Aziz Ghahary

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Open magnet resonance imaging : application of new technology to improve the evaluation of pelvic organ prolapse in women (2019)

Background: Weakness or damage to the pelvic floor muscles results in pelvicorgan prolapse (POP), which affects 50% of women >50 years old. Posture and gravityimpact organ position and symptom severity. Current limitations of clinical examinationand restriction to imaging in the supine position impact accurate diagnosis and diseasestaging. Open upright magnetic resonance imaging (MRO), allows images of patientssitting, standing, and supine. In this dissertation it is hypothesized that MRO images willallow improved detection of the presence and extent of prolapse.Methods: A Paramed Medical Systems 0.5 T upright Open MRI scanner (MRO)was used to obtain axial and sagittal T2-weighted pelvic scans in women when supine,sitting, and standing. Symptomatic women with POP and asymptomatic controls werestudied. The protocol developed obtains good quality images efficiently in all threepositions. Validated reference lines were used to identify where POP was present in thesame patient in different positions and to grade its severity. A manual segmentationmethodology was developed using Analyze 12.0 software to construct 3D models of thefemale pelvis from 2D images to enhance the visualization of complex pelvic anatomy.Results: Forty women (20 with POP and 20 asymptomatic controls) werestudied. Detection of POP in standing versus supine images in symptomatic womenwas 50% vs. 5% for cystocele and 35% vs. 25% for vaginal prolapse, indicatingimproved visualization of gravity-dependent POP with MRO standing imaging. Imagecomparison indicated that the extent of prolapse is best evaluated in the standingposition using the pubococcygeal reference line. These images better identifydownward movement in the anterior and posterior compartments. No appreciablebenefit was afforded by seated images.Conclusion: The findings support the hypothesis that MRO imaging of POPpatients is relevant to improving the detection and quantification of POP. The MROprotocol developed for standing images in women with POP demonstrated feasibilityallowing supine, sitting, and standing imaging of changes in pelvic floor anatomy inupright positioning. When compared with supine images, standing images better identifythe presence and extent of POP. 3D image modeling allows more comprehensivevisualization of complex female pelvic anatomy.

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Evaluating the therapeutic use of Indoleamine 2,3-dioxygenase (IDO) expressing allogenic dermal fibroblast populated within an acellular skin substitute as a wound coverage (2018)

Acute and chronic wounds contribute to increased morbidity and mortality in affected people and impose significant financial burdens on healthcare systems. Allogeneic cell-based skin substitutes have been proposed as ready-to-use wound coverage as an alternative to conventional split-thickness skin grafts, but the survival and usefulness of such cells after transplantation into an immunocompetent host remain controversial.We hypothesize that the application of an indoleamine 2,3-dioxygenase (IDO) expressing allogenic dermal fibroblast populated within an acellular dermal matrix (ADM) is sufficient to create an immune-privileged area within the wound to protect fibroblasts from rejection. Fibroblasts in the skin substitute can potentially assist the graft to restore its function by synthesizing extracellular matrix components and growth factors.In this study, ADMs were prepared using a novel detergent–free method, recellularized with IDO-expressing or control fibroblasts from C57/B6 mice, and were transplanted on splinted full-thickness skin wounds in Balb/c mice.Transplantation studies demonstrated that ADM significantly enhanced the wound-healing process but there was no demonstrable benefit when the ADM was recellularized with fibroblasts. Investigating the transplanted cells’ fate by bioluminescence in vivo imaging after intra-hypo-dermal injection of fibroblasts in to mouse skin revealed that both type of fibroblasts were rejected in allogeneic recipients while in immunodeficient NOD-SCID-Il2r gamma null (NSG) mice they were not. Allogeneic fibroblasts transplanted in natural killer T cell and gamma delta T (NKT) cell deficient mice were rejected as well. Depleting natural killer (NK) cells or CD4+ T cells could delay the rejection but not prevent it. Double depletion of CD4+ and CD8+ cells could partially prevent the rejection. Analyzing the infiltrated immune cells to graft region in time of cell rejection revealed the presence of high number of monocytes, macrophage and neutrophils. Our data indicate a clear immune response to allogeneic fibroblasts in which both innate and T-cell immune response are involved in targeting cells. Although it has been reported by other researchers that IDO-expressing cells exhibit strong immunosuppressive activity in vitro and in vivo, we documented rejection of these cells in our study, suggesting that the application of these cells in wound sites requires further improvements.

