Browsing by Author "Khan, Imran"

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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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    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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    Demographics and Clinicopathologic Profile of Pulmonary Sarcomatoid Carcinoma with Survival Analysis and Genomic Landscape
    (MDPI, 2023-04-26) Ullah, Asad; Ahmed, Asim; Yasinzai, Abdul Qahar Khan; Lee, Kue Tylor; Khan, Israr; Asif, Bina; Khan, Imran; Tareen, Bisma; Kakar, Kaleemullah; Andam, Gul; Heneidi, Saleh; Khan, Jaffar; Khan, Hina; Karki, Nabin R.; Del Rivero, Jaydira; Karim, Nagla Abdel; Pathology and Laboratory Medicine, School of Medicine
    Background: Pulmonary sarcomatoid carcinoma (PSC) is a rare subtype of non-small cell lung cancer (NSCLC) with an aggressive clinical nature and poor prognosis. With novel targeted therapeutics being developed, new ways to effectively treat PSC are emerging. In this study, we analyze demographics, tumor characteristics, treatment modalities, and outcomes of PSC and genetic mutations in PSC. Methods: Data from the Surveillance, Epidemiology, and End Results (SEER) database were reviewed to analyze cases of pulmonary sarcomatoid carcinoma from 2000 to 2018. The molecular data with the most common mutations in PSC were extracted from the Catalogue Of Somatic Mutations in Cancer (COSMIC) database. Results: A total of 5259 patients with PSC were identified. Most patients were between 70 and 79 years of age (32.2%), male (59.1%), and Caucasian (83.7%). The male-to-female ratio was 1.45:1. Most tumors were between 1 and 7 cm in size (69.4%) and poorly differentiated (grade III) (72.9%). The overall 5-year survival was 15.6% (95% confidence interval (95% CI) = 14.4-16.9)), and the cause-specific 5-year survival was 19.7% (95% CI = 18.3-21.1). The five-year survival for those treated with each modality were as follows: chemotherapy, 19.9% (95% CI = 17.7-22.2); surgery, 41.7% (95% CI = 38.9-44.6); radiation, 19.1% (95% CI = 15.1-23.5); and multimodality therapy (surgery and chemoradiation), 24.8% (95% CI = 17.6-32.7). On multivariable analysis, age, male gender, distant stage, tumor size, bone metastasis, brain metastasis, and liver metastasis were associated with increased mortality, and chemotherapy and surgery were associated with reduced mortality (p < 0.001). The best survival outcomes were achieved with surgery. The most common mutations identified in COSMIC data were TP53 31%, ARID1A 23%, NF1 17%, SMARCA4 16%, and KMT2D 9%. Conclusions: PSC is a rare and aggressive subtype of NSCLC, usually affecting Caucasian males between 70 and 79. Male gender, older age, and distant spread were associated with poor clinical outcomes. Treatment with surgery was associated with better survival outcomes.
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    A Murine Tail Lymphedema Model
    (JoVE, 2021) Hassanein, Aladdin H.; Sinha, Mithun; Neumann, Colby R.; Mohan, Ganesh; Khan, Imran; Sen, Chandan K.; Surgery, School of Medicine
    Lymphedema is extremity swelling caused by lymphatic dysfunction. The affected limb enlarges because of accumulation of fluid, adipose, and fibrosis. There is no cure for this disease. A mouse tail model that uses a focal full thickness skin excision near the base of the tail, resulting in tail swelling, has been used to study lymphedema. However, this model may result in vascular comprise and consequent tail necrosis and early tail swelling resolution, limiting its clinical translatability. The chronic murine tail lymphedema model induces sustained lymphedema over 15 weeks and a reliable perfusion to the tail. Enhancements of the traditional murine tail lymphedema model include 1) precise full thickness excision and lymphatic clipping using a surgical microscope, 2) confirmation of post-operative arterial and venous perfusion using high resolution laser speckle, and 3) functional assessment using indocyanine green near infrared laser lymphangiography. We also use tissue nanotransfection technology (TNT) for novel non-viral, transcutaneous, focal delivery of genetic cargo to the mouse tail vasculature.
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    Oxylipins in Breast Implant–Associated Systemic Symptoms
    (Oxford University Press, 2024) Khan, Imran; Timsina, Lava; Chauhan, Ruvi; Ingersol, Christopher; Wang, David R.; Rinne, Ethan; Muraru, Rodica; Mohan, Ganesh; Minto, Robert E.; Van Natta, Bruce W.; Hassanein, Aladdin H.; Kelley-Patteson, Christine; Sinha, Mithun; Surgery, School of Medicine
    Background: A subset of females with breast implants have reported a myriad of nonspecific systemic symptoms collectively termed systemic symptoms associated with breast implants (SSBI). SSBI symptoms are similar to manifestations associated with autoimmune and connective tissue disorders. Breast tissue is rich in adipose cells, comprised of lipids. Insertion of an implant creates an oxidative environment leading to lipid oxidation. Oxylipins can influence immune responses and inflammatory processes. Objectives: In this study we explored the abundance of a spectrum of oxylipins in the periprosthetic tissue surrounding the breast implant. Because oxylipins are immunogenic, we sought to determine if they were associated with the SSBI patients. We have also attempted to determine if the common manifestations exhibited by such patients have any association with oxylipin abundance. Methods: The study included 120 patients divided into 3 cohorts. We analyzed 46 patients with breast implants exhibiting manifestations associated with SSBI; 29 patients with breast implants not exhibiting manifestations associated with SSBI (control cohort I, non-SSBI); and 45 patients without implants (control cohort II, no-implant tissue). Lipid extraction and oxylipin quantification were performed with liquid chromatography mass spectrometry (LC-MS/MS). LC-MS/MS targeted analysis of the breast adipose tissue was performed. Results: Of the 15 oxylipins analyzed, 5 exhibited increased abundance in the SSBI cohort when compared to the non-SSBI and no-implant cohorts. Conclusions: The study documents the association of the oxylipins with each manifestation reported by the patient. This study provides an objective assessment of the subjective questionnaire, highlighting which symptoms may be more relevant than the others.