Browsing by Author "Gordillo, Gayle M."
Now showing 1 - 10 of 15
Results Per Page
Sort Options
Item 153. Quantification of Lymphangiogenesis in Murine Lymphedema Tail Model Using Intravital Microscopy(Wolters Kluwer, 2023-05-19) Mohan, Ganesh; Khan, Imran; Diaz, Stephanie M.; Neumann, Colby R.; Jorge, Miguel D.; Sinha, Mithun; Gordillo, Gayle M.; Sen, Chandan K.; Hassanein, Aladdin H.; Surgery, School of MedicinePURPOSE: Lymphedema is limb swelling caused by lymphatic dysfunction. It occurs in 30% of patients that undergo axillary lymph node dissection in the treatment of breast cancer. There is no cure for this disease. Understanding the mechanisms of lymphatic growth will play a pivotal role in developing therapeutic strategies against these conditions. Visualization of lymphangiogenesis and functional assessment remains a challenge. Intravital two-photon microscopy (IVM) is a powerful imaging tool for investigating various biological processes in live animals. Tissue nanotransfection technology (TNT) facilitates a direct, transcutaneous non-viral vector gene delivery using a chip with nanochannel poration in a rapid (<100ms) focused electric field. TNT was used in this study to deliver the genetic cargo in the murine tail lymphedema to assess the lymphangiogenesis. The purpose of this study is to experimentally evaluate the applicability of IVM to visualize and quantify lymphatics. METHODS: The murine tail model of lymphedema was utilized. A 3 mm full thickness skin excision and lymphatic vessel disruption was performed 20 mm from the base of the tail in twelve C57BL/6 mice. TNT was applied to the murine tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: Group I (control) was given pCMV6 (expression vector backbone alone) (n=6); Group II had pCMV6-Prox1 (n=6). Post-TNT (day 10), a 3 cm segment of murine tail was deskinned distal to the site of occlusion to optimize visualization. FITC-Dextran (2000 kD) injected intradermally at the distal tail region for lymphatic uptake. Lymphatic vessels are visualized at the second skin excision site with the Leica SP8 Confocal/Multiphoton Microscope and assessed for number of branching points to determine the newly formed lymphatics. Lymphatic vessel density was also observed by immunostaining with anti-Podoplanin antibody. RESULTS: The experimental group II exhibited increased branching points (3-fold) using filamentation analysis compared to control group I at the site of TNT treatment (n=6, p<0.05). Increased lymphatic vessel density was also observed with Podoplanin immunostaining post-TNT application. Intensity quantification of immunohistochemistry revealed greater expression of Podoplanin in Group II when compared to Group I (n=6, p<0.05). CONCLUSION: This study demonstrates a novel, powerful imaging tool for investigating lymphatic vessels in live murine tail model of lymphedema. Intravital microscopy can be utilized for functional assessment of lymphatics and visualization of lymphangiogenesis following gene-based therapy.Item Biofilm Derived Oxylipin Mediated Autoimmune Response in Breast Implant Subjects(medRxiv, 2020-11-20) Khan, Imran; Minto, Robert E.; Kelley-Patteson, Christine; Natta, Bruce W. Van; Neumann, Colby R.; Suh, Lily J.; Singh, Kanhaiya; Lester, Mary; VonDerHaar, R. Jason; Gordillo, Gayle M.; Hassanein, Aladdin H.; Sen, Chandan K.; Kadin, Marshall E.; Sinha, Mithun; Chemistry and Chemical Biology, School of ScienceOver 10 million women worldwide have breast implants for breast cancer/prophylactic reconstruction or cosmetic augmentation. In recent years, a number of patients have described a constellation of symptoms that are believed to be related to their breast implants. This constellation of symptoms has been named Breast Implant Illness (BII). The symptoms described include chronic fatigue, joint pain, muscle pain and a host of other manifestations often associated with autoimmune illnesses. In this work, we report that bacterial biofilm is associated with BII. We postulate that the pathogenesis of BII is mediated via a host-pathogen interaction whereby the biofilm bacteria Staphylococcus epidermidis interacts with breast lipids to form the oxylipin 10-HOME. The oxylipin 10-HOME was found to activate CD4+ T cells to Th1 subtype. An increased abundance of CD4+Th1 was observed in the breast tissue of BII subjects. The identification of a mechanism of immune activation associated with BII via a biofilm enabled pathway provides insight into the pathogenesis for implant-associated autoimmune symptoms.Item Biofilm Management in Wound Care(Wolters Kluwer, 2021) Sen, Chandan K.; Roy, Sashwati; Mathew-Steiner, Shomita S.; Gordillo, Gayle M.; Surgery, School of MedicineLearning objectives: After studying this article, the participant should be able to: 1. Understand the basics of biofilm infection and be able to distinguish between planktonic and biofilm modes of growth. 