Browsing by Author "Sinha, Mithun"

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    142. Optimization of the Murine Hindlimb Lymphedema Model
    (Wolters Kluwer, 2025-04-24) Ahmed, Shahnur; Mohan, Ganesh; Sullivan, Steven J.; Jorge, Miguel; Sinha, Mithun; Hassanein, Aladdin H.; Surgery, School of Medicine
    PURPOSE: Secondary lymphedema is limb swelling from lymphatic injury. It frequently occurs following lymph node dissection and radiation during the treatment of malignancies such as breast cancer or melanoma. The murine tail is the most commonly used model to study secondary lymphedema and involves full thickness tail skin excision and lymphatic vessel disruption. The murine hindlimb model, which has been less frequently used in the literature, offers a more clinically translatable method. However, there is inconsistency and variability, including the benefit of radiation, which have contributed to the model being less widely adapted than the tail model. The purpose of this study is to 1) optimize the murine hindlimb lymphedema to achieve consistent results and 2) assess the effect of radiation on outcome in the murine hindlimb model. METHODS: C57BL/6 mice either underwent 20 Gy irradiation of one hindlimb seven days prior to surgery (n=11) or no preoperative radiation (n=9). For all mice, a circumferential skin incision was created at the proximal hindlimb exposing the subcutaneous soft tissues. Lymphatics were identified with isosulfan blue dye injection into the paw and disrupted. Popliteal lymph nodes were excised. The skin was sutured leaving a 3 mm gap. The contralateral hindlimb served as the control. Paw thickness and calf thickness measurements were obtained at weekly intervals and indocyanine green (ICG) near-infrared laser lymphangiography was used to assess lymphatic function. RESULTS: For the irradiated mice, the average paw thickness of the operated hindlimb on postoperative day (POD) 14 was 3.5±0.3 cm compared to 2.1±0.05 cm on the contralateral limb (p=0.0001). At POD-90, the average paw thickness of the irradiated, operated hindlimb was 2.4±0.1 cm compared to 2.1±0.1 cm for the contralateral limb (p=0.01). ICG lymphangiography at 24-hours postinjection on POD-42 demonstrated an average signal intensity of 97.7±28.5 arbitrary fluorescent units (AFU) in the operated hindlimb compared to 33.6±6.2 AFU in the non-operated hindlimb (p=0.003). In the mice that did not undergo radiation, the average paw thickness was 2.5±0.2 cm on POD-42 was greater than the contralateral limb (2.1±0.1 cm) (p=0.0002) but smaller than hindlimbs that underwent radiation (3.2±0.1 cm) (p=0.0002). The nonradiated mice had greater paw thickness than the contralateral control until POD-56 whereas the radiated mice sustained significant paw thickness until Day 90. CONCLUSION: Radiation of the murine hindlimb model results in sustained lymphedema compared to non-irradiated mice. The murine hindlimb lymphedema model is clinically more translatable than the murine tail model and includes limb lymphatic vessel disruption, and popliteal lymphadenectomy and ideally radiation for consistent results with lymphedema sustained for 90 days.
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    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 Medicine
    PURPOSE: 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.
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    80. Lymphatic Preconditioning: Novel Investigation Of A “Lymphatic Delay Phenomenon”
    (Wolters Kluwer, 2024-04-19) Hulsman, Luci; Mohan, Ganesh; Ahmed, Shahnur; Jorge, Miguel D.; Sullivan, Steven J.; Mohammed, Imran; Sinha, Mithun; Hassanein, Aladdin H.; Surgery, School of Medicine
    Background: The vascular delay phenomenon is a well-described concept of flap physiology with many clinical applications used to increase flap viability. The approach employs a staged surgical procedure with selective partial disruption of the flap’s blood supply to increase the robustness of the remaining blood supply, followed by interval flap transfer 7-10 days later. While this vascular delay phenomenon has been thoroughly studied, no investigation has been performed to determine if a similar “delay phenomenon” exists for lymphatic vessels. Lymphedema frequently occurs following injury of lymphatics during lymph node dissection. The purpose of this study was to evaluate whether lymphatic preconditioning with staged disruption of lymphatics can be protective against lymphedema. Methods: The standard murine tail lymphedema model was utilized which involves creating a 3 mm circumferential skin excision by the base of the tail and surgically clipping two lymphatics. This standard model was used for a control (Group 1, Control A, n=6). A second control (Group 2, Control B, n=5) had 3 mm circumferential skin excision, one tail lymphatic clipped at that level, and an immediate second hemi-circumferential skin excision on the more proximal tail with disruption of the other remaining lymphatic. Group 3 (experimental lymphatic preconditioning, n=6), had a 3 mm full thickness skin excision by the base of the tail, disruption of one tail lymphatic at that level (leaving one lymphatic vessel intact) followed by staged hemi-circumferential skin excision with disruption of the remaining lymphatic vessel 7 days later. Tail volume was assessed with tail measurements using the truncated cone equation. Immunohistochemistry and histology was sent. Results: Group 3 (experimental lymphatic preconditioning) had a change in tail volume of 79.1 mm3 compared to Group 1 (Control A) 154.6 mm3 (p=0.03) and Group 2 (Control B) 126.6 mm3 (p=0.05) at 28 days post-lymphatic injury. Conclusion: Mice that underwent lymphatic preconditioning with partial lymphatic injury followed by staged completion of lymphatic disruption 7 days later exhibited less tail swelling. This study demonstrates evidence for a novel concept of “lymphatic delay phenomenon” parallel to the well-known vascular delay phenomenon. Lymphatic preconditioning has potential translational clinical applications for protective effects to minimize lymphatic dysfunction.
