Publications by authors named "Agustin P Dalmasso"

18 Publications

  • Page 1 of 1

New Model for the Assessment of Transcatheter Aortic Valve Replacement Devices in Sheep.

J Invest Surg 2020 Dec 28:1-10. Epub 2020 Dec 28.

Experimental Surgical Services Laboratory, Department of Surgery, University of Minnesota, Minneapolis, Minnesota, USA.

Background: Transcatheter aortic valve replacement (TAVR) is an effective therapy in treating high-risk patients suffering from aortic stenosis. Animal models used to evaluate safety and efficacy of TAVR devices prior to clinical use lack a stenotic aortic annulus, a critical impediment to long-term TAVR device evaluation. We sought to create a reproducible model of aortic stenosis using a modified aortic annuloplasty (MAA) procedure in sheep, followed by deployment and long-term evaluation of TAVR devices using this model.

Methods: Twelve sheep underwent the MAA procedure and were recovered. Transthoracic echocardiography (TTE) was used to monitor changes in the aortic annulus in the postoperative period. At 60 days post-MAA, Test group animals were anesthetized for TAVR insertion and Control animals underwent a necropsy. Test animals were recovered following TAVR insertion and observed for a postoperative period of 140 days.

Results: Twelve sheep survived the annuloplasty procedure and the 60-day recovery period. Gross examination of seven Control group animals revealed the implanted annuloplasty ring segments formed hard protrusions into the aortic annulus. Five sheep in the Test group underwent successful deployment of Abbott's experimental TAVR device without evidence of migration. Examination at 140 days post-TAVR insertion showed all devices tightly anchored within the modified aortic annulus.

Conclusions: The MAA procedure creates stenotic segments in the aortic annulus with adequate rigidity for anchorage and long-term evaluation of TAVR devices. This represents the first model that successfully mimics human aortic stenosis and provides a clinically relevant TAVR deployment platform for long-term evaluation in sheep.
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http://dx.doi.org/10.1080/08941939.2020.1864796DOI Listing
December 2020

Critical role of C5a in sickle cell disease.

Am J Hematol 2019 03 3;94(3):327-337. Epub 2019 Jan 3.

Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, Minnesota.

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD.
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http://dx.doi.org/10.1002/ajh.25384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986874PMC
March 2019

The Hancock® Valved Conduit for Right Ventricular Outflow Tract Reconstruction in Sheep for Assessing New Devices.

J Heart Valve Dis 2017 07;26(4):472-480

Division of Experimental Surgery, Department of Surgery, University of Minnesota School of Medicine, Minneapolis, Minnesota, USA. Electronic correspondence:

Background And Aim Of The Study: Xenograft conduits have been used successfully to repair congenital heart defects, but are prone to failure over time. Hence, in order to improve patient outcomes, better xenografts are being developed. When evaluating a conduit's performance and safety it must first be compared against a clinically available control in a large animal model. The study aim was to evaluate a clinically available xenograft conduit used in right ventricular outflow tract (RVOT) reconstruction in a sheep model.

Methods: RVOT reconstruction was performed in 13 adult and juvenile sheep, using the Medtronic Hancock® Bioprosthetic Valved Conduit (Hancock conduit). The method had previously been used on patients, and a newly modified variant termed 'RVOT Extraction' was employed to facilitate the surgical procedure. Animals were monitored over predetermined terms of 70 to 140 days. Serial transthoracic echocardiography, intracardiac pressure measurements and angiography were performed. On study completion the animals were euthanized and necropsies performed.

Results: Two animals died prior to their designated study term due to severe valvular stenosis and distal conduit narrowing, respectively. Thus, 11 animals survived the study term, with few or no complications. Generally, maximal and mean transvalvular pressure gradients across the implanted conduits were increased throughout the postoperative course. Among 11 full-term animals, seven conduits were patent with mild or no pseudointimal proliferation and with flexible leaflets maintaining the hemodynamic integrity of the valve.

