Pilot Study of Delayed ICOS/ICOS-L Blockade With alphaCD40 to Modulate Pathogenic Alloimmunity in a Primate Cardiac Allograft Model

Background: Inducible costimulator (ICOS) is rapidly upregulated with T-cell stimulation and may represent an escape pathway for T-cell costimulation in the setting of CD40/CD154 costimulation blockade. Induction treatment exhibited no efficacy in a primate renal allograft model, but rodent transplant models suggest that the addition of delayed ICOS/ICOS-L blockade may prolong allograft survival and prevent chronic rejection. Here, we ask whether ICOS-Ig treatment, timed to anticipate ICOS upregulation, prolongs NHP cardiac allograft survival or attenuates pathogenic alloimmunity. Methods: Cynomolgus monkey heterotopic cardiac allograft recipients were treated with alphaCD40 (2C10R4, d0-90) either alone or with the addition of delayed ICOS-Ig (d63-110). Results: Median allograft survival was similar between ICOS-Ig + alphaCD40 (120 days, 120-125 days) and alphaCD40 (124 days, 89-178 days) treated animals, and delayed ICOS-Ig treatment did not prevent allograft rejection in animals with complete CD40 receptor coverage. Although CD4(+) TEM cells were decreased in peripheral blood (115 +/- 24) and mLNs (49 +/- 1.9%) during ICOS-Ig treatment compared with monotherapy (214 +/- 27%, P = 0.01; 72 +/- 9.9%, P = 0.01, respectively), acute and chronic rejection scores and kinetics of alloAb elaboration were similar between groups. Conclusions: Delayed ICOS-Ig treatment with the reagent tested is probably ineffective in modulating pathogenic primate alloimmunity in this model

Third WHO Global Consultation on regulatory requirements for xenotransplantation clinical trials, Changsha, Hunan, China December 12-14, 2018: "The 2018 Changsha Communiqué" The 10-Year Anniversary of The International Consultation on Xenotransplantation

After feedback from the working parties, the final session focused on drafting proposed revisions of the WHO documents, and resulted in the formulation of the draft “Third WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials, The 2018 Changsha Communiqué.” This draft was submitted to WHO in February 2019 for WHO and World Health Assembly consideration. If approved, the 2018 Changsha Communiqué will then be posted on the websites of WHO, IXA, and TTS, and published in Xenotransplantation. This report includes summaries of the various sessions, followed by the abstracts of invited speakers from the update sessions

The antioxidant tempol attenuates pressure overload-induced cardiac hypertrophy and contractile dysfunction in mice fed a high-fructose diet

We have previously shown that high-sugar diets increase mortality and left ventricular (LV) dysfunction during pressure overload. The mechanisms behind these diet-induced alterations are unclear but may involve increased oxidative stress in the myocardium. The present study examined whether high-fructose feeding increased myocardial oxidative damage and exacerbated systolic dysfunction after transverse aortic constriction (TAC) and if this effect could be attenuated by treatment with the antioxidant tempol. Immediately after surgery, TAC and sham mice were assigned to a high-starch diet (58% of total energy intake as cornstarch and 10% fat) or high-fructose diet (61% fructose and 10% fat) with or without the addition of tempol [0.1% (wt/wt) in the chow] and maintained on the treatment for 8 wk. In response to TAC, fructose-fed mice had greater cardiac hypertrophy (55.1% increase in the heart weight-to-tibia length ratio) than starch-fed mice (22.3% increase in the heart weight-to-tibia length ratio). Treatment with tempol significantly attenuated cardiac hypertrophy in fructose-fed TAC mice (18.3% increase in the heart weight-to-tibia ratio). Similarly, fructose-fed TAC mice had a decreased LV area of fractional shortening (from 38 ± 2% in sham to 22 ± 4% in TAC), which was prevented by tempol treatment (33 ± 3%). Markers of lipid peroxidation in fructose-fed TAC hearts were also blunted by tempol. In conclusion, tempol significantly blunted markers of cardiac hypertrophy, LV remodeling, contractile dysfunction, and oxidative stress in fructose-fed TAC mice

Four-Dimensional Characterization of Thrombosis in a Live-Cell, Shear-Flow Assay: Development and Application to Xenotransplantation