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Anti-inflammatory role of IDO and tryptophan metabolites (2017)

Inflammation is essential to the establishment of homeostasis following injury and inflammatory cell and the cytokine network associate with tissue repair. However, sometimes inflammation can cause further inflammation; it can become self-perpetuating. One of the many possibilities of prolonged secretion of cytokines and growth factor is autoimmunity and delay in wound closure. The current anti-inflammatory treatment modalities vary however their adverse effects are common. Here we asked the question of whether Kynurenine (Kyn), one of the tryptophan (Trp) metabolite, could modulate the inflammation by altering the profile of the key pro-inflammatory cytokines as well as the proliferation of immune cells. We showed that Kyn treatment significantly reduced some pro-inflammatory cytokines and chemokines such as IL-17, IL-2, CXCL-9 and CXCL-10 in ConA⁺ Kyn-treated splenocytes. To validate our findings in a wound healing model, we also showed that topical application of Kyn cream resulted in fewer infiltration of CD3⁺ T cells at wound site. Further, in this study we used kynurenic acid (KynA) instead of Kyn as KynA is the end product and safer metabolites in the kynurenine pathway. The emphasis was given in evaluating the effect of KynA on expression of IL-17/IL-23 axis which has recently been identified to be very important in the immunopathogenesis of autoimmune diseases and inflammation such as psoriasis. Our findings have shown that KynA can modulate the frequency of IL-23 and IL-17 by DCs and CD4⁺ cells. Moreover, we showed that KynA suppresses the production of IL-23 in DCs through GPCR35 activation. We then evaluated the therapeutic use of intra-lesional injection of IDO-expressing fibroblasts, as a source of Kyn and KynA production in psoriasis, which is one of the most common recurrent chronic inflammatory diseases of the skin. The findings of this work demonstrated that IDO-expressing cells significantly improved thickness, erythema, and scaling scores in skin psoriatic like condition. Moreover, IDO-expressing fibroblasts reduce infiltration of IL-17⁺ CD4⁺, IL-17⁺ γδ⁺ T cells, IL-23⁺-activated dendritic cells and granulocytes in skin psoriatic like condition. The findings presented in this thesis collectively prove the potent local immunosuppressive activity of IDO-expressing dermal fibroblast and tryptophan metabolites in skin inflammatory conditions.

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Development and application of anti-fibrogenic dressings (2016)

It is well established that more than 15 million patients develop pathological scarring each year following elective operations, surgical procedures, deep trauma, thermal and electrical injuries. These scars, that cause major functional, cosmetic, psychological, and social consequences for the patients, impose a significant financial burden on health care systems. The current treatment modalities for these pathological conditions vary from topical application and intralesional injection of anti-scarring agents to surgical revisions and radiotherapy. The limited efficacy of these therapeutics for prevention of scar formation raised a great need for innovation within the wound care industry.Recently Kynurenine (Kyn), a tryptophan metabolite, has been identified as a potent anti-fibrotic agent. Kyn prevents scar formation by enhancing the expression of ECM degrading enzymes, matrix metalloproteinases (MMPs), and suppressing the expression of collagen. Although daily topical application of Kyn-cream improved the wound healing outcome in animal models, this method of drug delivery is not clinically practical in situations where dressings need to be kept on for 3-5 days.In this dissertation, it is hypothesized that topical application of a slow and controlled releasing Kyn or its metabolites from nanofiber dressing at the wound site improves and/or prevents dermal fibrosis by modulating the expression of the key ECM components involved in dermal fibrotic conditions. To test this hypothesis, three specific objectives were employed: (1) Evaluating and comparing the anti-fibrotic effects of Kynurenic acid (KynA) and Kyn, (2) Developing, characterizing and optimizing the nanofibrous dressings as a slow releasing drug delivery system for Kyn and KynA, (3) Examining the functionality of the developed anti-fibrotic dressings in open wounds in animal models. The findings of these specific objectives of this work demonstrated that topical application of the developed polymeric dressings, which slowly release Kyn/KynA over the course of 4 days, effectively reduces dermal fibrosis by modulating the key ECM components such as MMPs, collagen and fibronectin. The findings of this study support our hypothesis that development of an anti-fibrogenic dressing is feasible and as such its application would overcome the difficulties associated with development of hypertrophic scarring frequently seen in millions of patients worldwide.

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Cell- and matrix-based approaches for improvement of islet beta-cell survival and function in vitro and in vivo (2015)