Conclusions: RVOT reconstruction using the Hancock conduit was shown to be successful in sheep, with durable and efficient performances. With its extensive clinical use in patients, and ability for long-term use in sheep (as described in the present study) it can be concluded that the Hancock conduit is an excellent control device for the evaluation of new xenografts in future preclinical studies.
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July 2017

IL-4 and IL-13 induce protection from complement and melittin in endothelial cells despite initial loss of cytoplasmic proteins: membrane resealing impairs quantifying cytotoxicity with the lactate dehydrogenase permeability assay.

Xenotransplantation 2015 Jul-Aug;22(4):295-301. Epub 2015 Jun 1.

Department of Surgery, Medical School, University of Minnesota, Minneapolis, MN, USA.

Endothelial cell activation and injury by the terminal pathway of complement is important in various pathobiological processes, including xenograft rejection. Protection against injury by human complement can be induced in porcine endothelial cells (ECs) with IL-4 and IL-13 through metabolic activation. However, despite this resistance, the complement-treated ECs were found to lose membrane permeability control assessed with the small molecule calcein. Therefore, to define the apparent discrepancy of permeability changes vis-à-vis the protection from killing, we now investigated whether IL-4 and IL-13 influence the release of the large cytoplasmic protein lactate dehydrogenase (LDH) in ECs incubated with complement or the pore-forming protein melittin. Primary cultures of ECs were pre-treated with IL-4 or IL-13 and then incubated with human serum as source of antibody and complement or melittin. Cell death was assessed using neutral red. Membrane permeability was quantitated measuring LDH release. We found that IL-4-/IL-13-induced protection of ECs from killing by complement or melittin despite loss of LDH in amounts similar to control ECs. However, the cytokine-treated ECs that were protected from killing rapidly regained effective control of membrane permeability. Moreover, the viability of the protected ECs was maintained for at least 2 days. We conclude that the protection induced by IL-4/IL-13 in ECs against lethal attack by complement or melittin is effective and durable despite severe initial impairment of membrane permeability. The metabolic changes responsible for protection allow the cells to repair the membrane injury caused by complement or melittin.
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http://dx.doi.org/10.1111/xen.12172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519407PMC
May 2016

Interleukin-4 induces up-regulation of endothelial cell claudin-5 through activation of FoxO1: role in protection from complement-mediated injury.

J Biol Chem 2014 Jan 26;289(2):838-47. Epub 2013 Nov 26.

From the Departments of Surgery.

Injury to endothelial cells (ECs) often results in cell retraction and gap formation. When caused by antigen aggregation or complement, this injury can be prevented by pretreatment of the ECs with IL-4, suggesting that IL-4 modifies the intercellular junction. Therefore, we investigated the effects of IL-4 on expression of intercellular junction proteins and whether such effects are required for IL-4-induced resistance of ECs against complement-mediated injury. We found that IL-4 induces upregulation of the junction protein claudin-5 in porcine ECs through activation of Jak/STAT6 and phosphorylation and translocation of FoxO1 from the nucleus to the cytoplasm. Increased claudin-5 expression resulted in increased transmembrane electrical resistance of the endothelial monolayer and participated in IL-4-induced protection of the ECs from complement injury. Down-regulation of FoxO1 using siRNA by itself caused up-regulation of claudin-5 expression and partial protection from cytotoxicity. This protection was enhanced by stimulation with IL-4. We previously reported that increased phospholipid synthesis and mitochondrial protection were required for IL-4-induced resistance of ECs against complement injury and now we demonstrate a contribution of claudin-5 expression in IL-4-induced protection.
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http://dx.doi.org/10.1074/jbc.M113.455766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887209PMC
January 2014

On the intersections of basic and applied research in xenotransplantation.

Xenotransplantation 2012 May-Jun;19(3):137-43

Departments of Surgery and of Laboratory Medicine and Pathology, School of Medicine, University of Minnesota, Minneapolis, MN 55455, USA.

