Yau-Lin Tseng

Heart transplantation in baboons using α1,3-galactosyltransferase gene-knockout pigs as donors: Initial experience

Hearts from α1,3-galactosyltransferase knockout pigs (GalT-KO, n = 8) were transplanted heterotopically into baboons using an anti-CD154 monoclonal antibody–based regimen. The elimination of the galactose-α1,3-galactose epitope prevented hyperacute rejection and extended survival of pig hearts in baboons for 2–6 months (median, 78 d); the predominant lesion associated with graft failure was a thrombotic microangiopathy, with resulting ischemic injury. There were no infectious complications directly related to the immunosuppressive regimen. The transplantation of hearts from GalT-KO pigs increased graft survival over previous studies.

α1,3-galactosyltransferase gene-knockout pig heart transplantation in baboons with survival approaching 6 months

Background. The recent generation of α1,3-galactosyltransferase gene-knockout (GalT-KO) pigs has allowed investigation of the survival of GalT-KO pig organs in nonhuman primates. Methods. Heterotopic heart transplantation from GalT-KO pigs was carried out in baboons (n=8) using a human antihuman CD154 monoclonal antibody-based immunosuppressive regimen. Results. In six of the eight cases, graft survival extended to between approximately 2 and 6 months. All grafts developed thrombotic microangiopathy (TM). In particular, the clinical course of one baboon in which the graft functioned for 179 days is summarized. This baboon received aspirin (40 mg on alternate days) from day 4 in addition to heparin, which may have been a factor in the delay of onset and progression of TM and in prolonged graft survival. Maintenance therapy with anti-CD154 mAb, mycophenolate mofetil, and methylprednisolone was associated with persistently low numbers of CD3+CD4+ and CD3+CD8+ cells. Despite persisting depletion of these cells, no infectious complications occurred. Conclusions. It remains to be established whether TM is related to a very low level of natural preformed or T-cell-induced antibody deposition on the graft, inducing endothelial activation and injury, or to molecular incompatibilities in the coagulation mechanisms between pig and baboon, or to both. However, function of a pig organ in a baboon for a period approaching six months, which has not been reported previously, lends encouragement that the barriers to xenotransplantation will eventually be overcome.

Suppression of Natural and Elicited Antibodies in Pig-to-Baboon Heart Transplantation Using a Human Anti-Human CD154 mAb-Based Regimen

Natural and elicited antipig antibodies (Abs) lead to acute humoral xenograft rejection (AHXR). Ten baboons underwent heterotopic heart transplantation (Tx) from human decay‐accelerating factor (hDAF) pigs. Depletion of anti‐Galα1, 3Gal (Gal) Abs was achieved by the infusion of a Gal glycoconjugate from day – 1. Immunosuppression included induction of antithymocyte globulin, thymic irradiation, and cobra venom factor, and maintenance with a human antihuman CD154 mAb, mycophenolate mofetil, and methylprednisolone; heparin and prophylactic ganciclovir were also administered. Pig heart survival ranged from 4 to 139 (mean 37, median 27) days, with three functioning for >50 days. Graft failure (n = 8) was from classical AHXR [ 4], thrombotic microangiopathy [ 3], or intragraft thrombosis [ 1], with death (n = 2) from pneumonia [ 1], or possible drug toxicity (with features of thrombotic microangiopathy) [ 1]. Anti‐Gal Abs (in μg/mL) were depleted by Gal glycoconjugate before graft implantation from means of 41.3 to 6.3 (IgM) and 12.4–4.6 (IgG), respectively, and at graft excision were 6.3 and 1.7 μg/mL, respectively. No elicited Abs developed, and no cellular infiltration was seen. The treatment regimen was effective in maintaining low anti‐Gal Ab levels and in delaying or preventing AHXR. The combination of costimulatory blockade and heparin with Tx of a Gal‐negative pig organ may prolong graft survival further.