Islet transplantation provides a feasible approach for treatment of type 1 diabetes (T1D), in which islet β-cells are destroyed by autoimmune attack. However, its efficacy is currently limited by poor long-term islet graft survival. Loss of islet extracellular matrix (ECM) and formation of human islet amyloid polypeptide (hIAPP) aggregates are two important non-immunological factors contributing to β-cell destruction. In this study, we developed novel matrix- and cell-based approaches under 3 different objectives. We hypothesized that these new strategies can protect islet β-cells from the toxicity induced by non-immune or autoimmune factors thereby enhancing the viability and function of islet β-cells in both in vitro and in vivo diabetes models. To provide surrogate ECM materials for islets, three-dimensional scaffolds, collagen matrix (CM) alone and human fibroblasts-populated collagen matrix (FPCM) were developed in objective 1. Isolated human islets were then either embedded within the scaffolds or cultured in two-dimensional free-floating condition (control) for 7 days. The findings showed markedly lower formation of hIAPP aggregates in the scaffold-embedded islets as compared to control islets. The morphology, viability, and functionality of ECM-populated islets were significantly improved as compared to control islets. In objective 2, we evaluated the signaling pathways involved in the cytotoxicity induced by hIAPP aggregation. Protein kinase B (PKB), a key effector of the pro-survival phosphoinositide 3 (PI3)-kinase signaling pathway was studied. Our findings demonstrated that both exogenously applied and endogenously formed hIAPP aggregates reduce PKB activation in islet β-cells, likely via IL-1β signaling. Autoimmunity is another causative factor of β-cell destruction during islet transplantation and T1D. In objective 3, we aimed to prevent the progression of T1D in non-obese diabetic (NOD) mice. We used dermal fibroblasts expressing indoleamine 2,3-dioxygenase (IDO), which is an immuno-modulating enzyme. We found that intraperitoneal injection of 15 million dermal fibroblasts into NOD mice successfully reversed the progression of T1D through inhibiting the β-cell specific autoreactive T cells and Th17 cells, as well as inducing regulatory T cells. Collectively, the approaches developed in this study can reduce the destruction of islet β-cells by non-immune and autoimmune factors thereby enhancing viability and function of islet β-cells in diabetes.

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Development and application of an in-situ forming, biohybrid scaffold for wound repair (2015)

Tissue engineering has advanced rapidly over the past decade in an effort to address unmet medical needs in burn and chronic wound treatment. Each year, over 1 million patients seek medical attention for burn injuries in North America. Moreover, chronic wounds in the elderly and those with diabetes comprise the largest single segment of wound care. Without question current treatments are costly and challenging for healthcare professionals. When autografts are not possible, skin substitutes are often employed as alternative coverage. Although these strategies have dramatically improved healing in patients, they are limited by the time they take to fully integrate with surrounding tissue. Our idea to bridge the gap is to create a patient-ready skin substitute. Moving toward a skin substitute that is readily available for the patient, I developed an in-situ gelling scaffold that permits integration with surrounding tissue. Furthermore, it was my goal to create a system that resists cell mediated contracture and digestion, and provide an ideal environment for tissue repair. My hypothesis was that the fabrication of a composite matrix of collagen and a blended-polymer hydrogel would result in a material that could be lyophilized, reconstituted and gel rapidly in-situ. The objectives of this work are fourfold: (1) develop, characterize, optimize and evaluate the functionality of a reconstitutable in situ forming scaffold in vitro; (2) evaluate the efficacy of the scaffold to perform within an acute wound; (3) evaluate methods to tailor the scaffold to be used as a cell delivery vehicle; and (4) develop a prototype model product that could translated to the clinic. Satisfying the objectives of this work have demonstrated that with a biocompatible concentration of hydrogel collagen fibrillogenesis activation energy can be lowered, and the mechanical and physical properties of the resulting scaffold are enhanced. The resulting scaffold can be further lyophilized and reconstituted to form composite skin and other matrices both in vitro and in vivo. Assuming that regulatory requirements are met, the scaffold has the potential to improve the quality of life of patients with devastating chronic wounds and burns, and advance further knowledge in the field of tissue engineering.

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Development and application of a non-rejectable islet graft using indoleamine 2,3-dioxygenase (IDO) in diabetes. (2014)

Type 1 diabetes (T1D) is a devastating disease affecting more than 22 million people worldwide. Early diagnosis and treatment of T1D requires a deep understanding of the mechanisms underlying the progression of autoimmune diabetes. It has been shown that impaired IFNγ-induced indoleamine 2,3-dioxygenase (IDO) expression in dendritic cells of early prediabetic female nonobese diabetic (NOD) mice could contribute to their defective self-tolerance. IDO is an immunomodulatory and rate-limiting enzyme in tryptophan catabolism. Since fibroblasts are important components of islet extracellular matrix (ECM), we investigated IFNγ-induced IDO expression in NOD dermal fibroblasts. Our findings indicate that IFNγ fails to induce IDO expression in NOD dermal fibroblasts and moreover that the mechanism underlying this defect involves defective STAT1 phosphorylation in the IFNγ-induced-IDO signaling pathway. We further showed that an IFNγ-independent IDO expression pathway is operative in NOD mice fibroblasts. Islet transplantation, a promising strategy to restore efficient insulin regulation in T1D, is limited by poor post-transplant islet survival and toxicity of immunosuppressants. We developed a novel bioengineered cross-linked collagen matrix (CCM) to act as an ECM for the islets and found that islet function and survival significantly improved with this scaffold. We previously showed that in an IDO-mediated microenvironment, infiltrated immune cells but not islet cells cannot survive or proliferate. Here, we used IDO to generate a local immunomodulated environment, in which transplanted islets could remain viable and protected without compromising systemic immunity. We developed an improved islet allograft composed of stable IDO-expressing dermal fibroblasts (by lentiviral transduction) and allogeneic islets embedded within CCM in streptozotocin-induced diabetic mice. This IDO-expressing matrix did not compromise islet function or survival. IDO expression significantly suppressed the proliferation of alloantigen-stimulated splenocytes. Finally, we showed that local IDO expression delivered by a lentiviral vector significantly prolonged islet allograft survival by increasing the population of FOXP3+ cells at the graft site and preventing T cell infiltration. Overall, the studies in this thesis showed how defects in the IDO signaling pathway may underlie autoimmune diabetes and highlighted the therapeutic potential of IDO expression and improved matrices for enhancing survival and function of islet transplants in T1D.