I am very grateful to the Council and members of the International Xenotransplantation Association for this Honorary Membership. In accepting this prestigious award, I pay tribute to my mentors Antonio Oriol i Anguera, Carlos Martinez, Robert A. Good, and Hans Müller-Eberhard for their guidance and friendship as I was beginning my travels in biomedical research. I also thank the many gifted collaborators, students, and technical personnel, as well as the agencies and taxpayers, who funded our research and made our scientific contributions possible. Here I briefly mention some of these contributions, including early work on the immunobiology of the thymus, my short incursion in the immunology of Chagas disease, and what have been the dominant themes of my career: the mechanisms of complement injury, the role of complement in pathophysiology, and induction of cytoprotection in the vascular endothelium. I emphasize our contributions on the role of complement as related to understanding and overcoming xenograft injury, a work that has been personally very rewarding. Now it is exciting to see that the field of xenotransplantation research is moving forward vigorously, a time of great optimism suggesting that many potential clinical applications of xenotransplantation will come to fruition in the near future.
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http://dx.doi.org/10.1111/j.1399-3089.2012.00703.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457278PMC
October 2012

Protection of porcine endothelial cells against apoptosis with interleukin-4.

Xenotransplantation 2011 Nov-Dec;18(6):343-54

Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA.

Background: Apoptosis is crucial for tissue development and homeostasis, and insufficient apoptosis is pivotal in cancer pathogenesis. Apoptosis may also be important in tissue injury and in this case, it is of interest to induce protection against apoptosis. In organ transplantation, apoptosis has been implicated in acute vascular rejection (AVR); in xenotransplantation, the inducers of apoptosis of relevance in AVR, such as tumor necrosis factor-α (TNF-α), also cause endothelial cell (EC) activation. We have previously shown that interleukin (IL)-4 and IL-13 induced protection in porcine ECs against activation and apoptosis triggered by TNF-α. Now we define signaling processes activated by IL-4 in porcine ECs and mechanisms required for IL-4-induced protection against apoptosis.

Methods: Porcine aortic ECs were used as primary cultures or as virus-induced immortalized cells derived from galactosyl transferase-deficient (Gal(-/-) ) or wild-type pigs. ECs were stimulated with porcine IL-4, either extrinsically or transduced with recombinant adenovirus (adeno) IL-4, and analyzed using immunoblotting. Apoptosis was induced with TNF-α plus cycloheximide and assessed using neutral red uptake or flow cytometry. The role of various signaling proteins in IL-4-induced protection was established using pharmacologic inhibitors and siRNA downregulation of protein expression.

Results: IL-4 induced similar degrees of phosphorylation of STAT6 in all 3 types of ECs, and STAT6 was phosphorylated through Jak3. IL-4 induced phosphorylation of Bad through Jak3. Stimulation of ECs with IL-4 caused protection of ECs against apoptosis with an absolute requirement of Jak3/STAT6 activation and major participation of mammalian target of rapamycin complex 2 (mTORC2), Akt, and extracellular signal-regulated kinase 1/2. IL-4 caused no increase in EC levels of protective proteins hemoxygenase-1, inhibitor of apoptosis protein, heat shock protein 70, Bcl-2, and Bcl-xL. ECs transduced with adenoIL-4 exhibited strong and durable protection from apoptosis. Gal(-/-) ECs were as susceptible to induction of protection with IL-4 as wild-type ECs.

Conclusions: IL-4 induces activation of Jak3/STAT6 and phosphorylation of Bad in porcine ECs, ultimately resulting in effective protection of the ECs from apoptosis. Delineation of downstream signals activated by IL-4 that are required for induction of protection suggests possible sites of intervention to design effective therapeutic agents. This is of interest because substances such as IL-4 have pleiotropic effects and cannot be used directly due to potential deleterious effects. Inducing resistance to apoptosis in porcine vascular endothelium may be important to facilitate xenograft survival and accommodation.
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http://dx.doi.org/10.1111/j.1399-3089.2011.00678.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457391PMC
May 2012

IL-4 induces protection of vascular endothelial cells against killing by complement and melittin through lipid biosynthesis.

