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Galactosyl transferase knock-out pig lungs fail rapidly in baboons. Based on previously identified lung xenograft injury mechanisms, additional expression of human complement and coagulation pathway regulatory proteins, anti-inflammatory enzymes and self-recognition receptors, and knock-down of the β4Gal xenoantigen were tested in various combinations. Transient life-supporting GalTKO.hCD46 lung function was consistently observed in association with either hEPCR (n = 15), hTBM (n = 4), or hEPCR. hTFPI (n = 11), but the loss of vascular barrier function in the xenograft and systemic inflammation in the recipient typically occurred within 24 h. Co-expression of hEPCR and hTBM (n = 11) and additionally blocking multiple pro-inflammatory innate and adaptive immune mechanisms was more consistently associated with survival >1 day, with one recipient surviving for 31 days. Combining targeted genetic modifications to the lung xenograft with selective innate and adaptive immune suppression enables prolonged initial life-supporting lung function and extends lung xenograft recipient survival, and illustrates residual barriers and candidate treatment strategies that may enable the clinical application of other organ xenografts.

Antibody-mediated rejection has long been known to be one of the major organ failure mechanisms in xenotransplantation. In addition to the porcine α1,3-galactose (α1,3Gal) epitope, N-Glycolylneuraminic acid (Neu5Gc), a sialic acid, has been identified as an important porcine antigen against which most humans have pre-formed antibodies. Here we evaluate GalTKO.hCD46 lungs with an additional cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene knock-out (Neu5GcKO) in a xenogeneic ex vivo perfusion model METHODS: Eleven GalTKO.hCD46.Neu5GcKO pig lungs were perfused for up to 6 h with fresh heparinized human blood. Six of them were treated with histamine (H) blocker famotidine and 1-thromboxane synthase inhibitor Benzylimidazole (BIA) and five were left untreated. GalTKO.hCD46 lungs without Neu5GcKO (n = 18: eight untreated and 10 BIA+H treated) served as a reference. Functional parameters, blood, and tissue samples were collected at pre-defined time points throughout the perfusion RESULTS: All but one Neu5GcKO organs maintained adequate blood oxygenation and "survived" until elective termination at 6 h whereas two reference lungs failed before elective termination at 4 h. Human anti-Neu5Gc antibody serum levels decreased during the perfusion of GalTKO.hCD46 lungs by flow cytometry (∼40% IgM, 60% IgG), whereas antibody levels in Neu5GcKO lung perfusions did not fall (IgM p = .007; IgG p .001). Thromboxane elaboration, thrombin generation, and histamine levels were significantly reduced with Neu5GcKO lungs compared to reference in the untreated groups (p = .007, .005, and .037, respectively); treatment with BIA+H masked these changes. Activation of platelets, measured as CD62P expression on circulating platelets, was lower in Neu5GcKO experiments compared to reference lungs (p = .023), whereas complement activation (as C3a rise in plasma) was not altered. MCP-1 and lactotransferin level elevations were blunted in Neu5GcKO lung perfusions (p = .007 and .032, respectively). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated GalTKO.hCD46.Neu5GcKO lungs in comparison to the untreated GalTKO.hCD46 lungs (p = .003) CONCLUSION: Additional Neu5GcKO in GalTKO.hCD46 lungs significantly reduces parameters associated with antibody-mediated inflammation and activation of the coagulation cascade. Knock-out of the Neu5Gc sialic acid should be beneficial to reduce innate immune antigenicity of porcine lungs in future human recipients.

The phenomenon of diminishing hematocrit after in vivo liver and lung xenotransplantation and during ex vivo liver xenoperfusion has largely been attributed to action by resident liver porcine macrophages, which bind and destroy human erythrocytes. Porcine sialoadhesin (siglec-1) was implicated previously in this interaction. This study examines the effect of porcine genetic modifications, including knockout of the CMAH gene responsible for expression of Neu5Gc sialic acid, on the adhesion of human red blood cells (RBCs) to porcine macrophages. Wild-type (WT) porcine macrophages and macrophages from several strains of genetically engineered pigs, including CMAH gene knockout and several human transgenes (TKO+hTg), were incubated with human RBCs and "rosettes" (≥3 erythrocytes bound to one macrophage) were quantified by microscopy. Our results show that TKO+hTg genetic modifications significantly reduced rosette formation. The monoclonal antibody 1F1, which blocks porcine sialoadhesin, significantly reduced rosette formation by WT and TKO+hTg macrophages compared with an isotype control antibody. Further, desialation of human RBCs with neuraminidase before addition to WT or TKO+hTg macrophages resulted in near-complete abrogation of rosette formation, to a level not significantly different from porcine RBC rosette formation on porcine macrophages. These observations are consistent with rosette formation being mediated by binding of sialic acid on human RBCs to sialoadhesin on porcine macrophages. In conclusion, the data predict that TKO+hTg genetic modifications, coupled with targeting of porcine sialoadhesin by the 1F1 mAb, will attenuate erythrocyte sequestration and anemia during ex vivo xenoperfusion and following in vivo liver, lung, and potentially other organ xenotransplantation.

