Georg J. Furtmüller

A multiphase transitioning peptide hydrogel for suturing ultrasmall vessels

Many surgeries are complicated by the need to anastomose, or reconnect, micron-scale vessels. Although suturing remains the gold standard for anastomosing vessels, it is difficult to place sutures correctly through collapsed lumen, making the procedure prone to failure. Here, we report a multi-phase transitioning peptide hydrogel that can be injected into the lumen of vessels to facilitate suturing. The peptide, which contains a photocaged glutamic acid, forms a solid-like gel in a syringe and can be shear-thin delivered to the lumen of collapsed vessels (where it distends the vessel), and the space between two vessels (where it is used to approximate the vessel ends). Suturing is performed directly through the gel. Light is used to initiate the final gel-sol phase transition that disrupts the hydrogel network, allowing the gel to be removed and blood flow to resume. This gel adds a new tool to the armamentarium for micro- and supermicrosurgical procedures.

Mesenchymal Stem Cells Enhance Nerve Regeneration in a Rat Sciatic Nerve Repair and Hindlimb Transplant Model

This study investigates the efficacy of local and intravenous mesenchymal stem cell (MSC) administration to augment neuroregeneration in both a sciatic nerve cut-and-repair and rat hindlimb transplant model. Bone marrow-derived MSCs were harvested and purified from Brown-Norway (BN) rats. Sciatic nerve transections and repairs were performed in three groups of Lewis (LEW) rats: negative controls (n = 4), local MSCs (epineural) injection (n = 4), and systemic MSCs (intravenous) injection (n = 4). Syngeneic (LEW-LEW) (n = 4) and allogeneic (BN-LEW) (n = 4) hindlimb transplants were performed and assessed for neuroregeneration after local or systemic MSC treatment. Rats undergoing sciatic nerve cut-and-repair and treated with either local or systemic injection of MSCs had significant improvement in the speed of recovery of compound muscle action potential amplitudes and axon counts when compared with negative controls. Similarly, rats undergoing allogeneic hindlimb transplants treated with local injection of MSCs exhibited significantly increased axon counts. Similarly, systemic MSC treatment resulted in improved nerve regeneration following allogeneic hindlimb transplants. Systemic administration had a more pronounced effect on electromotor recovery while local injection was more effective at increasing fiber counts, suggesting different targets of action. Local and systemic MSC injections significantly improve the pace and degree of nerve regeneration after nerve injury and hindlimb transplantation.

Near-infrared lymphography as a minimally invasive modality for imaging lymphatic reconstitution in a rat orthotopic hind limb transplantation model

Wider application of vascularized composite allotransplantation (VCA) is limited by the need for chronic immunosuppression. Recent data suggest that the lymphatic system plays an important role in mediating rejection. This study used near‐infrared (NIR) lymphography to describe lymphatic reconstitution in a rat VCA model. Syngeneic (Lewis–Lewis) and allogeneic (Brown Norway–Lewis) rat orthotopic hind limb transplants were performed without immunosuppression. Animals were imaged pre‐ and postoperatively using indocyanine green (ICG) lymphography. Images were collected using an NIR imaging system. Co‐localization was achieved through use of an acrylic paint/hydrogen peroxide mixture. In all transplants, ICG first crossed graft suture lines on postoperative day (POD) 5. Clinical signs of rejection also appeared on POD 5 in allogeneic transplants, with most exhibiting Grade 3 rejection by POD 6. Injection of an acrylic paint/hydrogen peroxide mixture on POD 5 confirmed the existence of continuous lymphatic vessels crossing the suture line and draining into the inguinal lymph node. NIR lymphography is a minimally invasive imaging modality that can be used to study lymphatic vessels in a rat VCA model. In allogeneic transplants, lymphatic reconstitution correlated with clinical rejection. Lymphatic reconstitution may represent an early target for immunomodulation.

MEMS-Based Handheld Fourier Domain Doppler Optical Coherence Tomography for Intraoperative Microvascular Anastomosis Imaging

Purpose To demonstrate the feasibility of a miniature handheld optical coherence tomography (OCT) imager for real time intraoperative vascular patency evaluation in the setting of super-microsurgical vessel anastomosis. Methods A novel handheld imager Fourier domain Doppler optical coherence tomography based on a 1.3-µm central wavelength swept source for extravascular imaging was developed. The imager was minimized through the adoption of a 2.4-mm diameter microelectromechanical systems (MEMS) scanning mirror, additionally a 12.7-mm diameter lens system was designed and combined with the MEMS mirror to achieve a small form factor that optimize functionality as a handheld extravascular OCT imager. To evaluate in-vivo applicability, super-microsurgical vessel anastomosis was performed in a mouse femoral vessel cut and repair model employing conventional interrupted suture technique as well as a novel non-suture cuff technique. Vascular anastomosis patency after clinically successful repair was evaluated using the novel handheld OCT imager. Results With an adjustable lateral image field of view up to 1.5 mm by 1.5 mm, high-resolution simultaneous structural and flow imaging of the blood vessels were successfully acquired for BALB/C mouse after orthotopic hind limb transplantation using a non-suture cuff technique and BALB/C mouse after femoral artery anastomosis using a suture technique. We experimentally quantify the axial and lateral resolution of the OCT to be 12.6 µm in air and 17.5 µm respectively. The OCT has a sensitivity of 84 dB and sensitivity roll-off of 5.7 dB/mm over an imaging range of 5 mm. Imaging with a frame rate of 36 Hz for an image size of 1000(lateral)×512(axial) pixels using a 50,000 A-lines per second swept source was achieved. Quantitative vessel lumen patency, lumen narrowing and thrombosis analysis were performed based on acquired structure and Doppler images. Conclusions A miniature handheld OCT imager that can be used for intraoperative evaluation of microvascular anastomosis was successfully demonstrated.