Eur J Immunol 2010 Mar;40(3):803-12

Department of Surgery, Medical School, University of Minnesota, Minneapolis, MN 55455, USA.

We have shown previously that cytokines IL-4 and IL-13 induce protection in porcine vascular endothelial cells (EC) against killing by the membrane attack complex (MAC) of human complement. This protection is intrinsic, not due to changes in complement regulatory proteins, and requires activation of Akt and sterol receptor element binding protein-1 (SREBP-1), which regulates fatty acid and phospholipid synthesis. Here we report that, compared to EC incubated in medium, IL-4-treated EC had a profound reduction in complement-mediated ATP loss and in killing assessed by vital dye uptake, but only a slight reduction in permeability disruption measured by calcein release. While controls exposed to complement lost mitochondrial membrane potential and subsequently died, protected EC maintained mitochondrial morphology and membrane potential, and remained alive. SREBP-1 and fatty acid synthase activation were required for protection and fatty acid and phospholipid synthesis, including cardiolipin, were increased after IL-4 stimulation, without increase in cholesterol content or cell proliferation. IL-4 also induced protection of EC from killing by the channel forming protein melittin, similar to protection observed for the MAC. We conclude that IL-4 induced activation of Akt/SREBP-1/lipid biosynthesis in EC, resulting in protection against MAC and melittin, in association with mitochondrial protection.
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http://dx.doi.org/10.1002/eji.200939488DOI Listing
March 2010

Inhibition of the membrane attack complex of complement for induction of accommodation in the hamster-to-rat heart transplant model.

Xenotransplantation 2007 Nov;14(6):572-9

Department of Surgery, School of Medicine, University of Minnesota, Minneapolis, Minnisota, USA.

Background: To induce accommodation in the hamster-to-rat cardiac transplantation model, in addition to cyclosporin A (CSA) to inhibit T-cell-mediated graft rejection, cobra venom factor (CVF) is often used to prevent complement-mediated graft rejection. Although it is generally assumed that CVF makes accommodation possible because it inactivates the complement membrane attack complex (MAC), it is not known which complement components must be inactivated and whether complement activation products generated by CVF are also involved in the induction of accommodation. Therefore, to investigate mechanisms by which CVF contributes to accommodation, we studied induction of accommodation of hamster hearts grafted into rats with complement deficiencies of C6; these rats cannot assemble the MAC but, in contrast to CVF, retain in their native state all complement proteins that precede the MAC.

Methods: Golden Syrian hamster hearts were transplanted heterotopically into the abdomen of normocomplementemic and C6-deficient (C6D) PVG rats. Graft rejection was determined by cessation of palpable cardiac contractions. CSA, 10 mg/kg, was administered daily to all rats. Graft survival was compared in rats given CVF (60 U/kg 1-day pre-transplant and 20 U/kg/day for the next 9 days), C6D rats given no CVF, normocomplementemic rats given anti-C6 IgG or non-immune IgG but no CVF, and C6D rats reconstituted with normocomplementemic rat serum. Total complement and C6 serum levels were measured using hemolytic assays in rat peripheral blood.

Results: We found that hamster hearts transplanted into C6D rats receiving CSA but no CVF survived long-term, with histology typical of an accommodated heart. The accommodated hamster heart did not reconstitute C6 levels of the C6D recipient rats. Moreover, in normocomplementemic rats given anti-C6 antibodies (abs) to induce partial C6 deficiency, accommodation also developed without administration of CVF. Accommodation of the hamster heart failed to develop in C6D rats whose complement was reconstituted by administration of normocomplementemic rat serum given before and following transplantation.

Conclusions: These studies demonstrate that, in this model, inhibition of MAC-mediated graft rejection is sufficient to allow the development of accommodation. Inactivation of C3 or other complement proteins of the alternate pathway, or the presence of complement-derived biologically active fragments, is not needed for development of accommodation.
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http://dx.doi.org/10.1111/j.1399-3089.2007.00422.xDOI Listing
November 2007

Porcine endothelial cells and iliac arteries transduced with AdenoIL-4 are intrinsically protected, through Akt activation, against immediate injury caused by human complement.