Consistent survival of life-supporting pig heart xenograft recipients beyond 90 days was recently reported using genetically modified pigs and a clinically applicable drug treatment regimen. If this remarkable achievement proves reproducible, published benchmarks for clinical translation of cardiac xenografts appear to be within reach. Key mechanistic insights are summarized here that informed recent pig design and therapeutic choices, which together appear likely to enable early clinical translation.

The transplantation of organs across species offers the potential to solve the shortage of human organs. While activation of human platelets by human von Willebrand factor (vWF) requires vWF activation by shear stress, contact between human platelets and porcine vWF (pvWF) leads to spontaneous platelet adhesion and activation. This non-physiologic interaction may contribute to the thrombocytopenia and coagulation pathway dysregulation often associated with xenotransplantation of pig organs in nonhuman primates. Pigs genetically modified to decrease antibody and complement-dependent rejection (GTKO.hCD46) were engineered to express humanized pvWF (h*pvWF) by replacing a pvWF gene region that encodes the glycoprotein Ib-binding site with human cDNA orthologs. This modification corrected for non-physiologic human platelet aggregation on exposure to pig plasma, while preserving in vitro platelet activation by collagen. Organs from pigs with h*pvWF demonstrated reduced platelet sequestration during lung (p ≤ .01) and liver (p ≤ .038 within 4 h) perfusion ex vivo with human blood and after pig-to-baboon lung transplantation (p ≤ .007). Residual platelet sequestration and activation were not prevented by the blockade of canonical platelet adhesion pathways. The h*pvWF modification prevents physiologically inappropriate activation of human or baboon platelets by porcine vWF, addressing one cause of the thrombocytopenia and platelet activation observed with xenotransplantation.

The thoracic outlet is the space between the thorax and axilla through which the subclavian vein, subclavian artery, and brachial plexus travel from their central origins to their peripheral termini. Its bounds include the clavicle, first thoracic rib, insertion of the pectoralis minor muscle onto the coracoid process of the humerus, and the sternum. It contains three areas: the scalene triangle, the costoclavicular space, and the subcoracoid or pectoralis minor space. Aberrant anatomy is common in the thoracic outlet and may predispose patients to compression of the neurovascular bundle and development of clinical thoracic outlet syndrome (TOS). Much of this aberrancy is explained by the embryologic origins of the structures that comprise the thoracic outlet. A thorough understanding of this anatomy and embryology is therefore critical to the understanding of TOS.

Purpose In normal hemostasis, activated von Willebrand factor (vWF) stimulates platelet adhesion and aggregation, but porcine vWF (pvWF) is “always active” when exposed to human blood. We hypothesized that humanized vWF (h*pvWF) would reduce this nonphysiologic platelet adhesion in lung xenotransplantation models. Methods GTKO.hCD46.h*pvWF pig lungs were perfused with human blood ex vivo and compared to reference GTKO.hCD46 lungs. Anti-GPIb Fab, 1-BIA (thromboxane synthase inhibitor), and antihistamines were used in all perfusions. One lung from each pair (n=5 h*pvWF, n=5 ref) also received integrin (H52/IB4) and selectin (GMI-1271, PSGL-1) blockade. Lungs surviving 8 hrs of perfusion were terminated electively. Platelets were measured by flow cytometry. h*pvWF lungs were also transplanted into baboon recipients (n=3 h*pvWF, n=10 ref), with platelets measured by CBC. Results All ex vivo groups had similar survival, with most electively terminated. Pulmonary vascular resistance was very low and constant in all groups. Platelet sequestration was significantly reduced and delayed in h*pvWF lungs compared to pvWF (p Conclusion When perfused with human blood, h*pvWF lungs had reduced sequestration of platelets compared to pvWF lungs, with similar reductions in lung xenotransplant recipients. “Humanization” of porcine vWF attenuates the nonphysiologic platelet adhesion and aggregation associated with xenograft rejection. Further study will focus on Fc-receptors, adenosine and coagulation pathway dysregulation, and activated neutrophils through additional drugs and genetic modifications.

