Browsing by Author "Sen, Chandan K."
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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 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 Ad hoc Hybrid Synaptic Junctions to Detect Nerve Stimulation and its Application to Detect Onset of Diabetic Polyneuropathy(Elsevier, 2020-12) Gupta, Sujasha; Ghatak, Subhadip; Hery, Travis; Khanna, Savita; El-Masry, Mohamed; Sundaresan, Vishnu Baba; Sen, Chandan K.; Surgery, School of MedicineWe report a minimally invasive, synaptic transistor-based construct to monitor in vivo neuronal activity via a longitudinal study in mice and use depolarization time from measured data to predict the onset of polyneuropathy. The synaptic transistor is a three-terminal device in which ionic coupling between pre- and post-synaptic electrodes provides a framework for sensing low-power (sub μW) and high-bandwidth (0.1–0.5kHz) ionic currents. A validated first principles-based approach is discussed to demonstrate the significance of this sensing framework and we introduce a metric, referred to as synaptic efficiency to quantify structural and functional properties of the electrodes in sensing. The application of this framework for in vivo neuronal sensing requires a post-synaptic electrode and its reference electrode and the tissue becomes the pre-synaptic signal. The ionic coupling resembles axo-axonic junction and hence we refer to this framework as an ad hoc synaptic junction. We demonstrate that this arrangement can be applied to measure excitability of sciatic nerves due to a stimulation of the footpad in cohorts of m+/db and db/db mice for detecting loss in sensitivity and onset of polyneuropathy. The signal attributes were subsequently integrated with machine learning-based framework to identify the probability of polyneuropathy and to detect the onset of diabetic polyneuropathy.Item Adult skin fibroblast state change in murine wound healing(Springer Nature, 2023-01-17) Gharbia, Fatma Z.; Abouhashem, Ahmed S.; Moqidem, Yomna A.; Elbaz, Ahmed A.; Abdellatif, Ahmed; Singh, Kanhaiya; Sen, Chandan K.; Azzazy, Hassan M. E.; Surgery, School of MedicineWound healing is a well-organized dynamic process involving coordinated consecutive phases: homeostasis, inflammation, proliferation and resolution. Fibroblasts play major roles in skin wound healing such as in wound contraction and release of growth factors which are of importance in angiogenesis and tissue remodeling. Abnormal fibroblast phenotypes have been identified in patients with chronic wounds. In this work, we analyzed scRNA-seq datasets of normal and wounded skin from mice at day 4 post-wound to investigate fibroblast heterogeneity during the proliferative phase of wound healing. Compositional analysis revealed a specific subset of fibroblast (cluster 3) that primarily increased in wounded skin (14%) compared to normal skin (3.9%). This subset was characterized by a gene signature marked by the plasma membrane proteins Sfrp2 + Sfrp4 + Sfrp1 + and the transcription factors Ebf1 + Prrx1 + Maged1 + . Differential gene expression and enrichment analysis identified epithelial to mesenchymal transition (EMT) and angiogenesis to be upregulated in the emerging subset of fibroblasts of the wounded skin. Using two other datasets for murine wounded skin confirmed the increase in cluster 3-like fibroblasts at days 2, 7 and 14 post-wounding with a peak at day 7. By performing a similarity check between the differential gene expression profile between wounded and normal skin for this emerging fibroblast subset with drug signature from the ConnectivityMap database, we identified drugs capable of mimicking the observed gene expression change in fibroblasts during wound healing. TTNPB, verteprofin and nicotinic acid were identified as candidate drugs capable of inducing fibroblast gene expression profile necessary for wound healing. On the other hand, methocarbamol, ifosfamide and penbutolol were recognized to antagonize the identified fibroblast differential expression profile during wound healing which might cause delay in wound healing. Taken together, analysis of murine transcriptomic skin wound healing datasets suggested a subset of fibroblasts capable of inducing EMT and further inferred drugs that might be tested as potential candidates to induce wound closure.Item