J Immunol 2006 Nov;177(10):7355-63

Department of Surgery, University of Minnesota Medical School, Minneapolis, MN 55455, USA.

Vascular endothelial cells (ECs) can be injured in a variety of pathologic processes that involve activated complement. We reported previously that porcine ECs incubated with exogenous IL-4 or IL-13 are protected from cytotoxicity by human complement and also from apoptosis by TNF-alpha. The resistance to complement consists of an intrinsic mechanism that is lost a few days after cytokine removal. In our current study, we investigated whether transfer of the IL-4 gene into porcine ECs in vitro and into porcine vascular tissues in vivo would induce efficient and durable protection from human complement. We found that ECs transduced with adenoIL-4 or adenoIL-13 exhibited continuous production of the cytokine and prolonged protection from complement-mediated killing. IL-4 also protected ECs from activation: ECs incubated with IL-4 did not develop cell retraction and intercellular gaps upon stimulation with sublytic complement. The endothelium and subendothelium of pig iliac arteries that were transduced with the IL-4 gene were effectively protected from complement-dependent immediate injury after perfusion with human blood. However, after similar perfusion, the endothelium was immediately lost from arteries that were transduced with a control adenovirus. The protection was not due to up-regulation of the complement regulators decay accelerating factor, membrane cofactor protein, and CD59, or to reduced complement activation, but required the participation of Akt. Although our studies model protection in pig-to-primate xenotransplantation, our findings of IL-4 induction of Akt-mediated protection may be more broadly applicable to EC injury as manifested in ischemia-reperfusion, allotransplantation, and various vascular diseases.
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http://dx.doi.org/10.4049/jimmunol.177.10.7355DOI Listing
November 2006

Complement in asthma: sensitivity to activation and generation of C3a and C5a via the different complement pathways.

Transl Res 2006 Oct;148(4):157-63

Department of Medicine, School of Medicine, University of Minnesota, Minneapolis, MN 55455, USA.

Studies in rodent models suggested that complement may play a critical role in susceptibility to airway hyperresponsiveness (AHR) and as a mediator of bronchial obstruction and inflammation in asthma. Complement may participate in susceptibility to asthma because of an intrinsic abnormality in complement activation and generation of C3a, C5a, or other products that affect cellular responses, resulting in T(H)2 predominance and asthma susceptibility. Alternatively, an intrinsic abnormality in the cellular response to complement activation products could determine susceptibility to asthma. In this study, the authors investigated whether complement in patients with atopic asthma versus nonatopic controls possesses an increased propensity to become activated. Despite reports that total complement plasma levels in unchallenged asthmatics are normal, an abnormal sensitivity of complement to activation may exist if an isoform or a polymorphic variant of a complement protein was present and resulted in gain or loss of function without associated changes in total complement levels. Therefore, complement activation was induced in vitro in plasma of asthmatics and controls using activators of the classical, alternative, and lectin pathways and measured C3a, other C3 fragments, and C5a. For each pathway, similar amounts of generated fragments, as well as C3a/C3 and C5a/C5 ratios, were found in asthmatics and controls. Also, similar basal plasma levels of C3a and C5a were found in both groups; however, mannan-binding lectin (MBL) levels were moderately elevated in asthmatics. In conclusion, the results suggest that, in asthmatic patients, complement activation does not exhibit an abnormal sensitivity to activation by any of the known activation pathways.
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http://dx.doi.org/10.1016/j.trsl.2006.05.004DOI Listing
October 2006

IL-4 and IL-13 induce protection of porcine endothelial cells from killing by human complement and from apoptosis through activation of a phosphatidylinositide 3-kinase/Akt pathway.

J Immunol 2005 Aug;175(3):1903-10

Department of Surgery, University of Minnesota School of Medicine, Minneapolis, MN 55455, USA.

