Pig-to-baboon lung xenotransplantation: Extended survival with targeted genetic modifications and pharmacologic treatments.

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 ß4Gal 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.

Interleukin-8 mediates neutrophil-endothelial interactions in pig-to-human xenogeneic models.

BACKGROUND: Human neutrophils are sequestered by pig lung xenografts within minutes during ex vivo perfusion. This phenomenon is not prevented by pig genetic modifications that remove xeno-antigens or added human regulatory molecules intended to down-regulate activation of complement and coagulation pathways. This study investigated whether recipient and donor interleukin-8 (IL-8), a chemokine known to attract and activate neutrophils during inflammation, is elaborated in the context of xenogeneic injury, and whether human or pig IL-8 promote the adhesion of human neutrophils in in vitro xenograft models. METHODS: Plasma levels of pig, human or non-human primate (NHP) IL-8 from ex vivo pig lung perfusion experiments (n = 10) and in vivo pig-to-baboon lung transplantation in baboons (n = 22) were analysed by ELISA or Luminex. Human neutrophils stimulated with human or pig IL-8 were analysed for CD11b expression, CD18 activation, oxidative burst and adhesion to resting or TNF-activated endothelial cells (EC) evaluated under static and flow (Bioflux) conditions. For some experiments, human neutrophils were incubated with Reparixin (IL-8/CXCL8 receptor blocker) and then analysed as in the in vitro experiments mentioned above. RESULTS: Plasma levels of pig IL-8 (~6113 pg/mL) increased more than human (~1235 pg/mL) between one and four hours after initiation of ex vivo lung perfusion. However, pig IL-8 levels remained consistently low (<60 pg/mL) and NHP IL-8 plasma levels increased by ~2000 pg/mL after four hours in a pig-to-baboon lung xenotransplantation. In vitro, human neutrophils' CD11b expression, CD18 activation and oxidative burst all increased in a dose-dependent manner following exposure to either pig or human IL-8, which also were associated with increased adhesion to EC in both static and flow conditions. Reparixin inhibited human neutrophil activation by both pig and human IL-8 in a dose-dependent fashion. At 0.1 mg/mL, Reparixin inhibited the adhesion of IL-8-activated human neutrophils to pAECs by 84 ± 2.5%. CONCLUSIONS: Pig IL-8 increased in an ex vivo model of pig-to-human lung xenotransplantation but is not detected in vivo, whereas human or NHP IL-8 is elevated to a similar degree in both models. Both pig and human IL-8 activate human neutrophils and increase their adhesion to pig aortic ECs, a process significantly inhibited by the addition of Reparixin to human neutrophils. This work implicates IL-8, whether of pig or human origin, as a possible factor mediating in lung xenograft inflammation and injury and supports the evaluation of therapeutic targeting of this pathway in the context of xenotransplantation.

P- and E-selectin receptor antagonism prevents human leukocyte adhesion to activated porcine endothelial monolayers and attenuates porcine endothelial damage.

BACKGROUND: Alongside the need to develop more effective and less toxic immunosuppression, the shortage of human organs available for organ transplantation is one of the major hurdles facing the field. Research into xenotransplantation, as an alternative source of organs, has unveiled formidable challenges. Porcine lungs perfused with human blood rapidly sequester the majority of circulating neutrophils and platelets, which leads to inflammation and organ failure within hours, and is not significantly attenuated by genetic modifications to the pig targeted to diminish antibody binding and complement and coagulation cascade activation. METHODS: Here, we model the interaction of freshly isolated human leukocytes with xenotransplanted vasculature under physiologic flow conditions using microfluidic channels coated with porcine endothelial cells. Both isolated human neutrophils and whole human blood were perfused over transgenic pig aortic endothelial cells that had been activated with rhTNF-α or rhIL-4 using the BioFlux system. Novel compounds GMI-1271 and rPSGL1.Fc were tested as E- and P- selectin antagonists, respectively. Cellular adhesion and rolling events were tracked using FIJI (imageJ). RESULTS: Porcine endothelium activated with either rhTNF-α or rhIL-4 expressed high amounts of selectins, to which isolated human neutrophils readily rolled and tethered. Both E-and P-selectin antagonism significantly reduced the number of neutrophils rolling and rolling distance in a dose-dependent manner, with near total inhibition at higher doses (P < .001). Similarly, with whole human blood, selectin blocking compounds exhibited dose-dependent inhibition of prevalent leukocyte adhesion and severe endothelial injury (Untreated: 394 ± 97 PMNs/hpf, 57 ± 6% loss EC; GMI1271+rPSGL1.Fc: 23 ± 9 PMNs/hpf, 8 ± 6% loss EC P < .01). CONCLUSIONS: Selectin blockade may be useful as part of an integrated strategy to prevent neutrophil-mediated organ xenograft injury, especially during the early time points following reperfusion.