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Role of aminopeptidase N in wound healing (2012)

The dynamics and complexity of tissue repair are dominated by specific andintricately coordinated cellular events. Disruptions at the level of cellularcommunication are associated with imbalanced extracellular matrix (ECM)synthesis/degradation leading to fibrosis and chronic wounds. Our group hasdemonstrated that 14-3-3 sigma (also known as stratifin) functions as astimulator of matrix metalloproteinase-1 (MMP-1) through interactions withaminopeptidase N (APN) on the surface of dermal fibroblasts. In this doctoralresearch project, it is hypothesized that APN functions as a receptor forkeratinocyte-derived paracrine signals that control the expression of key ECMcomponents in dermal fibroblasts.Three specific objectives were accomplished in this project. Under Objective1, the nature of APN expression in an environment of active epithelial-stromalcommunication was examined using an in vitro keratinocyte-fibroblast crosstalkmodel. The fibroblast expression of APN was significantly upregulated in thepresence of keratinocyte-releasable soluble factors, of which stratifin was shownto be a potent stimulator. In light of the recent identification of APN as a receptorresponsible for stratifin-mediated p38 MAPK activation leading to upregulation ofMMP-1, the role of APN as a transmembrane mediator of signals that regulateECM remodeling was investigated in Objective 2. Comparative analysis of theexpression profiles of 118 ECM genes under conditions of keratinocytestimulation and APN gene silencing revealed a group of key matrix proteasesand adhesion molecules influenced by keratinocyte-derived signaling mediated through APN. The aim of Objective 3 was to explore the therapeutic potential of targeting APN in cutaneous tissue repair. Topical application of an APN-neutralizing antibody on full-thickness skin wounds in a murine model had a positive outcome in healing. Acceleration of wound closure was accompanied by increased collagen deposition and fibroblast contractility.Collectively, the findings presented herein confirmed our hypothesis that APN can be induced by keratinocytes and acts as a regulator of keratinocyte-derived stimuli in epidermal-dermal communication. Specifically, these findings support the receptor role of APN in mediating transmembrane signals derived from keratinocytes, and provide encouraging evidence for further investigations on the therapeutic use of APN agonist/antagonists in the field of tissue repair.

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Identification and mechanism of action of keratinocyte-releasable factors in regulation of extracellular matrix expression (2011)

The complexity of multi-cellular organisms demands a dynamic reciprocity between neighboring cells in any given tissue. The disruption in keratinocytes and fibroblasts cross-talk in skin has been linked to an imbalance in extracellular matrix (ECM) expression leading to the onset of fibrosis such as hypertrophic scarring. Our group has recently identified 14-3-3σ or stratifin (SFN) as a potent MMP-1 stimulatory factor in fibroblasts. In this doctoral research project, we hypothesized that SFN can modulate other ECM components and execute its transmembrane signaling through interaction with a receptor on the surface of fibroblasts. Three specific objectives were accomplished in this project. Under objective 1, ECM gene expression profile of fibroblasts treated with SFN or co-cultured with keratinocytes was characterized by an ECM-pathway specific gene array and revealed that SFN upregulates a wider range of MMPs such as MMP-3, 8, 10, and 24 other than MMP-1. As SFN was not responsible for the keratinocyte-mediated decrease in collagen expression, under objective 2 attempts were made to characterize the nature of a collagen inhibitory factor in keratinocyte-conditioned medium (KCM). Analysis of keratinocyte/fibroblast co-culture and KCM revealed a 30-50 kDa keratinocyte-derived collagen inhibitory factor with stable activity at high temperature (56 ºC) and acidic environment (pH=2, 30 min). Under objective 3, SFN’s transmembrane signaling mechanism was investigated by utilizing a combination of receptor ectodomain biotin labeling, serial affinity purification, and MS/MS to identify aminopeptidase N or CD13 (APN) as a potential SFN receptor in fibroblasts. APN/SFN binding was further confirmed by immunoprecipitation, cross-linking, and co-distribution studies. Expression of APN and SFN increased after wound closure in a rabbit ear fibrotic model as well as a longitudinal study in rats. The transient knockdown of APN blocked SFN-mediated p38 MAPK activation and MMP-1 expression. Collectively, the findings presented in this thesis provide further support for the importance of keratinocyte-releasable factors in the regulation of ECM and MMP expression in fibroblasts. We also identify APN as a novel cell surface receptor for SFN. Therefore, our findings may provide additional therapeutic tools for the regulation of MMP expression in dermal fibrosis and chronic wound healing disorders.