Vascular endothelial cells (EC) perform critical functions that require a balance of cell survival and cell death. EC death by apoptosis and EC activation and injury by the membrane attack complex of complement are important mechanisms in atherosclerosis and organ graft rejection. Although the effects of various cytokines on EC apoptosis have been studied, little is known about their effects on complement-mediated EC injury. Therefore, we studied the abilities of various cytokines to induce protection of porcine aortic EC against apoptosis and killing by human complement, a model of pig-to-human xenotransplantation. We found that porcine EC incubated with IL-4 or IL-13, but not with IL-10 or IL-11, became protected from killing by complement and apoptosis induced by TNF-alpha plus cycloheximide. Maximal protection required 10 ng/ml IL-4 or IL-13, developed progressively from 12 to 72 h of incubation, and lasted 48-72 h after cytokine removal. Protection from complement was not associated with reduced complement activation, C9 binding, or changes in CD59 expression. Inhibition of PI3K prevented development of protection; however, inhibition of p38 MAPK or p42/44 MAPK had no effect. IL-4 and IL-13 induced rapid phosphorylation of Akt. Although protection was inhibited by an Akt inhibitor and a dominant negative Akt mutant transduced into EC, it was induced by transduction of EC with the constitutively active Akt variant, myristylated Akt. We conclude that IL-4 and IL-13 can induce protection of porcine EC against killing by apoptosis and human complement through activation of the PI3K/Akt signaling pathway.
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http://dx.doi.org/10.4049/jimmunol.175.3.1903DOI Listing
August 2005

Alpha Gal ligation of pig endothelial cells induces protection from complement and apoptosis independently of NF-kappa B and inflammatory changes.

Am J Transplant 2005 Apr;5(4 Pt 1):712-9

Department of Surgery, School of Medicine, College of Veterinary Medicine, Minneapolis, MN, USA.

Cytoprotection of endothelial cells (EC) is important in EC biology and pathophysiology, including graft rejection. Using porcine aortic EC and human complement as an in vitro model of xenotransplantation, we have reported that ligation of EC Gal alpha (1-3)Gal epitopes (alpha Gal) with antibodies or lectins BS-I and IB4 induces EC resistance to injury by complement. However, before the protective response is observed, alpha Gal ligation induces an early, proinflammatory response. Using a similar model, we now investigated whether the early inflammatory response, as well as NF-kappa B activation, is required for induction of cytoprotection. Despite up-regulation of EC mRNA for many inflammatory cytokines rapidly after BS-I stimulation, recombinant cytokines or conditioned media from EC incubated with BS-I failed to induce protection when used to stimulate EC. While the lectin-induced inflammatory response was markedly reduced by inhibition of NF-kappa B, the protection from complement and apoptosis was unaffected. The lectins caused up-regulation of mRNA for protective genes A20, porcine inhibitor of apoptosis protein and hemoxygenase-1, which was not modified by NF-kappa B inhibition. These findings suggest that induction of cytoprotection in porcine EC by alpha Gal ligation results from activation of pathways that are largely independent of those that elicit NF-kappaB activation and the inflammatory response.
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http://dx.doi.org/10.1111/j.1600-6143.2005.00771.xDOI Listing
April 2005

Reversal of diabetes in non-immunosuppressed rhesus macaques by intraportal porcine islet xenografts precedes acute cellular rejection.

Xenotransplantation 2004 Sep;11(5):396-407

Diabetes Institute for Immunology and Transplantation, University of Minnesota, Minneapolis, MN 55455, USA.

Background: The functional response and immunobiology of primarily non-vascularized islet cell xenografts remain poorly defined in non-human primates.

Methods: We transplanted 20,000 adult porcine islet equivalents/kg (purified and cultured for 48-h) intraportally into six streptozotocin-diabetic and two non-diabetic rhesus macaques. Two recipients were killed at various intervals post-transplant for histologic examination of livers bearing xenografts.

