Erzsebet Szilagyi

Mesenchymal stem cells in the induction of transplantation tolerance.

Mesenchymal stem cells directly suppress ongoing immune responses. Through production of toleragenic cytokines, inhibition of lymphocyte proliferation, delivery of reparative and protective signals after reperfusion injury, and facilitation of hematopoietic chimerism, these cells demonstrate a wide-ranging potential for the development of multifaceted toleragenic strategies after transplantation.

Activated mesenchymal stem cells increase wound tensile strength in aged mouse model via macrophages

BACKGROUND Wound healing is impaired in the aged. Mesenchymal stem cells (MSCs) can exert beneficial effects in wounds; however, promoting healing in the challenging setting of aged skin may require additional potency. MSCs can enhance the production of pro-regenerative cytokines and growth factors when activated with interferon gamma. We hypothesized that the increased potency of activated MSC could be used to facilitate wound healing in the aged mice. METHODS Young and old C57BL6 mice underwent incisional wounds and were treated with naive MSCs, activated MSCs, or vehicle to examine MSC effects on tensile strength in the aged skin. To test whether the benefits of MSC treatment could be attributed to the participation of host macrophages, liposomal clodronate was used to deplete host macrophages. RESULTS In older mice, tensile strength of healing wounds was significantly lower than that in younger mice. Older mice treated with activated MSCs showed significant increases in tensile strength restoring the strength to that observed in younger mice. Macrophage depletion abrogated the beneficial effect of MSC. CONCLUSIONS Activated MSCs restored wound tensile strength in the aged mice, and this effect was dependent on host macrophage activity. These data provide encouraging support for the development of activated MSC therapies for enhanced tissue regeneration, especially for older population groups.

Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy

Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin–deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 106) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

Subject-Based versus Population-Based Care after Radiation Exposure

In a mass casualty radiation event situation, individualized therapy may overwhelm available resources and feasibility issues suggest a need for the development of population-based strategies. To investigate the efficacy of a population-based strategy, Chinese macaques (n = 46) underwent total-body irradiation and received preemptive antibiotics, IV hydration on predetermined postirradiation days and were then compared to macaques (n = 48) that received subject-based care in which blood transfusions, IV hydration, nutritional supplementation and antibiotic supportive measures were provided. Estimated radiation doses for LD30/60, LD50/60 and LD70/60 of animals with subject-based care: 6.83 Gy (6.21, 7.59), 7.44 Gy (6.99, 7.88) and 8.05 Gy (7.46, 8.64), respectively, and for population-based care: 5.61 Gy (5.28, 6.17), 6.62 Gy (6.13, 7.18) and 7.63 Gy (7.21, 8.20), respectively. Analysis of four time periods, 0–9, 10–15, 16–25 and 26–60 days postirradiation, identified significant mortality differences during the period of 10–15 days. A subset analysis of higher radiation doses (6.75–7.20 Gy, n = 32) indicated hydration, nutrition and septic status were not significantly different between treatments. Whole blood transfusion treatment, administered only in subject-supportive care, was associated with significantly higher platelet and absolute neutrophil counts. Median platelet counts greater than 5,670 cells/μl and absolute neutrophil counts greater than 26 cells/μl during this period correlated with survival. We observed that the population-based treatment increased the LD50/60 compared to nontreatment (6.62 Gy vs. 4.92 Gy) and may be further optimized during days 10–15, where strategic blood transfusions or other strategies to achieve increases in neutrophil and platelet counts may further increase survival rates in subjects exposed to high doses of radiation.

The Importance of Non-Human Primate Models for Pre-clinical Studies in Hematopoiesis

Of all living experimental models, murine studies of hematopoiesis represent the greatest number. Such models can be undeniably elegant reflecting the extensive technological tools available. While mice can be used to study specific genetic pedigrees, specific physiologic, and gene pathways using knockouts, and a variety of immune responses using immunologically deficient mice, murine models are limited in their applicability for clinical use. Rodents differ from humans in many aspects including their short lifespan, difference in the rate of doubling of hematopoietic stem/progenitor cells, and the responses of blood cells to the hematological stresses of radiation and cytotoxic agents [1-5].