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Keratinocyte-releasable factors modulate extracellular matrix components in dermal fibroblasts (2011)

A fine balance between the synthesis and degradation of extracellularmatrix (ECM) is required in maintaining the structural integrity of healing tissue.An imbalance in ECM expression leads to fibrotic conditions such ashypertrophic scars (HS). It has been demonstrated that keratinocyte-releasablefactors can function as ECM modulating factors (MMP-1 and type I collagen) infibroblasts. We have shown that Stratifin (SFN) is an MMP-1 stimulatory factor infibroblasts that failed to suppress the expression of type I collagen in fibroblasts.SFN is an intracellular protein that lacks a signal peptide. As such, it is critical toexplore its mechanism of release and identify the keratinocyte-derived collageninhibitingfactor(s) for dermal fibroblasts. In this doctoral research project Ihypothesize that keratinocyte-releasable factor(s) function as a stop signal(s) forwound healing by modulating the expression of key ECM components such asMMP-1 and type I collagen in fibroblasts.Two specific objectives were accomplished to address these issues.Under objective 1, the mechanism by which SFN is released has been explored.The findings demonstrate that SFN is released via exosomes in a Ca²⁺dependant fashion. Moreover, only differentiated keratinocytes release SFN.Exosome-associated SFN exhibits a potent MMP-1 stimulatory effect. Underobjective 2, using a series of systematic protein purification methods followed bymass spectroscopy, two proteins (SPARC and SFN) that inhibited collagenproduction by dermal fibroblasts were identified in keratinocyte-conditionedmedia. Using co-immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby regulating type I collagen expression infibroblasts. The levels of these proteins in fibrotic tissues (animal and human)were also evaluated and a differential expression of these proteins betweennormal and fibrotic tissue confirmed their potential role in development of fibroticcondition.In conclusion, the identification of the mechanism of release of SFN andthe identification of SPARC/SFN complex contribute to the understanding of howthese factors are involved in the wound healing process. Also, SPARC/SFNcomplex provides us with another anti-fibrogenic factor that may be used togenerate an effective therapeutic agent to treat HS frequently developedfollowing burn injury, deep trauma and/or surgical incisions.

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Mechanisms of selective immunosuppressive effects of indoleamine 2, 3-dioxygenase and borrelidin (2011)

No abstract available.

Topical and local controlled release of stratifin for the improvement of hypertrophic scarring in open and surgically closed wounds (2011)