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    97. Focal Gene Delivery in the Murine Lymphedema Tail Model Using Tissue Nanotransfection Technology (TNT)
    (Wolters Kluwer, 2022) Mohan, Ganesh; Khan, Imran; Sinha, Mithun; Gordillo, Gayle M.; Sen, Chandan K.; Hassanein, Aladdin H.; Surgery, School of Medicine
    Background: Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. It affects an estimated 5 million Americans. There is no cure for this disease. Experimental gene-based therapeutic approaches (e.g., using viral vectors) have had limited translational applicability. Tissue nanotransfection technology (TNT) utilizes a direct, transcutaneous non-viral vector, gene delivery using a chip with nanochannel poration in a rapid (<100ms) focused electric field. This platform technology has been used for various applications in tissue reprogramming. The ability to deliver genetic cargo at a focal, non-global site would have practical clinical potential in lymphedema treatment. The purpose of this study is to experimentally evaluate the applicability of TNT for lymphedema. 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). TNT was applied with square wave pulse electric stimulation (10x10ms pulses, 250 V, 10 mA). The efficiency of gene delivery was assessed through qRT-PCR using primers with SYBR Green fluorescence quantification and immunostaining with anti-Prox1 antibody. Results: The experimental Group II exhibited four-fold increased expression of Prox1 using qRT-PCR compared to control Group I at the site of TNT treatment (P<0.05). Increased expression of Prox1 was also observed with immunohistochemistry 3 days post-TNT application. Intensity quantification of immunohistochemistry revealed greater expression of Prox1 in Group II when compared to Group I (P<0.05). Conclusion: This study demonstrates a novel, focal nanotechnology approach for local genetic cargo delivery in murine tail lymphedema model without the use of viral vectors for transfection. TNT can rapidly and effectively be applied for potentially therapeutic delivery of factors locally at the site of lymphedema.
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    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 Science
    Over 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.
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    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 and Chemical Biology, School of Science
    This 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.
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    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 Medicine
    This 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.
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    Breast Implant-Associated Immunological Disorders
    (Hindawi, 2022-05-04) Suh, Lily J.; Khan, Imran; Kelley-Patteson, Christine; Mohan, Ganesh; Hassanein, Aladdin H.; Sinha, Mithun; Surgery, School of Medicine
    Background: Breast implants are commonly placed postbreast cancer reconstruction, cosmetic augmentation, and gender-affirming surgery. Breast implant illness (BII) is a systemic complication associated with breast implants. Patients with BII may experience autoimmune symptoms including fatigue, difficulty concentrating, hair loss, weight change, and depression. BII is poorly understood, and the etiology is unknown. The purpose of this literature review is to characterize BII autoimmune disorders and determine possible causes for its etiology. Methods: The PubMed, Google Scholar, Embase, Web of Science, and OVID databases were interrogated from 2010 to 2020 using a query strategy including search term combinations of "implants," "breast implant illness," "autoimmune," and "systemic illness." Results: BII includes a spectrum of autoimmune symptoms such as fatigue, myalgias/arthralgias, dry eyes/mouth, and rash. A review of epidemiological studies in the past ten years exhibited evidence affirming an association between breast implants and autoimmune diseases. The most commonly recognized were Sjogren's syndrome, rheumatoid arthritis, systemic sclerosis, chronic fatigue syndrome, and Raynaud's syndrome. Explantation resulted in alleviation of symptoms in over 50% of patients, strengthening the hypothesis linking breast implants to BII. Studies have shown that silicone is a biologically inert material and unlikely to be the cause of these symptoms. This is supported by the fact that increased risk of autoimmune disease was also reported in patients with other implantable biomaterials such as orthopedic implants. Recent studies shed light on a possible role of bacterial biofilm and subsequent host-pathogen interactions as a confounding factor to this problem. Conclusion: BII could be dependent on biofilm infection and the microenvironment around the implants. The true pathophysiology behind these complaints must be further investigated so that alternative treatment regimens other than explantation can be developed. Translational significance of these studies is not limited to breast implants but extends to other implants as well.
