Mohamed S.; Zeng, Xuyao; Song, Woran; Das, Amitava; Khandelwal, Puneet; Clark, Andrew; Singh, Kanhaiya; Guda, Poornachander R.; Gorain, Mahadeo; Timsina, Lava; Xuan, Yi; Jacobson, Stephen C.; Novotny, Milos V.; Roy, Sashwati; Agarwal, Mangilal; Lee, Robert J.; Sen, Chandan K.; Clemmer, David E.; Ghatak, Subhadip; Surgery, School of MedicineBidirectional cell–cell communication involving exosome-borne cargo such as miRNA has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte–macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP-reporter (Exoκ-GFP) using tissue nanotransfection (TNT), and they were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Unlike skin exosome, wound-edge Exoκ-GFP demonstrated characteristic N-glycan ions with abundance of low-base-pair RNA and was selectively engulfed by wound macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exoκ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exoκ-GFPin vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNPκ) were designed with 94.3% encapsulation efficiency. Application of TLNPκ/si-hnRNPA2B1 to the murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to the TLNPκ/si-control group. Although no significant difference in wound closure or re-epithelialization was observed, the TLNPκ/si-hnRNPA2B1 treated group showed a significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to the TLNPκ/si-control group. Furthermore, TLNPκ/si-hnRNPA2B1 treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exoκ-GFP is recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties postinjury.Item Mesenchymal stem cells promote mesenteric vasodilation through hydrogen sulfide and endothelial nitric oxide(American Physiological Society, 2019-09-24) Te Winkel, Jan; John, Quincy E.; Hosfield, Brian D.; Drucker, Natalie A.; Das, Amitava; Olson, Ken R.; Markel, Troy A.; Surgery, School of MedicineMesenteric ischemia is a devastating process that can result in intestinal necrosis. Mesenchymal stem cells (MSCs) are becoming a promising treatment modality. We hypothesized that 1) MSCs would promote vasodilation of mesenteric arterioles, 2) hydrogen sulfide (H2S) would be a critical paracrine factor of stem cell-mediated vasodilation, 3) mesenteric vasodilation would be impaired in the absence of endothelial nitric oxide synthase (eNOS) within the host tissue, and 4) MSCs would improve the resistin-to-adiponectin ratio in mesenteric vessels. H2S was measured with a specific fluorophore (7-azido-3-methylcoumarin) in intact MSCs and in cells with the H2S-producing enzyme cystathionine β synthase (CBS) knocked down with siRNA. Mechanical responses of isolated second- and third-order mesenteric arteries (MAs) from wild-type and eNOS knockout (eNOSKO) mice were monitored with pressure myography, after which the vessels were snap frozen and later analyzed for resistin and adiponectin via multiplex beaded assay. Addition of MSCs to the myograph bath significantly increased vasodilation of norepinephrine-precontracted MAs. Knockdown of CBS in MSCs decreased H2S production by MSCs and also decreased MSC-initiated MA dilation. MSC-initiated vasodilation was further reduced in eNOSKO vessels. The MA resistin-to-adiponectin ratio was higher in eNOSKO vessels compared with wild-type. These results show that MSC treatment promotes dilation of MAs by an H2S-dependent mechanism. Furthermore, functional eNOS within the host mesenteric bed appears to be essential for maximum stem cell therapeutic benefit, which may be attributable, in part, to modifications in the resistin-to-adiponectin ratio. NEW & NOTEWORTHY Stem cells have been shown to improve survival, mesenteric perfusion, and histological injury scores following intestinal ischemia. These benefits may be due to the paracrine release of hydrogen sulfide. In an ex vivo pressure myography model, we observed that mesenteric arterial dilation improved with stem cell treatment. Hydrogen sulfide release from stem cells and endothelial nitric oxide synthase within the vessels were critical components of optimizing stem cell-mediated mesenteric artery dilation.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 Skin Transcriptome of Middle-Aged Women Supplemented With Natural Herbo-mineral Shilajit Shows Induction of Microvascular and Extracellular Matrix Mechanisms(Taylor & Francis, 2019-06-04) Das, Amitava; Masry, Mohamed S. El; Gnyawali, Surya C.; Ghatak, Subhadip; Singh, Kanhaiya; Stewart, Richard; Lewis, Madeline; Saha, Abhijoy; Gordillo, Gayle; Khanna, Savita; Surgery, School of MedicineObjective: Shilajit is a pale-brown to blackish-brown organic mineral substance available from Himalayan rocks. We demonstrated that in type I obese humans, shilajit supplementation significantly upregulated extracellular matrix (ECM)–related genes in the skeletal muscle. Such an effect was highly synergistic with exercise. The present study (clinicaltrials.gov ) aimed to evaluate the effects of shilajit supplementation on skin gene expression profile and microperfusion in healthy adult females. Methods: The study design comprised six total study visits including a baseline visit (V1) and a final 14-week visit (V6) following oral shilajit supplementation (125 or 250 mg bid). A skin biopsy of the left inner upper arm of each subject was collected at visit 2 and visit 6 for gene expression profiling using Affymetrix Clariom™ D Assay. Skin perfusion was determined by MATLAB processing of dermascopic images. Transcriptome data were normalized and subjected to statistical analysis. The differentially regulated genes were subjected to Ingenuity Pathway Analysis (IPA®). The expression of the differentially regulated genes identified by IPA® were verified using real-time polymerasechain reaction (RT-PCR). Results: Supplementation with shilajit for 14 weeks was not associated with any reported adverse effect within this period. At a higher dose (250 mg bid), shilajit improved skin perfusion when compared to baseline or the placebo. Pathway analysis identified shilajit-inducible genes relevant to endothelial cell migration, growth of blood vessels, and ECM which were validated by quantitative real-time polymerasechain reaction (RT-PCR) analysis. Conclusions: This work provides maiden evidence demonstrating that oral shilajit supplementation in adult healthy women induced genes relevant to endothelial cell migration and growth of blood vessels. Shilajit supplementation improved skin microperfusion.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 A surfactant polymer wound dressing protects human keratinocytes from inducible necroptosis(Springer Nature, 2021-02-23) Khandelwal, Puneet; Das, Amitava; Sen, Chandan K.; Srinivas, Sangly P.; Roy, Sashwati; Khanna, Savita; Surgery, School of MedicineChronic wounds show necroptosis from which keratinocytes must be protected to enable appropriate wound re-epithelialization and closure. Poloxamers, a class of synthetic triblock copolymers, are known to be effective against plasma membrane damage (PMD). The purpose of this study is to evaluate the efficacy of a specific poloxamer, surfactant polymer dressing (SPD), which is currently used clinically as wound care dressing, against PMD in keratinocytes. Triton X-100 (TX100) at sub-lytic concentrations caused PMD as demonstrated by the efflux of calcein and by the influx of propidium iodide and FM1-43. TX100, an inducer of necroptosis, led to mitochondrial fragmentation, depletion of nuclear HMGB1, and activation of signaling complex associated with necroptosis (i.e., activation of RIP3 and phosphorylation of MLKL). All responses following exposure of human keratinocytes to TX100 were attenuated by pre- or co-treatment with SPD (100 mg/ml). The activation and translocation of phospho-MLKL to the plasma membrane, taken together with depletion of nuclear HMGB1, characterized the observed cell death as necroptosis. Thus, our findings show that TX100-induced plasma membrane damage and death by necroptosis were both attenuated by SPD, allowing keratinocyte survival. The significance of such protective effects of SPD on keratinocytes in wound re-epithelialization and closure warrant further studies.Item Urolithin A augments angiogenic pathways in skeletal muscle by bolstering NAD+ and SIRT1(NPG, 2020-11-11) Ghosh, Nandini; Das, Amitava; Biswas, Nirupam; Gnyawali, Surya; Singh, Kanhaiya; Gorain, Mahadeo; Polcyn, Carly; Khanna, Savita; Roy, Sashwati; Sen, Chandan K.; Surgery, School of MedicineUrolithin A (UA) is a natural compound that is known to improve muscle function. In this work we sought to evaluate the effect of UA on muscle angiogenesis and identify the underlying molecular mechanisms. C57BL/6 mice were administered with UA (10 mg/body weight) for 12–16 weeks. ATP levels and NAD+ levels were measured using in vivo 31P NMR and HPLC, respectively. UA significantly increased ATP and NAD+ levels in mice skeletal muscle. Unbiased transcriptomics analysis followed by Ingenuity Pathway Analysis (IPA) revealed upregulation of angiogenic pathways upon UA supplementation in murine muscle. The expression of the differentially regulated genes were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). Angiogenic markers such as VEGFA and CDH5 which were blunted in skeletal muscles of 28 week old mice were found to be upregulated upon UA supplementation. Such augmentation of skeletal muscle vascularization was found to be bolstered via Silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1α) pathway. Inhibition of SIRT1 by selisistat EX527 blunted UA-induced angiogenic markers in C2C12 cells. Thus this work provides maiden evidence demonstrating that UA supplementation bolsters skeletal muscle ATP and NAD+ levels causing upregulated angiogenic pathways via a SIRT1-PGC-1α pathway.