Hypertrophic scar (HS), which results from the uncontrolled synthesis andexcessive deposition of extracellular matrix (ECM) at sites of dermal injury, representsan undesirable endpoint of wound healing. Stratifin (SFN) was recently identified as apotent matrix methaloproteinase-1 (MMP-1) stimulatory factor in dermal fibroblasts. Inthis research project, it is hypothesized that stratifin can modulate the deposition of ECMcomponents and prevent HS formation when it is applied topically to open wounds orlocally delivered to surgically closed wounds.Four specific objectives were accomplished in this project. Under objective 1, ahydrogel /microsphere dermal implant was fabricated, specifically to be placed insurgical wounds before closure and locally deliver stratifin in a controlled manner toreduce HS formation. Microencapsulating stratifin complexed chitosan particles intoPLGA microspheres allowed bioactive stratifin to be controllably released over 30 dayswith a reduced burst release. Under objective 2, use of a rat surgical wound modelshowed that the local controlled delivery of stratifin markedly reduced fibrosis andinflammation induced by drug-free implants, confirmed by a reduced collagen deposition,total tissue cellularity, and infiltrated CDHypertrophic scar (HS), which results from the uncontrolled synthesis andexcessive deposition of extracellular matrix (ECM) at sites of dermal injury, representsan undesirable endpoint of wound healing. Stratifin (SFN) was recently identified as apotent matrix methaloproteinase-1 (MMP-1) stimulatory factor in dermal fibroblasts. Inthis research project, it is hypothesized that stratifin can modulate the deposition of ECMcomponents and prevent HS formation when it is applied topically to open wounds orlocally delivered to surgically closed wounds.Four specific objectives were accomplished in this project. Under objective 1, ahydrogel /microsphere dermal implant was fabricated, specifically to be placed insurgical wounds before closure and locally deliver stratifin in a controlled manner toreduce HS formation. Microencapsulating stratifin complexed chitosan particles intoPLGA microspheres allowed bioactive stratifin to be controllably released over 30 dayswith a reduced burst release. Under objective 2, use of a rat surgical wound modelshowed that the local controlled delivery of stratifin markedly reduced fibrosis andinflammation induced by drug-free implants, confirmed by a reduced collagen deposition,total tissue cellularity, and infiltrated CD³⁺ immune cells.Under objective 3, the anti-fibrogenic effect of topically applied stratifin wasinvestigated on open wounds created in a rabbit ear fibrotic model. Qualitative woundassessment showed a reduced HS in wounds treated with stratifin-impregnated CMC gel(0.002%, w/w) applied twice a day, confirmed with reduced scar volume includingdeposited collagen and total tissue cellularity. Under objective 4, CMC gel was modifiedinto a thermoreversible emulgel, which demonstrated a significant effect on scarreduction through its occlusive effect. Using this emulgel and combining stratifin withacetylsalicylic acid significantly reduced HS, even with once a day application.In conclusion, the findings presented in this thesis provide further support for theimportance and feasibility of using stratifin as an MMP-1 stimulatory factor for theimprovement/prevention of dermal fibrosis in open and surgically closed wounds. Thesefindings also provide additional information regarding controlled delivery of proteins toany kind of wounds, such as those resulting from surgical or burns. immune cells.Under objective 3, the anti-fibrogenic effect of topically applied stratifin wasinvestigated on open wounds created in a rabbit ear fibrotic model. Qualitative woundassessment showed a reduced HS in wounds treated with stratifin-impregnated CMC gel(0.002%, w/w) applied twice a day, confirmed with reduced scar volume includingdeposited collagen and total tissue cellularity. Under objective 4, CMC gel was modifiedinto a thermoreversible emulgel, which demonstrated a significant effect on scarreduction through its occlusive effect. Using this emulgel and combining stratifin withacetylsalicylic acid significantly reduced HS, even with once a day application.In conclusion, the findings presented in this thesis provide further support for theimportance and feasibility of using stratifin as an MMP-1 stimulatory factor for theimprovement/prevention of dermal fibrosis in open and surgically closed wounds. Thesefindings also provide additional information regarding controlled delivery of proteins toany kind of wounds, such as those resulting from surgical or burns.

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Circulating Bone Marrow-derived Precursor Cells Modulate the Wound Healing Outcome by Cell Transdifferentiation (2009)

No abstract available.

Development and application of a non-rejectable composite pancreatic islet allograft using indoleamine 2, 3 dioxygenase in a diabetic mouse model (2009)

Success of transplantation of pancreatic islets as a promising therapeutic method for restoring efficient regulated insulin secretion in type 1 diabetes depends on lifelong use of immunosuppressive drugs. With the goal of eliminating the necessity of systemic immunosuppressive agents after islet transplantation, in this doctoral research project we hypothesized that a novel non-rejectable islet graft through employing a local immunosuppressive factor, indoleamine 2, 3 dioxygenase (IDO) can be developed and applied. IDO is a tryptophan degrading enzyme and functions as a potent immunomodulatory factor. To address this hypothesis, we engineered a three-dimensional composite islet graft equipped with IDO expressing bystander cells as local immunosuppressive system. In this composite graft, expression of IDO in syngeneic fibroblasts provided a low tryptophan microenvironment within which T-cells could not proliferate and infiltrate islets.Three specific aims were accomplished in this study. We first showed that local expression of IDO in syngeneic bystander fibroblasts efficiently suppressed in vitro proliferation of lymphocytes stimulated with allogeneic pancreatic islets. In the next phase, the impact of IDO on viability and function of mouse islets embedded within IDO-expressing fibroblast-populated collagen matrix was investigated. The result showed no reduction in islet cells viability and comparable insulin content and secretion in IDO-expressing versus control preparations. In contrast to lymphocytes, a nutrient deficiency stress-responsive pathway was not activated in islets co-cultured with IDO-expressing fibroblasts confirming the selective suppressive effect of IDO on immune versus islet cells. Furthermore, when allogeneic immune response was eliminated by using a syngeneic transplant model, IDO-expressing composite islet grafts were functional in vivo for up to 100 days. Finally, to address the last specific aim, composite allogeneic islet grafts were transplanted into renal subcapsular space of streptozotocin-induced diabetic immunocompetent mice. IDO-expressing grafts survived significantly longer than controls without using any systemic immunosuppressive agent (41.2±1.64 vs. 12.9±0.73 days, p
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Development and application of non-rejectable skin substitute to improve wound healing (2009)