Synthetic liver function is detectable in transgenic porcine livers perfused with human blood.

In addition to immune barriers, molecular incompatibilities between species are predicted to limit pig liver survival in primate xenotransplantation models. Assessment and measurement of synthetic function of genetically modified porcine livers after ex vivo perfusion with human blood have not previously been described. Eight porcine livers from α1,3-galactosyl transferase knockout and human membrane cofactor (GalTKO.hCD46), six livers from GalTKO.hCD46 and N-glycolylneuraminic acid knockout (GalTKO.hCD46.Neu5GcKO), and six livers from GalTKO.hCD46 with humanized decay-accelerating factor (hCD55), endothelial protein C receptor (hEPCR), tissue factor pathway inhibitor (hTFPI), and integrin-associated protein (hCD47) (GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47) pigs were perfused with human blood under physiologic conditions. Timed blood samples were tested for liver enzymes and for pig-specific albumin production via Western blot. Porcine albumin levels increased with time in all experiments. By densitometry, GalTKO.hCD46.Neu5GcKO livers had the highest albumin levels, measured both as total produced, and when controlled for perfusion duration, compared to GalTKO.hCD46 (P = .068) and GalTKO.hCD46.hCD55.hEPCR.hTFPI.hCD47 livers (P = .04). Porcine livers perfused with human blood demonstrated the synthetic ability to produce albumin in all cases. GalTKO.hCD46.Neu5GcKO pig livers demonstrated the most robust albumin production. This suggests that the Neu5GcKO phenotype provides a protective effect on the graft due to decreased human antibody recognition and graft injury.

Comparative Evaluation of αCD40 (2C10R4) and αCD154 (5C8H1 and IDEC-131) in a Nonhuman Primate Cardiac Allotransplant Model.

BACKGROUND: Specific blockade of T cell costimulation pathway is a promising immunomodulatory approach being developed to replace our current clinical immunosuppression therapies. The goal of this study is to compare results associated with 3 monoclonal antibodies directed against the CD40/CD154 T cell costimulation pathway. METHODS: Cynomolgus monkey heterotopic cardiac allograft recipients were treated with either IDEC-131 (humanized αCD154, n = 9), 5C8H1 (mouse-human chimeric αCD154, n = 5), or 2C10R4 (mouse-rhesus chimeric αCD40, n = 6) monotherapy using a consistent, comparable dosing regimen for 3 months after transplant. RESULTS: Relative to the previously reported IDEC-131-treated allografts, median survival time (35 ± 31 days) was significantly prolonged in both 5C8H1-treated (142 ± 26, P < 0.002) and 2C10R4-treated (124 ± 37, P < 0.020) allografts. IDEC-131-treated grafts had higher cardiac allograft vasculopathy severity scores during treatment relative to either 5C8H1 (P = 0.008) or 2C10R4 (P = 0.0002). Both 5C8H1 (5 of 5 animals, P = 0.02) and 2C10R4 (6/6, P = 0.007), but not IDEC-131 (2/9), completely attenuated IgM antidonor alloantibody (alloAb) production during treatment; 5C8H1 (5/5) more consistently attenuated IgG alloAb production compared to 2C10R4 (4/6) and IDEC-131 (0/9). All evaluable explanted grafts experienced antibody-mediated rejection. Only 2C10R4-treated animals exhibited a modest, transient drop in CD20 lymphocytes from baseline at day 14 after transplant (-457 ± 152 cells/µL) compared with 5C8H1-treated animals (16 ± 25, P = 0.037), and the resurgent B cells were primarily of a naive phenotype. CONCLUSIONS: In this model, CD154/CD40 axis blockade using IDEC-131 is an inferior immunomodulatory treatment than 5C8H1 or 2C10R4, which have similar efficacy to prolong graft survival and to delay cardiac allograft vasculopathy development and antidonor alloAb production during treatment.