Analysis of Keratinocytic Exosomes from Diabetic and Nondiabetic Mice by Charge Detection Mass Spectrometry(American Chemical Society, 2022) Brown, Brooke A.; Guda, Poornachander R.; Zeng, Xuyao; Anthony, Adam; Couse, Andrew; Barnes, Lauren F.; Sharon, Edie M.; Trinidad, Jonathan C.; Sen, Chandan K.; Jarrold, Martin F.; Ghatak, Subhadip; Clemmer, David E.; Surgery, School of MedicineUnresolved inflammation compromises diabetic wound healing. Recently, we reported that inadequate RNA packaging in murine wound-edge keratinocyte-originated exosomes (Exoκ) leads to persistent inflammation [Zhou, X. ACS Nano 2020, 14(10), 12732-12748]. Herein, we use charge detection mass spectrometry (CDMS) to analyze intact Exoκ isolated from a 5 day old wound-edge tissue of diabetic mice and a heterozygous nondiabetic littermate control group. In CDMS, the charge (z) and mass-to-charge ratio (m/z) of individual exosome particles are measured simultaneously, enabling the direct analysis of masses in the 1-200 MDa range anticipated for exosomes. These measurements reveal a broad mass range for Exoκ from ∼10 to >100 MDa. The m and z values for these exosomes appear to fall into families (subpopulations); a statistical modeling analysis partially resolves ∼10-20 Exoκ subpopulations. Complementary proteomics, immunofluorescence, and electron microscopy studies support the CDMS results that Exoκ from diabetic and nondiabetic mice vary substantially. Subpopulations having high z (>650) and high m (>44 MDa) are more abundant in nondiabetic animals. We propose that these high m and z particles may arise from differences in cargo packaging. The veracity of this idea is discussed in light of other recent CDMS results involving genome packaging in vaccines, as well as exosome imaging experiments. Characterization of intact exosome particles based on the physical properties of m and z provides a new means of investigating wound healing and suggests that CDMS may be useful for other pathologies.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 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 Bone marrow- or adipose-mesenchymal stromal cell secretome preserves myocardial transcriptome profile and ameliorates cardiac damage following ex vivo cold storage(Elsevier, 2022) Scott, Susan R.; March, Keith L.; Wang, I-Wen; Singh, Kanhaiya; Liu, Jianyun; Turrentin, Mark; Sen, Chandan K.; Wang, Meijing; Surgery, School of MedicineBackground: Heart transplantation, a life-saving approach for patients with end-stage heart disease, is limited by shortage of donor organs. While prolonged storage provides more organs, it increases the extent of ischemia. Therefore, we seek to understand molecular mechanisms underlying pathophysiological changes of donor hearts during prolonged storage. Additionally, considering mesenchymal stromal cell (MSC)-derived paracrine protection, we aim to test if MSC secretome preserves myocardial transcriptome profile and whether MSC secretome from a certain source provides the optimal protection in donor hearts during cold storage. Methods and results: Isolated mouse hearts were divided into: no cold storage (control), 6 h cold storage (6 h-I), 6 h-I + conditioned media from bone marrow MSCs (BM-MSC CM), and 6 h-I + adipose-MSC CM (Ad-MSC CM). Deep RNA sequencing analysis revealed that compared to control, 6 h-I led to 266 differentially expressed genes, many of which were implicated in modulating mitochondrial performance, oxidative stress response, myocardial function, and apoptosis. BM-MSC CM and Ad-MSC CM restored these gene expression towards control. They also improved 6 h-I-induced myocardial functional depression, reduced inflammatory cytokine production, decreased apoptosis, and reduced myocardial H2O2. However, neither MSC-exosomes nor exosome-depleted CM recapitulated MSC CM-ameliorated apoptosis and CM-improved mitochondrial preservation during cold ischemia. Knockdown of Per2 by specific siRNA abolished MSC CM-mediated these protective effects in cardiomyocytes following 6 h cold storage. Conclusions: Our results demonstrated that using MSC secretome (BM-MSCs and Ad-MSCs) during prolonged cold storage confers preservation of the normal transcriptional "fingerprint", and reduces donor heart damage. MSC-released soluble factors and exosomes may synergistically act for donor heart protection.