Maria Madajka

A novel role for platelet secretion in angiogenesis: mediating bone marrow-derived cell mobilization and homing

Angiogenesis alleviates hypoxic stress in ischemic tissues or during tumor progression. In addition to endothelial cell proliferation and migration, the angiogenic process requires bone marrow-derived cell (BMDC) recruitment to sites of neovascularization. However, the mechanism of communication between hypoxic tissues and the BM remains unknown. Using 2 models of hypoxia-induced angiogenesis (ischemic hindlimb surgery and subcutaneous tumor growth), we show that platelet infusion promotes BMDC mobilization into the circulation, BMDC recruitment into growing neovasculature, tumor vascularization, and blood flow restoration in ischemic limbs, whereas platelet depletion inhibits these effects. Thus, platelets are required for BMDC recruitment into ischemia-induced vasculature. Secretion of platelet α-granules, but neither dense granules nor platelet aggregation is crucial for BMDC homing and subsequent angiogenesis, as determined using VAMP-8(-/-), Pearl, and integrin Beta 3(-/-) platelets. Finally, platelets sequester tumor-derived promoters of angiogenesis and BMDC mobilization, which are counterbalanced by the antiangiogenic factor thrombospondin-1. A lack of thrombospondin-1 in platelets leads to an imbalance in proangiogenic and antiangiogenic factors and accelerates tumor growth and vascularization. Our data demonstrate that platelets stimulate BMDC homing in a VAMP-8-dependent manner, revealing a previously unknown role for platelets as key mediators between hypoxic tissues and the bone marrow during angiogenesis.

Natural conduits for bridging a 15-mm nerve defect: Comparison of the vein supported by muscle and bone marrow stromal cells with a nerve autograft

OBJECT The gold standard for reconstructing large nerve defects, the nerve autograft, results in donor-site morbidity. This detrimental consequence drives the search for alternatives. We used a vein filled with a small piece of fresh muscle to prevent the vein from collapsing and with bone marrow stromal cells (BMSCs) to enhance regeneration. METHODS In 60 rats, a 15-mm sciatic nerve defect was bridged with a nerve autograft, a vein filled with muscle or a vein filled with muscle and BMSCs. Toe spread and pinprick were used to evaluate motor and sensory function. Compound muscle action potentials (CMAPs) and the gastrocnemius muscle index (GMI) were recorded to assess conduction properties and denervation atrophy. Extensive histology was performed to confirm presence of BMSCs and to evaluate regeneration by staining neural tissue for Schwann cells and neural growth factor. RESULTS After 12 weeks, all animals responded with toe spread and pinprick reaction; significant differences were found between groups. Six weeks post grafting no difference was found comparing the GMI between the groups. Group I had a significant increase in GMI at 12 weeks compared to group II and group III. The CMAP measurements showed comparable results at 6 weeks post grafting. Twelve weeks after reconstruction, group I had significantly better results compared to group II and group III. Group III showed a tendency to outperform group II at 12 weeks postoperatively. Immunofluorescence analysis showed an increased number of Schwann cells in group III compared to group II. The BMSCs were visible 6 and 12 weeks postoperatively. CONCLUSIONS This study is a step forward in the search for an alternative to the nerve autograft because it demonstrates the beneficial effect of BMSCs to a conduit. However, our data do not demonstrate sufficient benefit to warrant clinical implementation at this stage.

Long-term survival of composite hemiface/mandible/tongue allografts correlates with multilineage chimerism development in the lymphoid and myeloid compartments of recipients

Background. To maximize aesthetic and functional outcomes for complex craniofacial defects, application of composite tissue allografts opens unique one-stage reconstructive option. We have assessed role of different components of the hemiface/mandible/tongue allograft on induction and maintenance of donor-specific chimerism, in correlation with allograft survival. Methods. Twenty-two composite hemiface/mandible/tongue transplantations were performed in three groups: group 1 (n=8)—isotransplantations between Lewis (LEW) rats (RT11)—served as controls. Group 2 (n=8)—allograft rejection controls—hemiface/mandible/tongue transplants performed across major histocompatibility complex (MHC) barrier between semiallogeneic LEW-Brown-Norway (RT11+n) donors and LEW (RT1l) recipients without immunosuppression. Allografts in group 3 (n=6) received tapered cyclosporine A monotherapy. Assessments included monitoring of rejection, flow cytometry for donor-specific chimerism of major histocompatibility complex class I (RT1n) antigen, immunohistochemistry for engraftment of donor-origin cells into lymphoid organs and bone marrow (BM) compartment, and histology for hemiface/mandible/tongue architecture. Results. Isograft controls survived indefinitely; in allografts without treatment, rejection started within 5 to 7 days. Treated hemiface/mandible/tongue allotransplants survived up to 385 days, without signs of rejection or graft loss. Flap angiography confirmed intact vascularity, and computer tomography scan and histology confirmed bone viability. Donor-specific chimerism at day 125 was present for T cells (3.3% CD4/RT1n, 1.1% CD8/RT1n) and B cells (6.7% CD45RA/RT1n). Engraftment of donor-origin cells was confirmed into BM compartment and lymphoid organs of recipients. Conclusions. We introduced a new multitissue model of composite hemiface/mandible/tongue allograft containing lymphoid and vascularized BM components. Long-term allograft survival correlated with development and maintenance of donor-specific chimerism in lymphoid organs and BM compartment.