Results: Plasma glucose levels in diabetic recipients averaged 94 mg/dl at 12 h, 92 mg/dl at 24 h, 147 mg/dl at 48 h, and 157 mg/dl at 72 h post-transplant. Serum porcine C-peptide was present in eight of eight recipients at 12 h, in five of six at 24 h, in four of four at 48 h, and in one of two at 72 h post-transplant. C3a and SC5b-9 plasma levels increased at 12 h post-transplant and returned to pre-transplant levels by 24 h. IgG, IgM anti-pig and anti-Gal IgG serum antibody levels did not increase post-transplant. Rejection was initiated by IgM and complement deposition on islets. Neutrophils dominated the cellular infiltrate at 12 h; CD4+ and CD8+ T cells were the main infiltrating cells at 24, 48, and 72 h; and macrophages increasingly infiltrated xenografts starting at 24 h post-transplant. Numerous xenoislets were present at all time points; their proportion without intraislet infiltrates decreased from 65% at 24 h to 17% at 72 h post-transplant.

Conclusions: Pig-to-primate intraportal islet xenografts reverse diabetes and the majority of intraportally transplanted xenogeneic islets are not subject to hyperacute rejection. They undergo acute cellular rejection mediated by CD4+- and CD8+ T cells and macrophages.
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http://dx.doi.org/10.1111/j.1399-3089.2004.00157.xDOI Listing
September 2004

Characteristics of CD59 up-regulation induced in porcine endothelial cells by alphaGal ligation and its association with protection from complement.

Xenotransplantation 2003 Sep;10(5):387-97

Department of Surgery, School of Medicine, University of Minnesota, Minneapolis, MN 55455, USA.

Background: Activation of endothelial cells may result in proinflammatory and procoagulant changes, or in changes that protect the endothelial cells (EC) from injurious insults. Stimulation of porcine EC with human anti-porcine antibodies, or lectins from Bandeiraea simplicifolia that bind terminal Galalpha(1-3)Gal (abbreviated alphaGal), can induce EC protection from cytotoxicity by human complement. These EC also exhibit up-regulation of CD59 protein and mRNA expression. Porcine CD59 has been reported to protect porcine cells from human complement. Therefore we investigated the specificity requirements and other characteristics of the induced CD59 up-regulation, as well as the role of up-regulated CD59 in lectin-induced protection of EC from human complement.

Methods: Aortic EC were incubated in vitro with alphaGal-binding lectins B. simplicifolia lectin I isolectin B4 (IB4) and B. simplicifolia lectin I (BS-I) and CD59 expression was assessed by flow cytometry and enzyme linked immunosorbent assay (ELISA). Binding requirement was studied using disaccharides containing either alphagalactosyl or betagalactosyl moieties to inhibit CD59 up-regulation. Protection from complement killing was assessed after incubation of EC with human serum as a source of anti-porcine antibodies and complement. The role of CD59 in lectin-induced protection was studied in the presence of an anti-pig CD59 antibody and after removal of CD59 using phosphatidylinositol (PI)-specific phospholipase C (PI-PLC).

Results: We found that induction of CD59 up-regulation required specific binding of the lectin to terminal alphaGal and was not induced either by soluble factors that may be released from EC by stimulation with the lectin or by TNF-alpha, IFN-gamma, or IL-1alpha. Unstimulated or BS-I-treated EC showed little or no expression of decay accelerator factor (DAF). Removal of membrane-associated CD59 (and other proteins that are associated with the membrane through PI linkage) with PI-PLC from EC that had been exposed to lectin restored their complement sensitivity to various degrees, depending on the extent of lectin-induced protection. Cytotoxicity was completely restored in cells that exhibited partial protection induced with lectin at low doses or for a short period of time. However, EC that were fully resistant to complement did not regain sensitivity to complement after removal of CD59. Changes in CD59 expression did not modify the degree of C9 binding.