N-(Pivaloyloxy)alkoxy-carbonyl Prodrugs of the Glutamine Antagonist 6-Diazo-5-oxo-l-norleucine (DON) as a Potential Treatment for HIV Associated Neurocognitive Disorders

Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate synthesis in HIV-infected microglia/macrophages, offering therapeutic potential for HAND. We show that 14 prevents manifestation of spatial memory deficits in chimeric EcoHIV-infected mice, a model of HAND. 14 is not clinically available, however, because its development was hampered by peripheral toxicities. We describe the synthesis of several substituted N-(pivaloyloxy)alkoxy-carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed to 14 in the brain. The lead prodrug, isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and human plasma but liberated 14 in swine brain homogenate. When dosed systemically in swine, 13d provided a 15-fold enhanced CSF-to-plasma ratio and a 9-fold enhanced brain-to-plasma ratio relative to 14, opening a possible clinical path for the treatment of HAND.

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Functional Reconstruction of Urogentital Tissue Defects Using Vascularized Composite Allotransplantation: A Novel Microsurgical Penis Transplant Model and Clinical & Histological Rejection Classification

Introduction Defects of the male urogenital structures subsequent to major trauma are associated with impaired urinary and sexual function and result in a significantly reduced quality-of-life. Modern reconstructive methods employ autologous tissue-based reconstructions and implant placement to reconstruct a functional phallus, but outcomes are frequently unsatisfactory. Penis transplantation has been successfully employed in the recent past and represents an exciting avenue for restoration of male urogenitalia and function by using “like-with-like” tissue, when no conventional options are available. However, only little is currently known about the immunological features of these unique tissue grafts. Thus, to fill this critical void, we established a new animal model and clinical & histological rejection classification. Materials and Methods In male 8-week old BN & Lewis rats the penis was dissected to design a penile graft based on the internal pudendal artery and dorsal penile vein including prepuce skin. A non-suture cuff technique was employed to perform end-to-end anastomosis of the graft vessels to the recipient inferior epigastric vessels. 30 syngeneic and allogeneic transplants were performed. Grafts were followed clinically and histologically at post-operative days (POD) 3,5,7,9,11,13,14,16 and 18. Results The graft design using anastomosis of the dorsal penile vein and the distal internal pudendal artery at the bifurcation into dorsal and deep penile arteries ensures optimal perfusion of the entire superficial and deep graft tissues. The non-suture cuff technique allows for successful microvascular anastomosis by a single surgeon in an average of 2.5 hours, at a 91% surgical success rate. Long-term graft survival (>POD 45) was observed in syngeneic transplants. Graft rejection follows a 4-stage clinical progression, indicating comparability to other skin containing VCAs. Graft rejection, however appears to be minimally delayed compared to hind limb allografts, with all untreated allografts fully rejected by POD 16. Histological analysis allowed for the development of a specific 4-grade rejection classification in analogy to the 2007 BANFF criteria for hand transplantation. Of note, graft skin and urethral lining tissue are first targets of rejection, which follows a distal to proximal pattern. Conclusion We established a robust and reproducible murine model to study the immunobiology of urogenital tissue in the context of reconstructive transplantation. The graft design ensures vascular perfusion of all penile tissues. Heterotopic inset allows for standardized visual monitoring of graft viability. We propose a novel 4-grade histological rejection scale based on graft skin and urethral lining as the main targets of rejection.

Ex vivo Expanded Regulatory T cells Combined with Short-term Costimulation Blockade Prevent Rejection of Vascularized Composite Allografts