Effectiveness of topical immunosuppressants in prevention and treatment of rejection in face allotransplantation.

Background The use of topical immunosuppressants has been anecdotally reported for the treatment of rejection in vascularized composite allotransplantation. The aim of this study was to evaluate the effectiveness of topical tacrolimus and clobetasol in the prevention and treatment of rejection. Methods Seventy-six hemiface allotransplants, between ACI (RT1a) donors and Lewis (RT1l) recipients, were performed in 11 groups and treated with topical tacrolimus or clobetasol, or in combination with systemic cyclosporine A and anti–&agr;&bgr;–T-cell receptor antibody for 1 week. Topical treatment increased the survival of the allograft in all groups. Results Best outcomes were obtained in the groups treated with systemic therapy and topical tacrolimus. Expression of proinflammatory cytokines interleukin 2, interferon &ggr;, tumor necrosis factor &agr;, and transforming growth factor &bgr; correlated with clinical signs of rejection and the final outcomes. Clobetasol application was associated with a marked depletion of lymphocytic populations, and dermal and epidermal atrophy. Conclusions Both topical tacrolimus and clobetasol were effective in treating episodes of acute rejection, and the best outcomes were achieved when their application was initiated after systemic immunosuppression. Topical tacrolimus proved to be a preferable adjunct agent to the systemic therapy by preventing both the local and systemic complications.

Application of cell-based therapies in facial transplantation

Abstract The main purpose of cellular therapy application in face transplantation is the continuous need for the development of new strategies that would eliminate the use of toxic immunosuppressive protocols. Cellular therapy in transplantation can significantly benefit allograft survival and shorten healing time. Cells used for a therapeutic purpose are isolated mostly from bone marrow and adipose tissues. They have the ability to proliferate and differentiate in the transplanted tissue and have immunomodulatory activity. Most of the cellular therapies such as T-regulatory, dendritic, and chimeric cells are still in the experimental stage. Molecular characterization of these cells and the mechanism of their participation in allograft acceptance and rejection are not well established and will contribute to the future of modern transplantology.

A new vascularized cervical lymph node transplantation model: an anatomic study in rats.

IntroductionVascularized lymph node transfer is of high interest for the treatment of lymphedema. Currently, there are few experimental small animal models of vascularized lymph node transplantation. In this article, our aim was to describe a new vascularized cervical lymph node transplantation model in rats. Materials and MethodsTen male Sprague-Dawley rats weighing 200 to 250 g were used in this study. The anatomic features of the neck lymph nodes in rats were explored. Anatomic neck dissections were performed, and lymph node flaps were harvested. The common carotid artery and the jugular vein were used as the vascular pedicles of the lymph node flap. Methylene blue dye was injected into the arterial pedicle. Lymph nodes were identified, and their structure was confirmed by histological evaluation. Laser-assisted indocyanine green angiography was used to confirm perfusion of the lymph node flap. ResultsAn adequate perfusion was observed in the lymph node flap. The dye disseminated evenly within the lymph nodes, indicating that the flap had a well-established vascular network and an adequate blood supply. Macroscopically, perfusion of 5 to 6 lymph nodes was observed. Histological examination of tissue samples confirmed well-defined lymph nodes. After indocyanine green administration, fluorescence was observed throughout the lymph node flap and within the venous pedicle of the flap. ConclusionsTo the best of our knowledge, this is the first report describing vascularized lymph node flap in the head and neck region of a rat. Our lymph node flap preparation technique confirmed the presence of 5 to 6 lymph nodes within the flap. The presented vascularized lymph node flap can be applied to transplantation studies, lymphedema studies, and to studies on immunological mechanism of tolerance and rejection.