Transgenic expression of human leukocyte antigen-E attenuates GalKO.hCD46 porcine lung xenograft injury.

BACKGROUND: Lung xenografts remain susceptible to loss of vascular barrier function within hours in spite of significant incremental advances based on genetic engineering to remove the Gal 1,3-αGal antigen (GalTKO) and express human membrane cofactor protein (hCD46). Natural killer cells rapidly disappear from the blood during perfusion of GalTKO.hCD46 porcine lungs with human blood and presumably are sequestered within the lung vasculature. Here we asked whether porcine expression of the human NK cell inhibitory ligand HLA-E and ß2 microglobulin inhibits GalTKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models. METHODS: Lungs from pigs modified to express GalTKO.hCD46 (n=37) and GalTKO.hCD46.HLA-E (n=5) were harvested and perfused with human blood until failure or elective termination at 4 hours. Airway pressures and pulmonary artery hemodynamics were recorded in real time. Blood samples were also collected throughout the experiment for analysis. Porcine aortic endothelial cells (PAECs) from each genotype were cultured in monolayers in microfluidic channels and used in fluorescent cytotoxicity assays using human NK cells. RESULTS: HLA-E expression on GalTKO.hCD46 PAECs was associated with significantly decreased antibody-dependent and antibody-independent NK-mediated cytotoxicity under in vitro conditions simulating physiologic shear stress. Relative to GalTKO.hCD46 pig lungs perfused with human blood on an ex vivo platform, additional expression of HLA-E increased median lung survival (>4 hours, vs 162 minutes, P=.012), and was associated with attenuated rise in pulmonary vascular resistance, and decreased platelet activation and histamine elaboration. As expected, HLA-E expression was not associated with a significant difference in NK cell adhesion to endothelial cells in vitro, or NK cell and neutrophil sequestration during organ perfusion. CONCLUSIONS: We conclude human NK cell activation contributes significantly to GalTKO.hCD46 pig endothelial injury and lung inflammation and show that expression of HLA-E is associated with physiologically meaningful protection of GalTKO.hCD46 cells and organs exposed to human blood.

Hip range of motion in children: what is the norm?

BACKGROUND: Abnormal range of motion (ROM) is a common sign of pathology in the pediatric hip, yet there are little data in the literature defining what the normal hip ROM is in children. The purpose of this study was to establish normative values for hip ROM in children of varying ages. METHODS: We performed an Institutional Review Board approved, prospective study of otherwise healthy patients receiving fracture care at our institution. Inclusion criteria were boys and girls aged 2 to 17, who were being treated for an isolated upper extremity injury and who had no underlying musculoskeletal condition, history of lower extremity injury, or other systemic diagnosis. All patients were evaluated with a standard measurement technique using the same double-long-armed goniometer. Supine abduction, adduction, and hip flexion were measured with care taken to stabilize the pelvis. Internal and external rotation in flexion were assessed with both the hip and knee flexed to 90 degrees. In the prone position, hip extension was recorded as was internal and external rotation in extension. Left and right measurements were averaged to produce a single data point for each index. On the basis of a power analysis (to detect a minimal detectable difference of 6 degrees), 2 separate cohorts of 23 patients were randomly selected for the assessment of intraobserver and interobserver reliability. RESULTS: We measured 504 hips in 252 pediatric patients, including 163 boys and 89 girls. We found a decreasing trend in ROM for almost all indices with advancing age, although this decline was less apparent among girls. Intraobserver reliability demonstrated excellent agreement (intra class correlation coefficient>0.81) for all indices. Interobserver assessments revealed excellent agreement for abduction, external rotation in flexion, internal rotation in extension, and external rotation in extension. Substantial agreement (intra class correlation coefficient, 0.61 to 0.8) was found for adduction, flexion, extension, and internal rotation in flexion. CONCLUSIONS: Normative values for hip ROM in children of varying ages have been established with acceptable intraobserver and interobserver reliability. LEVEL OF EVIDENCE: Level II (Diagnostic).