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    CD30 Lateral Flow and Enzyme-Linked Immunosorbent Assays for Detection of BIA-ALCL: A Pilot Study
    (MDPI, 2023-10-25) Zeyl, Victoria G.; Xu, Haiying; Khan, Imran; Machan, Jason T.; Clemens, Mark W.; Hu, Honghua; Deva, Anand; Glicksman, Caroline; McGuire, Patricia; Adams, William P., Jr.; Sieber, David; Sinha, Mithun; Kadin, Marshall E.; Surgery, School of Medicine
    Introduction: Breast Implant-Associated Anaplastic Large Cell Lymphoma (BIA-ALCL) commonly presents as a peri-implant effusion (seroma). CD30 (TNFRSF8) is a consistent marker of tumor cells but also can be expressed by activated lymphocytes in benign seromas. Diagnosis of BIA-ALCL currently includes cytology and detection of CD30 by immunohistochemistry or flow cytometry, but these studies require specialized equipment and pathologists' interpretation. We hypothesized that a CD30 lateral flow assay (LFA) could provide a less costly rapid test for soluble CD30 that eventually could be used by non-specialized personnel for point-of-care diagnosis of BIA-ALCL. Methods: We performed LFA for CD30 and enzyme-linked immunosorbent assay (ELISA) for 15 patients with pathologically confirmed BIA-ALCL and 10 patients with benign seromas. To determine the dynamic range of CD30 detection by LFA, we added recombinant CD30 protein to universal buffer at seven different concentrations ranging from 125 pg/mL to 10,000 pg/mL. We then performed LFA for CD30 on cryopreserved seromas of 10 patients with pathologically confirmed BIA-ALCL and 10 patients with benign seromas. Results: Recombinant CD30 protein added to universal buffer produced a distinct test line at concentrations higher than 1000 pg/mL and faint test lines at 250-500 pg/mL. LFA produced a positive test line for all BIA-ALCL seromas undiluted and for 8 of 10 malignant seromas at 1:10 dilution, whereas 3 of 10 benign seromas were positive undiluted but all were negative at 1:10 dilution. Undiluted CD30 LFA had a sensitivity of 100.00%, specificity of 70.00%, positive predictive value of 76.92%, and negative predictive value of 100.00% for BIA-ALCL. When specimens were diluted 1:10, sensitivity was reduced to 80.00% but specificity and positive predictive values increased to 100.00%, while negative predictive value was reduced to 88.33%. When measured by ELISA, CD30 was below 1200 pg/mL in each of six benign seromas, whereas seven BIA-ALCL seromas contained CD30 levels > 2300 pg/mL, in all but one case calculated from dilutions of 1:10 or 1:50. Conclusions: BIA-ALCL seromas can be distinguished from benign seromas by CD30 ELISA and LFA, but LFA requires less time (<20 min) and can be performed without special equipment by non-specialized personnel, suggesting future point-of-care testing for BIA-ALCL may be feasible.
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    Comparison of Breast Reconstruction Outcomes Using Oxychlorosene versus Triple Antibiotic Solution for Pocket Irrigation
    (Wolters Kluwer, 2022-08-18) Bamba, Ravinder; Tran, Phu C.; Mailey, Brian A.; Lin, Jenny; DeBrock, William; Dawson, Steven; Sinha, Mithun; Hartman, Brett C.; Hadad, Ivan; Lester, Mary E.; Hassanein, Aladdin H.; Surgery, School of Medicine
    Background: Breast pocket irrigation with antiseptic solutions is performed to reduce contamination with breast implants. The optimal antiseptic irrigation solution and the efficacy of individual practices are unclear. Oxychlorosene sodium is frequently used at our institution. Oxychlorosene is bactericidal with a mechanism of action of oxidation and hypochlorination. The purpose of our study was to compare the outcomes of oxychlorosene sodium irrigation with triple antibiotic solution (TAS) in implant-based breast reconstruction. Methods: All patients who underwent implant-based reconstruction after mastectomy were reviewed. The primary predictive variable was type of solution used for pocket irrigation (TAS or oxychlorosene). Outcome variables included surgical site infection, device removal, and wound complications. Results: Between 2013 and 2018, 331 implant-based breast reconstructions were performed. Of these, 62% (n = 206) received oxychlorosene for surgical pocket irrigation (group I), and 38% (n = 125) received TAS (group II). Group I had an 11.7% (n = 24) 90-day surgical site infection rate, with 4.9% (n = 10) requiring oral antibiotics, 2.4% (n = 5) requiring intravenous antibiotics without device removal, and 4.4% (n = 9) requiring prosthetic removal. Group II had an 11.2% (n = 14) 90-day infection rate, with 5.6% (n = 7) requiring oral antibiotics, 2.4% (n = 3) requiring intravenous antibiotics without device removal, and 3.2% (n = 4) requiring removal (P = 0.90). When comparing the cost of oxychlorosene irrigation with TAS irrigation, oxychlorosene was less expensive. Conclusions: Oxychlorosene and TAS have similar surgical site infection rates in prosthetic breast reconstruction. Ease of preparation and cost make oxychlorosene a more favorable option for antibiotic irrigation in reconstructive breast surgery with prosthetic devices.