<div><p>Background</p><p>Porcine xenografts are a promising source of scarce transplantable organs, but stimulate intense thrombosis of human blood despite targeted genetic and pharmacologic interventions. Current experimental models do not enable study of the blood/endothelial interface to investigate adhesive interactions and thrombosis at the cellular level under physiologic conditions. The purpose of this study was to develop and validate a live-cell, shear-flow based thrombosis assay relevant to general thrombosis research, and demonstrate its potential in xenotransplantation applications.</p><p>Methodology/Principal Findings</p><p>Confluent wild-type (WT, n = 48) and Gal transferase knock-out (GalTKO, which resist hyperacute rejection; n = 11) porcine endothelia were cultured in microfluidic channels. To mimic microcirculatory flow, channels were perfused at 5 dynes/cm² and 37°C with human blood stained to fluorescently label platelets. Serial fluorescent imaging visualized percent surface area coverage (SA, for adhesion of labeled cells) and total fluorescence (a metric of clot volume). Aggregation was calculated by the fluorescence/SA ratio (FR). WT endothelia stimulated diffuse platelet adhesion (SA 65 ± 2%) and aggregation (FR 120 ± 1 a.u.), indicating high-grade thrombosis consistent with the rapid platelet activation and consumption seen in whole-organ lung xenotransplantation models. Experiments with antibody blockade of platelet aggregation, and perfusion of syngeneic and allo-incompatible endothelium was used to verify the biologic specificity and validity of the assay. Finally, with GalTKO endothelia thrombus volume decreased by 60%, due primarily to a 58% reduction in adhesion (P < 0.0001 each); importantly, aggregation was only marginally affected (11% reduction, P < 0.0001).</p><p>Conclusions/Significance</p><p>This novel, high-throughput assay enabled dynamic modeling of whole-blood thrombosis on intact endothelium under physiologic conditions, and allowed mechanistic characterization of endothelial and platelet interactions. Applied to xenogeneic thrombosis, it enables future studies regarding the effect of modifying the porcine genotype on sheer-stress-dependent events that characterize xenograft injury. This in-vitro platform is likely to prove broadly useful to study thrombosis and endothelial interactions under dynamic physiologic conditions.</p></div

Life-supporting function of genetically modified swine lungs in baboons

Objective: During ex vivo perfusion with human blood, homozygous galactosyl transferase knockout swine lungs exhibit prolonged survival (∼2 hours) relative to wild-type (\u3c15 minutes) and swine lungs expressing human decay accelerating factor (\u3c1 hour). In this study, the in vivo behavior of galactosyl transferase knockout lungs was evaluated. Methods: Three galactosyl transferase knockout swine left lungs were transplanted into baboons in a life-supporting model. One baboon lung allograft and two swine lung xenografts transgenic for human membrane cofactor protein (CD46) served as controls. Results: Whereas two membrane cofactor protein lungs exhibited high pulmonary vascular resistance (\u3e500 mm Hg · min/L) and failed to support life within 21 minutes, two of three galactosyl transferase knockout lungs supported life, for 90 and 215 minutes, and displayed low peripheral vascular resistance (48 ± 12 mm Hg · min/L at 60 minutes), similar to the allogeneic control. Complement activation (delta C3a \u3c 250 ng/mL through 60 minutes) and C5b-9 deposition were minimal in both galactosyl transferase knockout and membrane cofactor protein lungs. Neutrophils, monocytes, and platelets were rapidly sequestered in galactosyl transferase knockout and human membrane cofactor protein lung recipients, unlike the allogeneic control (\u3c20%); and thrombin formation (delta plasma fraction 1+2 \u3e 0.5 nmol/L) was seen in the galactosyl transferase knockout recipients. Platelet activation (β-thromboglobulin rise \u3e 200) and appearance of capillary congestion and vessel thrombosis confirmed coagulation activation associated with galactosyl transferase knockout lung failure. Conclusions: Galactosyl transferase knockout swine lungs are significantly protected in vivo from the physiologic consequences (increased pulmonary vascular resistance, capillary leak) associated with hyperacute lung rejection. As during ex vivo perfusion, dysregulated coagulation-thrombin elaboration, platelet activation, and intravascular thrombosis-mediates galactosyl transferase knockout lung xenograft injury. © 2007 The American Association for Thoracic Surgery

A—C. Representative volumetric thrombus models.

<p>3D surface renderings of end-perfusion images (<i>t</i> = 50 minutes) for WT and GalTKO endothelia perfused with heparinized human blood (A & B, respectively) and WT endothelium perfused with heparinized human blood treated with 0.5 μg/mL abciximab (C). Panel A demonstrates significant adhesion and aggregation. In Panel B adhesion is reduced but aggregation is mostly intact, while in Panel C aggregation is ablated. Source images were taken at 100x magnification using a 100-millisecond exposure and rendered using ImageJ.</p

Results from perfusing porcine endothelia with syngeneic and allo-incompatible porcine blood.

<p>*All experiments were performed with heparinized blood. a.u., arbitrary units; TV, thrombus volume; Δ, relative change; SA, percent surface area coverage; FR, fluorescence ratio; T₅₀, time to 50% maximal surface area coverage. All Δ & <i>P</i> values are versus WT controls, and are expressed as mean ± SEM.</p><p>Results from perfusing porcine endothelia with syngeneic and allo-incompatible porcine blood.</p

Results from xenoperfusion of porcine endothelia with human blood.

<p>*All experiments were performed with heparinized blood. a.u., arbitrary units; TV, thrombus volume; Δ, relative change; SA, percent surface area coverage; FR, fluorescence ratio; T₅₀, time to 50% maximal surface area coverage. All Δ & <i>P</i> values are versus WT controls, and are expressed as mean ± SEM.</p><p>Results from xenoperfusion of porcine endothelia with human blood.</p