Repair of the peripheral nerve gap with epineural sheath conduit to prevent muscle denervation atrophy in the diabetic rat model

UNLABELLED Muscle denervation atrophy is a result of lower motor neuron injury, thus an early restitution of muscle stimulation is essential in prevention of atrophic changes. THE AIM OF THE STUDY To evaluate the new application of naturally occurring epineural sheath conduit in repair of the peripheral nerve gap to prevent development of muscle denervation atrophy. MATERIAL AND METHODS We used the model of 20 mm sciatic nerve gap, resulting in denervation atrophy of the gastrocnemius muscle in the diabetic rats (DM type 2, n=42, Zucker Diabetic Fatty strain). We applied the epineural sheath conduit created from the autologous sciatic nerve for gap repair. Muscle atrophy was assessed with the Gastrocnemius Muscle Index (GMI) and microscopic muscle morphometry (mean fiber area) at 6 and 12 postoperative week. Muscle regeneration in the experimental group was compared to the gold-standard technique of autologous nerve grafting for the repair of created nerve gap. RESULTS The GMI evaluation revealed comparable muscle mass restoration in groups with nerve repair using both epineural sheath and standard autologous nerve grafting (reaching 28 and 35% of contralateral muscle mass at 12 postoperative week, respectively, p=0.1), and significantly better restoration when compared to the negative control group (no repair, 20%, p<0.01). Micromorphometry confirmed significantly larger area of the regenerated muscle fibers in groups with both nerve grafting and epineural sheath conduit repair (reaching for both ca. 42% of the non-operated side), when compared to severe atrophic outcome when no nerve repair was performed (14% of the control fiber area, p<0.0001). The effectiveness of epineural conduit technique in muscle mass restoration was observed between 6 and 12 weeks after nerve repair--when gastrocnemius muscle mass increased by 12%. CONCLUSIONS Peripheral nerve gap repair with naturally occurring epineural sheath conduit is effective in prevention of muscle denervation atrophy. This method is applicable in diabetic model conditions, showing results of regeneration which are comparable to the autologous nerve graft repair.

Vascularized axillary lymph node transfer: A novel model in the rat.

Vascularized lymph node transfer (VLNT) is a promising microvascular free flap technique for the surgical treatment of lymphedema. To date, few experimental animal models for VLNT have been described and the viability of lymph nodes after the transfer tested. We aimed to evaluate the feasibility of axillary VLNT in the rat. Lymph node containing flaps were harvested from the axillary region in 10 Lewis rats based on the axillary vessels. Flaps were transferred to the ipsilateral groin and end‐to‐side microanastomosis was performed to the femoral vessels using 10‐0 sutures. Indocyanine green (ICG) angiography was used to confirm flap perfusion. On postoperative day 7, flaps were elevated to assess their structure and vessel patency. Hematoxylin and eosin staining was used to confirm the presence and survival of lymph nodes. All animals tolerated the procedure well. Immediate post‐procedure ICG angiography confirmed flap perfusion. No signs of ischemia or necrosis were observed in donor extremities. At postoperative day 7, all flaps remained viable with patent vascular pedicles. Gross examination and histology confirmed the presence of 3.6 ± 0.5 lymph nodes in each flap without any signs of necrosis. This study showed that the transfer of axillary lymph nodes based on the axillary vessels is feasible. The flap can be used without the need for donor animals and it contains a consistent number of lymph nodes. This reliable VLNT can be further utilized in studies involving lymphedema, transplantation, and induction of immunologic tolerance.

Bone Marrow-Derived Ex Vivo Created Hematopoietic Chimeric Cells to Support Engraftment and Maintain Long-Term Graft Survival in Reconstructive Transplantation

Vascularized composite allotransplantation (VCA) introduces a promising alternative approach to standard reconstructive procedures for severely disfigured patients. Currently, VCA research is focusing on the development of immunomodulatory protocols for tolerance induction. The ultimate goal is to reduce or preferably eliminate the need for toxic, lifelong immunosuppression and to prevent both acute and chronic rejection.

Pyrimidine synthesis inhibition enhances cutaneous defenses against antibiotic resistant bacteria through activation of NOD2 signaling

Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior antibacterial strategies directly target pathogen growth or viability. Host-directed strategies to increase antimicrobial defenses may be an effective alternative to antibiotics and reduce development of resistant strains. In this study, we demonstrated the efficacy of a pyrimidine synthesis inhibitor, N-phosphonacetyl-l-aspartate (PALA), to enhance clearance of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Acinetobacter baumannii strains by primary human dermal fibroblasts in vitro. PALA did not have a direct bactericidal effect, but enhanced cellular secretion of the antimicrobial peptides human β-defensin 2 (HBD2) and HBD3 from fibroblasts. When tested in porcine and human skin explant models, a topical PALA formulation was efficacious to enhance MRSA, P. aeruginosa, and A. baumannii clearance. Topical PALA treatment of human skin explants also resulted in increased HBD2 and cathelicidin (LL-37) production. The antimicrobial actions of PALA required expression of nucleotide-binding, oligomerization domain 2 (NOD2), receptor-interacting serine/threonine-protein kinase 2 (RIP2), and carbamoyl phosphatase synthase II/aspartate transcarbamylase/dihydroorotase (CAD). Our results indicate that PALA may be a new option to combat multidrug-resistant bacterial infections of the skin through enhancement of an integral pathway of the cutaneous innate immune defense system.