2585: The effect of size discrepancy of human nerve allograft derived epineural conduit on the functional outcomes of nerve regeneration in athymic nude rat model

2585: The effect of size discrepancy of human nerve allograft derived epineural conduit on the functional outcomes of nerve regeneration in athymic nude rat model Marcin Strojny, Rafal Gendek, Joanna Cwykiel, Erzsebet Szilagyi, Malgorzata Cyran, Wojciech Malewski, Husein Karagoz, and Maria Siemionow University of Illinois at Chicago, Chicago, IL, USA Background Nerve autograft is a gold standard in peripheral nerve regeneration However, it is challenged by limited availability, donor site morbidity and scarring Nerve allografts provide an unlimited source of nerve tissue, which can be matched to the recipient’s injured nerve to support nerve recovery This study aimed to assess the effect of human Epineural Sheath Conduit (hESC) adjusted with tissue adhesive or suture on restoration of long nerve defect in an athymic nude rat model. Methods Restoration of 20 mm of nude rat sciatic nerve defect with hESC filled with saline was performed in 5 groups: Group 1: autograft controls (n D 4), Group 2: mismatched size diameter hESC (n D 2), Group 3: hESC with diameter adjusted with tissue adhesive (n D 4), Group 4: hESC with diameter adjusted with nylon 10-0 suture (n D 2), and Group 5: matched diameter hESC (n D 4) Toe-spread and pinprick analyses were performed at 1, 3, 6, 9, 12 weeks Nerve samples for toluidine blue staining and for fluorescent immunostaining for GFAP, NGF, S-100, laminin B, CD3 and CD4 were harvested at 12 weeks post-surgery Muscle denervation atrophy was assessed by Gastrocnemius Muscle Index (GMI). Results Macroscopic evaluation of nerve conduits at 12 weeks showed no tissue adhesion or local signs of inflammation and good vascularization in all groups Additionally, the shape and integrity of the conduit were preserved The best sensory and motor recovery following hESC application was observed in groups with hESC diameter adjusted with tissue adhesive (Group 3) and matched diameter hESC (Group 5), pinprick 175 and 175; toe-spread 05 and 075, respectively hESC without diameter adjustment (Group 2) and hESC adjusted with sutures (Group 4) showed the worst regeneration GMI measurements were the highest for autograft group (Group 1–035) followed by matched diameter hESC (Group 5–032) and hESC adjusted with tissue adhesive (Group 3–0277). Conclusions We confirmed the feasibility of hESC creation and diameter adjustment hESC conduit with adjusted diameter using tissue adhesive showed sensory and motor recovery comparable to hESC conduit with matched diameter to the rat sciatic nerve. Our new hESC conduit provides an alternative option to the autograft nerve gap repair. CONTACT Marcin Strojny mstrojnymd@gmail.com

2597: Assessment of engraftment and safety of a new tolerance inducing therapy of human cord blood derived ex-vivo created di-chimeric cells in NOD SCID mouse model

2597: Assessment of engraftment and safety of a new tolerance inducing therapy of human cord blood derived ex-vivo created di-chimeric cells in NOD SCID mouse model Joanna Cwykiel, MSc, Natalia Filipek, BSc, Malgorzata Cyran, Can Emre Bas, Erzsebet Szilagyi, Ewa Bryndza Tfaily, Medhat Askar, MD, PhD, and Maria Siemionow University of Illinois at Chicago, Chicago, IL, USA; Cleveland Clinic, Cleveland, OH, USA Background The aim of this study was to evaluate the phenotype, engraftment and safety of ex-vivo fused human cord blood derived di-chimeric cell therapy (DCC) in the NOD SCID mouse model. Methods A total of 36 fusions of human umbilical cord blood (UCB) mononuclear clls were performed UCB from 2 unrelated donors were separately stained with PKH26 and PKH67 dyes Fused with polyethylene glycol, double (PKH26/PKH67) stained DCC were sorted and subjected to the in vitro evaluations (15 fusions): lymphocytotoxicity (LCT) test, PCR-rSSOP, STR-PCR, viability, apoptosis, colony forming unit (CFU) assay and COMET assay DCC (3–5 £ 10 cells) from 18 fusions (n D 6/Group) were delivered: Group 1: intraosseous, Group 2: intramuscular, and Group 3: subcutaneous to the NOD SCID recipient mice Control mice in Groups 4, 5, and 6 (n D 6) received 3 £ 10UCB utilizing the same 3 delivery sites Mice were observed daily for 90 days for changes in weight, activity, posture and hair loss Moreover, mice were evaluated bi-weekly by palpation and at 90 days by magnetic resonance imaging (MRI) The presence of DCC in the blood was determined by complete blood count (CBC) and flow cytometry (FC) The migratory pathways of DCC, peripheral blood, bone marrow and lymphoid organs were assessed at 3 months after delivery using immunofluorescent staining Furthermore, H&E staining was performed to assess harvested tissues for tumor growth. Results The presence of HLA class I and II from both UCB donors was confirmed by LCT, PCR-rSSOP, and STR COMET assay showed no damage to the DNA of DCC following fusion procedure Migratory properties of DCC were confirmed at 24 hours after cell delivery Human derived cells (CD45C, CD19C, HLA class I and CD4C) were detected at a level up to 2% in the peripheral blood as tested by CBC and flow cytometry at 90 days following delivery No DCC derived tumorlike growth was observed. Conclusions Phenotype, engraftment and safety of DCC were characterized The unique concept of supportive therapy of DCC introducing cells presenting phenotype characteristics of both transplant donor and recipient is a new promising approach for tolerance induction and prolonging VCA survival. CONTACT Joanna Cwykiel, MSc jcwykiel@uic.edu