Skin substitutes consist of dermal and epidermal components are very beneficial to improve the current strategies of wound healing. However, these substitutes are not yet a routine treatment for burns or non-healing wounds mainly due to the difficulty of timely obtaining autologous skin cells. The aim of this study is to investigate the immunoprotective role of IDO in bilayer skin substitutes made out of non-autologous cells. Toaddress this aim, first we asked the question of whether tryptophan deficient environmentcaused by IDO expression is safe for the survival of nonimmune cells, mainly primary skin cells. The results of our study showed a significant activation of apoptotic pathway as well as GCN2 kinase pathway in T cells, but not in skin cells, in response to the tryptophan deficient environment mediated by IDO expression. We then studied whether there is any differences between the main T cell populations in response to IDO mediated low tryptophan environment. Our results showed a marked immunosuppressive effect ofIDO expression on human T cells with more suppressive effect on proliferation ofCD8⁺ compared to that of CD4⁺ T cells which is, at least in part, due to differences in thelevel of GCN2 kinase pathway activation between these two sets of immune cells. Thereafter, we developed a bilayer skin substitute equipped with IDO expression ability. We found that IDO expressed by treated fibroblasts of this skin substitute suppress the proliferation of bystander lymphocytes in vitro considerably. Further, in our in vivo experiments, we found that the expression of IDO by cells of skin substitute significantlyimproved the wound healing rate, reduce the number of infiltrating T cells, and induced more revascularization in the wounds received IDO expressing skin substitutes compared to the non-IDO expressing ones. In conclusion, the results of this thesis research revealedthat IDO expression can improve the efficacy of non-autologous bilayer skin substitutes to be used not oniy as a wound coverage, but also as a source of wound healing process improvement. A better revascularization seen in IDO grafted skin substitute further improved the graft take and survival.

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Master's Student Supervision (2010 - 2018)
Evaluating the application of a shelf-ready reconstitutable liquid skin substitute for treatment of full-thickness wounds in a porcine model (2018)

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Deploying the tolerogenic effects of IDO enzyme and skin fibroblasts in prevention of graft rejection (2017)

Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme with tolerogenic effects on different immune cells. Our group has previously shown that co-transplantation of IDO-expressing fibroblasts with donor tissues can delay immune rejection by inducing local immunosuppression. We first asked a question whether we can improve this effect by delivering the IDO-fibroblasts through a systemic intraperitoneal approach, instead of local co-transplantation, and secondly whether this effect is only delivered by the immunosuppressive effects of IDO or the fibroblast cells have additional immunosuppressive effects.We employed a systemic approach to improve allograft survival without using any immunosuppressive medication. To achieve this, 10 million lentiviral transduced IDO-expressing donor derived fibroblasts were injected into the peritoneal cavity of allograft recipients. We showed that IDO-fibroblast therapy increases the survival of both islets and skin allografts and decreases the infiltration of immune cells in subcutaneous transplanted skins. Indirect pathway of allo-reactive T cell activation was suppressed more than the direct pathway. Injected IDO-fibroblasts were found in peritoneal cavity and mesenteric lymph nodes of the recipient mice. In conclusion, fibroblasts have tolerogenic effects on DCs and IDO-expressing fibroblast therapy proved to be a novel approach in improving the allogeneic graft survival.There is controversy about the immunomodulatory effect of fibroblasts on dendritic cells (DCs). In a mouse model, we showed that intra- peritoneal injection (IP) of both syngeneic and allogeneic fibroblasts significantly increased the expression level of co-inhibitory and co-stimulatory molecules on DCs. Priming of DCs with syngeneic and allogeneic fibroblasts reduced the proliferation of CD4+ and CD8+ T cells. Even activation of fibroblast-primed DCs failed to restore their ability to induce T-cell proliferation. Likewise, priming of DCs with fibroblasts blocked the ability of ovalbumin-pulsed DCs to induce proliferation of ovalbumin-specific CD4+ T cells. Compared with non-activated DCs, fibroblast-primed DCs had significantly higher expression levels of interleukin-10 and IDO. Fibroblast-primed DCs had a significantly reduced interleukin- 12 expression level compared with that of activated DCs. After priming with fibroblasts, DCs were able to migrate to lymphatic tissues and present fibroblast-derived antigens (ovalbumin).