Conclusions: Induction of CD59 expression required specific binding of the lectin to terminal alphaGal and was not induced by soluble factors that may be released from EC by lectin stimulation. Increased CD59 expression may contribute to this form of protection from complement; however, mechanisms other than CD59 up-regulation appear to be essential for the development of full protection.
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http://dx.doi.org/10.1034/j.1399-3089.2003.02088.xDOI Listing
September 2003

Transgenic pigs designed to express human CD59 and H-transferase to avoid humoral xenograft rejection.

Xenotransplantation 2002 Jan;9(1):45-57

Department of Molecular Sciences, Alexion Pharmaceuticals, Inc, Cheshire, CT 06410, USA.

Research in pig-to-primate xenotransplantation aims to solve the increasing shortage of organs for human allotransplantation and develop new cell- and tissue-based therapies. Progress towards its clinical application has been hampered by the presence of xenoreactive natural antibodies that bind to the foreign cell surface and activate complement, causing humoral graft rejection. Genetic engineering of donor cells and animals to express human complement inhibitors such as hCD59 significantly prolonged graft survival. Strategies to decrease the deposition of natural antibodies were also developed. Expression of human alpha1,2-fucosyltransferase (H transferase, HT) in pigs modifies the cell-surface carbohydrate phenotype resulting in reduced Galalpha1,3-Gal expression and decreased antibody binding. We have developed transgenic pigs that coexpress hCD59 and HT in various cells and tissues to address both natural antibody binding and complement activation. Functional studies with peripheral blood mononuclear cells and aortic endothelial cells isolated from the double transgenic pigs showed that coexpression of hCD59 and HT markedly increased their resistance to human serum-mediated lysis. This resistance was greater than with cells transgenic for either hCD59 or HT alone. Moreover, transgene expression was enhanced and protection maintained in pig endothelial cells that were exposed for 24 h to pro-inflammatory cytokines. These studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograft survival and improving the prospect of clinical xenotransplantation.
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http://dx.doi.org/10.1034/j.1399-3089.2002.0o142.xDOI Listing
January 2002

A soluble chimeric inhibitor of C3 and C5 convertases, complement activation blocker-2, prolongs graft survival in pig-to-rhesus monkey heart transplantation.

Xenotransplantation 2002 Mar;9(2):125-34

Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA.

Complement plays a critical role in many pathologic processes and in xenograft rejection. Therefore, effective complement inhibitors are of great interest. In pig-to-primate organ transplantation, hyperacute rejection results from antibody deposition and complement activation. Complement activation blocker-2 (CAB-2), a recombinant soluble chimeric protein derived from human decay accelerating factor (DAF) and membrane cofactor protein, inhibits C3 and C5 convertases of both classical and alternative pathways. CAB-2 reduces complement-mediated tissue injury of a pig heart perfused ex vivo with human blood. Therefore, we studied the efficacy of CAB-2 when a pig heart is transplanted heterotopically into rhesus monkeys receiving no immunosuppression. Graft survival in three control monkeys was 1.26 +/- 0.2 h; it was markedly prolonged in eight monkeys that received CAB-2. Of the six monkeys that received a single dose of CAB-2 (15 mg/kg i.v.), four had graft survivals of 21, 95, 96, and 108 h, and two died at 7 to 11 h post-transplant with a beating graft, as a result of technical complications. The two monkeys given multiple doses of CAB-2 had graft survivals of 95 and 96 h. CAB-2 markedly inhibited complement activation, as shown by a strong reduction in generation of C3a and SC5b-9. At graft rejection, tissue deposition of iC3b, C4 and C9 was similar or slightly reduced from controls, and deposition of IgG, IgM, C1q and fibrin did not change. Thus, complement inhibition with CAB-2 abrogates hyperacute rejection of pig hearts transplanted into rhesus monkeys, but does not prevent delayed/acute vascular rejection. These studies demonstrate that the beneficial effects of complement inhibition on survival of a pig heart xenograft in rhesus monkeys are similar to those in other primate species and that CAB-2 may be useful in xenotransplantation and other complement-mediated conditions.
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http://dx.doi.org/10.1034/j.1399-3089.2002.1o042.xDOI Listing
March 2002