Purpose Antibody-mediated xenograft rejection is inhibited by knock-out (KO) of porcine carbohydrates (CHO), including Galα1,3Gal. The β4Gal epitope was discovered using serum from xenograft-sensitized baboons. However, it remains controversial if humans and baboons have detectable titers of preformed anti-β4Gal antibody (as humans do for Neu5Gc), and whether anti-β4Gal antibody modulates lung xenograft injury. Methods In serum from 14 baboons, pre-lung transplant and post-op day1 anti-pig Ab binding against GalTKO versus GalTKO.β4GalKO (double KO (DKO)) porcine aortic endothelial cells (PAECs) was measured in animals that had received a lung from a GalTKO pig and compared to those in which the donor animal had β4GalKO (or knock down), with (TKO) or without (DKO) additional Neu5GcKO. To evaluate Ab binding to pig tissue, lung biopsies from wildtype, GalTKO, DKO and TKO pigs after 30min of ex vivo xeno-perfusion with human blood were stained for IgG and IgM binding. In one case Ab binding to GalTKO and DKO cells was also measured at 1 and 31d in a long-surviving xenolung recipient. Results IgM Ab binding in naive baboons was consistently lower on GalTKO.β4GalKO relative to GalTKO PAECs. Decline in anti-non-Gal Ab levels on post-op d1 in vivo was consistently less using GalTKO.β4GalKO lungs (31%±16%, n=3) when compared to single KO lungs (58%±16%, n=7). In a long-surviving LTX recipient animal, sensitized anti-non Gal antibody binding was almost completely abrogated when using TKO cells. Lungs with multiple CHO KOs appear to have decreased IgM tissue deposition relative to GalTKO lungs after perfusion with human blood when analyzed by Immunohistochemistry. Conclusion β4Gal is a dominant antigen after lung xenotransplantation. Binding of preformed antibody (IgM>IgG) in humans and baboons is reduced by β4GalKO. Since the β4Gal epitope presumably contributes to pig lung xenograft injury, including the β4GalKO modification in donor pigs will likely help to better control Ab-mediated xenorejection pathways and advance xenotransplantation of the lung and other organs towards the clinic.

Purpose In baboons, lungs from galactosyl transferase knock-out (GTKO) pigs expressing human complement regulatory proteins (hCPRP: hCD46, hCD55) exhibit elevated pulmonary vascular resistance and loss of vascular barrier function within hours. Here we summarize efforts over ten years to understand and prevent these phenomena. Methods Orthotopic left single lung transplantation was performed using GTKO.hCPRP lungs with additional expression of one or more thromboregulatory (hEPCR, hTBM, hTFPI, hCD39) and anti-inflammatory genes (HO-1, HLA-E, hCD47), and β4Gal carbohydrate xenoantigen knock-down (KD), in various combinations. Anti-inflammatory drugs (steroids, sC1Inh, anti-histamine, anti-thromboxane, A1AT, sTNFR, αIL-6, αIL-8, PSGL-1, αMac1), antibody adsorption (AbAds) using donor pig kidneys, and donor pretreatment with chlodronate liposomes were progressively added in recent years. Results Transient life-supporting GTKO.hCD46 lung function was consistently observed with either hEPCR (alone [n=6] or with hCD55 [6], HO-1 [2], or hCD47 [1]); with hTBM (alone [3] or with HO-1 [1] or CD47 [1]); or with hCD55.hTFPI.CD47 [11]. Although recipient survival was occasionally extended to 2 [n=6] or even 8 days [1], loss of xenograft vascular barrier function and systemic inflammation typically occurred within 24-48 hours. Consistent recipient survival beyond 12 hours was only observed with hEPCR and hTBM co-expression [n=12] in combination with one or more additional genetic modifications (HO-1 [2], CD47.HO-1 [3], hCD55 [2], hCD55.hCD39 [3], or β4GalKD.hCD47 [2]), including 7 and 9 day survivals with selectin/integrin blockade, A1AT, and liposomes. β4GalKD.hCD47, liposome pretreatment, recipient splenectomy, and AbAds were associated with prolonged recipient survival to 31 days in one instance. Graft demise beyond 2 days was association with elaboration of anti-non-Gal antibody whether using conventional or αCD40-based immunosuppression. Conclusion Consistent life-supporting lung xenograft function and increasingly frequent recipient survival >2d reflect significant progress in understanding the remaining barriers to clinical translation. We identify a pivotal role for dysregulated coagulation and multiple proinflammatory mechanisms in the loss of vascular barrier function which is associated with lung xenograft failure, and the need for more effective immunosuppression.

Sixty children, ranging in age from 5 to 11 years, looked at a series of congruous (i.e., normal) pictures (animals and other objects) and at pictures of incongruous objects formed by mismatched combinations of heads and bodies of the congruous objects. Afterwards, the children were asked to give names to all the pictures. The results disclosed longer looking times, more sustained expression changes, and greater likelihood of expression change to the incongruous than to the congruous pictures. Further, older children were more likely than younger children to recognize and respond to incongruity according to these measures and also were more likely to name both components of the incongruous pictures.

Summary Between 1929 and 1958 American college students showed an increase in the severity of their moral judgments (7). An analysis of the change scores (1958–1929) in terms of “prescriptive” (“ought to”) and “proscriptive” (“ought not to”) items indicated a significantly greater increase (p < .01) in severity of judgments to prescriptive items than to proscriptive items. The changes are considered in terms of the increased use of love-oriented socialization techniques over the years studied and students' perceptions of their parents' child rearing practices. Some limitations of methodology are discussed.