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Crosstalk between keratinocytes and dermal fibroblasts modulates the release of critical wound healing factors (2013)

Epidermal-mesenchymal communication plays a critical role in cutaneousintegrity during both normal and pathological wound healing processes. We havepreviously demonstrated that crosstalk between keratinocytes and dermalfibroblasts has the capacity to influence the expression of key extracellular matrix(ECM) components including members of the matrix metalloproteinase (MMP)and 14-3-3 protein families. In this Masters project, we hypothesized thatbidirectional communication between these cell types plays a central role inregulating local expression of critical wound healing factors.To address this question, two specific objectives were accomplished.Under Objective 1, we investigated whether fibroblasts are capable of modulatingthe expression of keratinocyte-releasable anti-fibrogenic 14-3-3 proteins byexamining the levels of four 14-3-3 family members (β, η, γ, σ) by western blotand quantitative real-time polymerase chain-reaction (qPCR). Gene analysisrevealed upregulation of all four 14-3-3 isoforms of interest in co-culturedcompared to mono-cultured keratinocytes. Additionally, co-cultured keratinocytesexpressed significantly higher levels of intracellular 14-3-3 γ and σ proteins.Under Objective 2, we examined the impact of keratinocyte-releasablefactors upon the fibroblast expression profile of key cytokines and growth factorsusing DNA microarray analysis. Fibroblasts co-cultured with keratinocytesdemonstrated significantly upregulated colony-stimulating factor 3 (CSF3)expression, which was further verified by RT-PCR and western blot.Keratinocyte-conditioned medium (KCM) from undifferentiated keratinocytes was observed to have higher CSF3-stimulatory potential than that of differentiatedcells. Partial-characterization of the CSF3-stimulatory factor(s) was achieved bytreating fibroblasts with KCM subjected to thermal stability and ammoniumsulfate precipitation analyses. Additionally, to investigate the CSF3-stimulatorypotential of interleukin-1 (IL-1) upon fibroblasts, we utilized an IL-1 receptorantagonist (IL-1ra) and revealed that keratinocyte-releasable IL-1 may be one ofthe factors responsible for stimulating CSF3 expression in fibroblasts.Our studies concluded that dermal fibroblasts regulate the expression ofseveral 14-3-3 isoforms (notably γ and σ) in keratinocytes, and conversely, thatkeratinocyte-releasable factors such as IL-1 modulate the expression of CSF3 infibroblasts. These findings underline the critical nature of crosstalk betweenkeratinocytes and fibroblasts, suggesting that bidirectional communicationbetween these cells in vivo plays a prominent regulatory role in the cytokineproduction required for wound healing and maintaining a normal skinenvironment.

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Expansion of antigen specific regulatory T cells in an IDO expressing fibroblast co-culture (2012)

Cadaver skin grafts are commonly utilised as dermal replacement treatments for large burn injuries when an autologous donor tissue shortage exists. The limitation of this therapy is immune rejection. The adverse side effects of immune suppressive drugs make them unsuitable for this subset of individuals with large surface area burns, who are often clinically unstable. Thus, a gap in treatment exists to prolong survival of these skin grafts. The goal of this Masters research was to address this gap and induce tolerance to specific skin antigens in vitro. We hypothesized that we could expand a specific population of Treg in an allogeneic indoleamine 2,3-dioxygenase (IDO) fibroblast co-culture, on the basis of specific MHC class II expression by the fibroblasts and that the IDO generated micro-environment afford selective survival of regulatory T cells (Treg) over effector T cells.Three specific aims were accomplished in this study. (1)We first showed that Treg could be expanded in an allogeneic IDO expressing fibroblast co-culture. (2) As a side project to this, we showed that naïve CD4 T cells could be converted to Treg in the same co-culture conditions. (3) In the next phase, the suppressive potential of the expanded and converted Treg populations were confirmed in a CD8 T cell suppression assay. We found that the Treg isolated from the allogeneic IDO expressing fibroblast co-culture, specifically suppressed CD8 proliferation to the allogeneic fibroblast antigens but not third party antigens. In contrast, the Treg from the control group did not suppress CD8 proliferation.The findings presented in this thesis collectively prove expansion of an antigen specific Treg population and conversion of naïve CD4 T cells to antigen specific Treg, in an allogeneic IDO expressing fibroblast co-culture, in vitro.

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The role of red blood cells in wound healing (2011)

Wound healing is a complex & intricate process that involves the coordinated efforts of an enormous number of unique tissues & cell lineages. Regardless of the cause, defects in this well-orchestrated process often lead to dramatic morbidity and treatments that are among the most costly, from an economic perspective. In an effort to understand the wound healing mechanisms and to establish more effective treatments, the roles of many underlying factors and cell types have been extensively studied. In spite of their ubiquitous presence and involvement in coagulation, thrombosis, and inflammation, the role(s) of red blood cells (RBC) in wound healing remains to be elucidated - RBCs are generally thought of as inert bystanders in wound healing. Recent evidence regarding RBCs novel secondary functions and their ability to actively participate in dynamic biological processes such as inflammation, led us to investigate their role in wound healing. In order to explore RBCs involvement in wound healing, three specific objectives were successfully accomplished in this research project: (1) utilizing Western blot and proteomic analyses the presence of five erythrocytic 14-3-3 isoforms were confirmed, (2) it was established that treatment of dermal fibroblasts with RBC lysate for 24 hours results in significant (p
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