2594: In vitro and in vivo characterization of human hematopoietic chimeric cells - A novel strategy to induce tolerance in transplantation

2594: In vitro and in vivo characterization of human hematopoietic chimeric cells A novel strategy to induce tolerance in transplantation Ewa Bryndza Tfaily, PhD, Malgorzata Cyran, MD, Rafal Gendek, MD, PhD, Erzsebet Szilagyi, MD, PhD, Jason Le, MD, Anna Domaszewska-Szostek, PhD, Joanna Cwykiel, MSc, and Maria Siemionow, MD, PhD, DSc University of Illinois at Chicago, Chicago, IL, USA; Polish Academy of Science, Warszawa, Poland Background Cell-based therapies represent a new promising approach for tolerance induction in transplantation One of the methods for immune response modulation in solid organ and vascularized composite allograft (VCA) recipients is donor bone marrow (BM) transplantation We propose a new cellular therapy based on application of the ex vivo created donor-recipient chimeric cells as an alternative approach to BM-based therapies in support of solid organ and VCA transplantation The aim of this study was further characterization of human hematopoietic chimeric cells (HHCC).

2550: Chimeric cell transplant for the treatment of duchenne muscular dystrophy

2550: Chimeric cell transplant for the treatment of duchenne muscular dystrophy Erzsebet Szilagyi, Joanna Cwykiel, Anna Domaszewska-Szostek, and Maria Siemionow University of Illinois at Chicago, Chicago, IL, USA Background Duchenne Muscular Dystrophy (DMD), is a progressive lethal disease, caused by X-linked mutations affecting dystrophin production in the muscle cells. Allogeneic stem cell therapies are aiming to restore dystrophin in affected muscles, however, are challenged by rejection and limited engraftment. Chimeric Cells (CC), created via ex vivofusion of donor and recipient cells, represent a promising therapy option in the field of tissue regeneration and Vascularized Composite Allotransplant (VCA), as eliminate the need of life-long immunosuppression. The aim of this study was to test the feasibility of Chimeric Cell therapy of mesenchymal stem cell (MSC) and myoblast origin through in vitro characterization of human and murine CC and through in vivo assessment of survival, engraftment and efficacy of human and murine CC in DMD mdx/scid and mdx mice models. Methods Twelve ex vivo fusions of allogenic human myoblasts-myoblast and myoblast-MSC were performed, using polyethylene glycol technique. CC phenotype was evaluated by flow cytometry and confocal microscopy. CC were cultured for 30 d to test proliferation capacity and myogenic differentiation was induced in specific culture conditions. To test efficacy, mdx/scid mice (nD 4) received 0.5£ 10 human myoblast/MSC and myoblast/myoblast CC, while mdx mice received 0.5 £ 10 healthy snj myoblast/mdx MSC and snj myoblast/mdx myoblast CC through local injections to gastrocnemius muscle. Therapeutic effect was monitored by muscle function tests (grip strength and wire hanging). Muscle characteristics (weight, inflammation, fibrosis, dystrophin expression as well as in situ contraction ability) were assessed at day 7, 30 and 90 after transplant. Results After successful fusion procedure, CC expressed antigens of both parent cells, maintained proliferative capacity in long-term cultures and differentiated toward mature skeletal myocytes. Human CC treated recipients showed CC engraftment in gastrocnemius muscle and locally increased dystrophin expression (12%), at 7 day after cell delivery. Both human and murine CC recipients showed increased motor function and dystrophin expression that was maintained through the 1 to 3-months follow-up period. Conclusion This study confirmed feasibility and efficacy of CC therapy, which may represent a novel, universal approach for treatment of muscular dystrophies and can be applicable to restore muscle components of VCA. CONTACT Erzsebet Szilagyi eszilagy@uic.edu

Response to the “Letter to the Editor” by Dant, Stricklin and Millage on our Article: “Subject-Based versus Population- Based Care after Radiation Exposure”

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