Background Reconstructive transplantation represents a valid therapeutic option after devastating tissue loss. Routine clinical application, however, is hampered by the toxicity of long-term maintenance immunosuppression. The current study investigated a novel approach using ex vivo expanded regulatory T cells combined with a short-term immunomodulatory strategy in a murine hind limb transplantation model. Methods Fully MHC-mismatched orthotopic hind limb transplants were performed from Balb/C to C57BL/6 mice. Recipients in the experimental groups received a combination regimen consisting of 0.5mg CTLA4 Ig on day 0, 2, 4 and 6 post-transplant, 20mg/kg anti-Thy 1.2 mAb on POD-1, and 1mg/kg Rapamycin (POD 0-9), and in one group, 1 wk expanded CD4+CD25+ Treg cells. Allograft survival was monitored and flow cytometric analysis was performed to evaluate mixed chimerism and clonal deletion of alloreactive T cells. Treg activity was assessed in vitro using suppression assays in order to support and supplement in vivo data. Results Combination of T cell depletion and CTLA4-Ig plus short-course of Rapamycin increased VCA survival significantly while untreated controls rejected allografts (MST 105 days; Untreated, MST 9 days; CTLA4 Ig only, MST 17 days, Rapamycin, MST 20 days; T cell depletion, 20 days; p<0.01[GB1]). [GB2] Mixed chimerism was detected in recipients receiving this combined treatment protocol with 5.013 ± 1.23 % of CD11b+ cells being donor-derived on POD 55. V&bgr; – TCR staining profiles in recipients after full treatment showed 1.570 ± 0.3700 % of &ngr;&bgr;5+CD4+ T cells, while naïve C57BL/6 express 3.567 ± 0.3690 % of &ngr;&bgr;5+CD4+ T cells, suggesting the actuation of central deletion of developing donor-reactive T cells. In order to further prolong allograft survival, one week expanded Tregs were then included in the combination therapy. The suppressive activity of the CD4+CD25+ Tregs was confirmed with in vitro suppression assays. The addition of ex vivo expanded regulatory T cells further increased VCA survival to >200 days and induced long-term stable mixed chimerism[GB3] with 16.7±1.5 % of CD11b cells being donor-derived on POD 55 after administration of expanded Treg cells. Conclusion The combination of T cell depletion, costimulation blockade, and a short-course of Rapamycin prevents VCA rejection and significantly prolongs graft survival without the need for myeloablative conditioning or maintenance therapy. Moreover, regulatory T cells added in the early post transplant period further optimize immune regulation by inducing sustained mixed chimerism.

Desensitization and prevention of antibody-mediated rejection in vascularized composite allotransplantation by syngeneic hematopoietic stem cell transplantation

Background Candidates for vascularized composite allotransplantation (VCA) are frequently sensitized, putting them at risk for antibody-mediated rejection. Current desensitization strategies are imperfect and require a living-donor setting. Here we investigated the impact of sensitization on and the efficacy of a desensitization protocol utilizing syngeneic hematopoietic stem cell transplantation (HSCT) to prevent antibody-mediated rejection in VCA. Methods Skin transplants from Dark Agouti to Lewis rats were performed for sensitization. Orthotopic hind limb transplants from Dark Agouti donors were performed to sensitized and nonsensitized recipients, and the animals were treated with either daily tacrolimus or no immunosuppression. A desensitization protocol consisting of total body irradiation, fludarabine, and syngeneic HSCT was applied to sensitized animals. Graft rejection was monitored by clinical assessment and histological analysis. Serum levels of donor-specific antibodies (DSA IgG) were measured using flow cytometry. Results Sensitized recipients exhibited accelerated rejection by 5.5 ± 1.2 days without immunosuppression and 10.2 ± 3.6 days with daily tacrolimus compared with 8.7 ± 1.2 days and longer than 30 days in nonsensitized recipients, respectively. Serum levels of DSA IgG were markedly elevated (37.3 ± 3.34-fold from baseline) in sensitized recipients after VCA and correlated with histologic evidence of rejection and C4d deposition. Desensitization significantly reduced DSA compared with sensitized controls (2.6 ± 0.5-fold vs 6.0 ± 1.2-fold, P < 0.01) and along with daily tacrolimus led to improved VCA survival longer than 30 days without evidence of C4d deposition (n = 6). Conclusions In summary, sensitization leads to accelerated rejection of VCA, and syngeneic HSCT combined with conventional immunosuppression effectively reduces DSA and improves allograft survival in sensitized rats.

Targeted delivery of immune therapeutics to lymph nodes prolongs cardiac allograft survival

The targeted delivery of therapeutic drugs to lymph nodes (LNs) provides an unprecedented opportunity to improve the outcomes of transplantation and immune-mediated diseases. The high endothelial venule is a specialized segment of LN vasculature that uniquely expresses peripheral node addressin (PNAd) molecules. PNAd is recognized by MECA79 mAb. We previously generated a MECA79 mAb–coated microparticle (MP) that carries tacrolimus. Although this MP trafficked to LNs, it demonstrated limited therapeutic efficacy in our transplant model. Here, we have synthesized a nanoparticle (NP) as a carrier of anti-CD3, and optimized the conjugation strategy to coat the NP surface with MECA79 mAb (MECA79-anti-CD3-NP) to enhance LN accumulation. As compared with nonconjugated NPs, a significantly higher quantity of MECA79-NPs accumulated in the draining lymph node (DLN). Many MECA79-NPs underwent internalization by T cells and dendritic cells within the LNs. Short-term treatment of murine cardiac allograft recipients with MECA79-anti-CD3-NP resulted in significantly prolonged allograft survival in comparison with the control groups. Prolonged graft survival following treatment with MECA79-anti-CD3-NP was characterized by a significant increase in intragraft and DLN Treg populations. Treg depletion abrogated the prolongation of heart allograft survival. We believe this targeted approach of drug delivery could redefine the methods of administering immune therapeutics in transplantation.