2. Have a working knowledge of conventional and emerging antibiofilm therapies and their modes of action as they pertain to wound care. 3. Understand the challenges associated with testing and marketing antibiofilm strategies and the context within which these strategies may have effective value. Summary: The Centers for Disease Control and Prevention estimate for human infectious diseases caused by bacteria with a biofilm phenotype is 65 percent and the National Institutes of Health estimate is closer to 80 percent. Biofilms are hostile microbial aggregates because, within their polymeric matrix cocoons, they are protected from antimicrobial therapy and attack from host defenses. Biofilm-infected wounds, even when closed, show functional deficits such as deficient extracellular matrix and impaired barrier function, which are likely to cause wound recidivism. The management of invasive wound infection often includes systemic antimicrobial therapy in combination with débridement of wounds to a healthy tissue bed as determined by the surgeon who has no way of visualizing the biofilm. The exceedingly high incidence of false-negative cultures for bacteria in a biofilm state leads to missed diagnoses of wound infection. The use of topical and parenteral antimicrobial therapy without wound débridement have had limited impact on decreasing biofilm infection, which remains a major problem in wound care. Current claims to manage wound biofilm infection rest on limited early-stage data. In most cases, such data originate from limited experimental systems that lack host immune defense. In making decisions on the choice of commercial products to manage wound biofilm infection, it is important to critically appreciate the mechanism of action and significance of the relevant experimental system. In this work, the authors critically review different categories of antibiofilm products, with emphasis on their strengths and limitations as evident from the published literature.Item Biofilm-derived oxylipin 10-HOME–mediated immune response in women with breast implants(The American Society for Clinical Investigation, 2023-11-30) Khan, Imran; Minto, Robert E.; Kelley-Patteson, Christine; Singh, Kanhaiya; Timsina, Lava; Suh, Lily J.; Rinne, Ethan; Van Natta, Bruce W.; Neumann, Colby R.; Mohan, Ganesh; Lester, Mary; VonDerHaar, R. Jason; German, Rana; Marino, Natascia; Hassanein, Aladdin H.; Gordillo, Gayle M.; Kaplan, Mark H.; Sen, Chandan K.; Kadin, Marshall E.; Sinha, Mithun; Surgery, School of MedicineThis study investigates a mechanistic link of bacterial biofilm–mediated host-pathogen interaction leading to immunological complications associated with breast implant illness (BII). Over 10 million women worldwide have breast implants. In recent years, women have described a constellation of immunological symptoms believed to be related to their breast implants. We report that periprosthetic breast tissue of participants with symptoms associated with BII had increased abundance of biofilm and biofilm-derived oxylipin 10-HOME compared with participants with implants who are without symptoms (non-BII) and participants without implants. S. epidermidis biofilm was observed to be higher in the BII group compared with the non-BII group and the normal tissue group. Oxylipin 10-HOME was found to be immunogenically capable of polarizing naive CD4+ T cells with a resulting Th1 subtype in vitro and in vivo. Consistently, an abundance of CD4+Th1 subtype was observed in the periprosthetic breast tissue and blood of people in the BII group. Mice injected with 10-HOME also had increased Th1 subtype in their blood, akin to patients with BII, and demonstrated fatigue-like symptoms. The identification of an oxylipin-mediated mechanism of immune activation induced by local bacterial biofilm provides insight into the possible pathogenesis of the implant-associated immune symptoms of BII.Item Biofilm-derived oxylipin 10-HOME–mediated immune response in women with breast implants(ASCI, 2024-02) Khan, Imran; Minto, Robert E.; Kelley-Patteson, Christine; Singh, Kanhaiya; Timsina, Lava; Suh, Lily J.; Rinne, Ethan; Van Natta, Bruce W.; Neumann, Colby R.; Mohan, Ganesh; Lester, Mary; VonDerHaar, R. Jason; German, Rana; Marino, Natascia; Hassanein, Aladdin H.; Gordillo, Gayle M.; Kaplan, Mark H.; Sen, Chandan K.; Kadin, Marshall E.; Sinha, Mithun; Chemistry, School of ScienceThis study investigates a mechanistic link of bacterial biofilm–mediated host-pathogen interaction leading to immunological complications associated with breast implant illness (BII). Over 10 million women worldwide have breast implants. In recent years, women have described a constellation of immunological symptoms believed to be related to their breast implants. We report that periprosthetic breast tissue of participants with symptoms associated with BII had increased abundance of biofilm and biofilm-derived oxylipin 10-HOME compared with participants with implants who are without symptoms (non-BII) and participants without implants. S. epidermidis biofilm was observed to be higher in the BII group compared with the non-BII group and the normal tissue group. Oxylipin 10-HOME was found to be immunogenically capable of polarizing naive CD4+ T cells with a resulting Th1 subtype in vitro and in vivo. Consistently, an abundance of CD4+Th1 subtype was observed in the periprosthetic breast tissue and blood of people in the BII group. Mice injected with 10-HOME also had increased Th1 subtype in their blood, akin to patients with BII, and demonstrated fatigue-like symptoms. The identification of an oxylipin-mediated mechanism of immune activation induced by local bacterial biofilm provides insight into the possible pathogenesis of the implant-associated immune symptoms of BII.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 Endothelial Phospholipase Cγ2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy(American Diabetes Association, 2022) Rustagi, Yashika; Abouhashem, Ahmed S.; Verma, Priyanka; Verma, Sumit S.; Hernandez, Edward; Liu, Sheng; Kumar, Manishekhar; Guda, Poornachander R.; Srivastava, Rajneesh; Mohanty, Sujit K.; Kacar, Sedat; Mahajan, Sanskruti; Wanczyk, Kristen E.; Khanna, Savita; Murphy, Michael P.; Gordillo, Gayle M.; Roy, Sashwati; Wan, Jun; Sen, Chandan K.; Singh, Kanhaiya; Medicine, School of MedicineTherapeutic vascular endothelial growth factor (VEGF) replenishment has met with limited success for the management of critical limb-threatening ischemia. To improve outcomes of VEGF therapy, we applied single-cell RNA sequencing (scRNA-seq) technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using the Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic versus nondiabetic skin, phospholipase Cγ2 (PLCγ2) was downregulated. The significance of PLCγ2 in VEGF-mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In these cells, VEGF enhanced mitochondrial function, as indicated by elevation in oxygen consumption rate and extracellular acidification rate. The VEGF-dependent increase in cell metabolism was blunted in response to PLCγ2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCγ2 in type 1 (streptozotocin-injected) and type 2 (db/db) diabetic ischemic tissue. Nonviral topical tissue nanotransfection technology (TNT) delivery of CDH5 promoter-driven PLCγ2 open reading frame promoted the rescue of hindlimb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with proangiogenic gene therapies. Taken together, our study demonstrates that TNT-mediated delivery of endothelial PLCγ2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.Item Genome-wide DNA hypermethylation opposes healing in patients with chronic wounds by impairing epithelial-mesenchymal transition(The American Society for Clinical Investigation, 2022) Singh, Kanhaiya; Rustagi, Yashika; Abouhashem, Ahmed S.; Tabasum, Saba; Verma, Priyanka; Hernandez, Edward; Pal, Durba; Khona, Dolly K.; Mohanty, Sujit K.; Kumar, Manishekhar; Srivastava, Rajneesh; Guda, Poornachander R.; Verma, Sumit S.; Mahajan, Sanskruti; Killian, Jackson A.; Walker, Logan A.; Ghatak, Subhadip; Mathew-Steiner, Shomita S.; Wanczyk, Kristen E.; Liu, Sheng; Wan, Jun; Yan, Pearlly; Bundschuh, Ralf; Khanna, Savita; Gordillo, Gayle M.; Murphy, Michael P.; Roy, Sashwati; Sen, Chandan K.; Surgery, School of MedicineAn extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1, and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces wound-edge gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure.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 Mitochondria as Target for Tumor Management of Hemangioendothelioma(Liebert, 2020) Gordillo, Gayle M.; Biswas, Ayan; Singh, Kanhaiya; Sen, Abhishek; Guda, Poornachander R.; Miller, Caroline; Pan, Xueliang; Khanna, Savita; Cadenas, Enrique; Sen, Chandan K.; Surgery, School of MedicineAims: Hemangioendothelioma (HE) may be benign or malignant. Mouse hemangioendothelioma endothelial (EOMA) cells are validated to study mechanisms in HE. This work demonstrates that EOMA cells heavily rely on mitochondria to thrive. Thus, a combination therapy, including weak X-ray therapy (XRT, 0.5 Gy) and a standardized natural berry extract (NBE) was tested. This NBE is known to be effective in managing experimental HE and has been awarded with the Food and Drug Administration Investigational New Drug (FDA-IND) number 140318 for clinical studies on infantile hemangioma. Results: NBE treatment alone selectively attenuated basal oxygen consumption rate of EOMA cells. NBE specifically sensitized EOMA, but not murine aortic endothelial cells to XRT-dependent attenuation of mitochondrial respiration and adenosine triphosphate (ATP) production. Combination treatment, selectively and potently, influenced mitochondrial dynamics in EOMA cells such that fission was augmented. This was achieved by lowering of mitochondrial sirtuin 3 (SIRT3) causing increased phosphorylation of AMP-activated protein kinase (AMPK). A key role of SIRT3 in loss of EOMA cell viability caused by the combination therapy was evident when pyrroloquinoline quinone, an inducer of SIRT3, pretreatment rescued these cells. Innovation and Conclusion: Mitochondria-targeting NBE significantly extended survival of HE-affected mice. The beneficial effect of NBE in combination with weak X-ray therapy was, however, far more potent with threefold increase in murine survival. The observation that safe natural products may target tumor cell mitochondria and sharply lower radiation dosage required for tumor management warrants clinical testing.