Thrombotic microangiopathy associated with humoral rejection of cardiac xenografts from α1,3-galactosyltransferase gene-knockout pigs in baboons

Heterotopic cardiac xenotransplantation from alpha1,3-galactosyltransferase gene-knockout (GalT-KO) swine to baboons was performed to characterize immunological reaction to the xenograft in the absence of anti-Gal antibody-mediated rejection. Eight baboons received heterotopic cardiac xenografts from GalT-KO porcine donors. All baboons were treated with chronic immunosuppressive therapy. Both histological and immunohistochemical studies were performed on biopsy and graftectomy samples. No hyperacute rejection was observed. Three baboons were euthanized or died 16 to 56 days after transplantation. The other five grafts ceased beating between days 59 and 179 (median, 78 days). All failing grafts exhibited thrombotic microangiopathy (TM) with platelet-rich fibrin thrombi in the microvasculature, myocardial ischemia and necrosis, and focal interstitial hemorrhage. TM developed in parallel with increases in immunoglobulin (IgM and IgG) and complement (C3, C4d, and C5b-9) deposition, as well as with subsequent increases in both TUNEL(+) endothelial cell death and procoagulant activation (increased expression of both tissue factor and von Willebrand factor and decreased expression of CD39). CD3(+) T-cell infiltration occurred in all grafts and weakly correlated with the development of TM. In conclusion, although the use of GalT-KO swine donors prevented hyperacute rejection and prolonged graft survival, slowly progressive humoral rejection--probably associated with non-Gal antibodies to the xenograft--and disordered thromboregulation represent major immunological barriers to long-term xenograft survival.

Thymic carcinoma: Involvement of great vessels indicates poor prognosis

BACKGROUND Thymic carcinoma is a rare, indolent, and invasive cancer. This study investigated the treatment results of thymic carcinoma and clinical prognostic factors. METHODS From June 1988 to January 2002, 38 patients were enrolled in this study with the diagnosis of thymic carcinoma in the Cheng-Kung University Hospital based on Rosais and Muller-Hermelinks classification. Clinical and pathologic data were retrospectively reviewed. Survival analysis was performed using the Kaplan-Meier, log rank, and Wilcoxon tests. Statistical significance was defined as p < 0.05. RESULTS Pathology revealed 14 poorly differentiated, 6 moderately differentiated, and 8 well-differentiated squamous cell carcinomas; 8 lymphoepithelioma-like carcinomas; and 2 other carcinomas. Pathologic staging using the Masaoka system included 6 stage II, 23 stage III, and 9 stage IV patients. Six biopsies, five debulkings, and 27 complete resections were performed. All patients were followed from 15 months to 10 years 9 months, with an average of 53.8 months. Median survival time was 81 months, and median recurrence time was 52 months. Eighteen patients are still alive, and 7 are alive with disease. Well-differentiated squamous cell carcinoma had better prognosis than other carcinomas (p = 0.022). Complete resection significantly increased survival rate (p < 0.001). Tumor invasion of the superior vena cava, pulmonary vessels, or aorta were significant predictors for poor prognosis (p = 0.016, 0.002, and 0.002, respectively). CONCLUSIONS Only patients with thymic carcinoma who underwent complete resection had long-term survival. Prognosis of thymic carcinoma seemed mainly dependent on tumor invasion of the great vessels.

Rejection of Cardiac Xenografts Transplanted from α1,3-Galactosyltransferase Gene-Knockout (GalT-KO) Pigs to Baboons

The use of α1,3‐galactosyltransferase gene‐knockout (GalT‐KO) swine donors in discordant xenotransplantation has extended the survival of cardiac xenografts in baboons following transplantation. Eight baboons received heterotopic cardiac xenografts from GalT‐KO swine and were treated with a chronic immunosuppressive regimen. The pathologic features of acute humoral xenograft rejection (AHXR), acute cellular xenograft rejection (ACXR) and chronic rejection were assessed in the grafts. No hyperacute rejection developed and one graft survived up to 6 months after transplantation. However, all GalT‐KO heart grafts underwent graft failure with AHXR, ACXR and/or chronic rejection. AHXR was characterized by interstitial hemorrhage and multiple thrombi in vessels of various sizes. ACXR was characterized by TUNEL+ graft cell injury with the infiltration of T cells (including CD3 and TIA‐1+ cytotoxic T cells), CD4+ cells, CD8+ cells, macrophages and a small number of B and NK cells. Chronic xenograft vasculopathy, a manifestation of chronic rejection, was characterized by arterial intimal thickening with TUNEL+ dead cells, antibody and complement deposition, and/or cytotoxic T‐cell infiltration. In conclusion, despite the absence of the Gal epitope, acute and chronic antibody and cell‐mediated rejection developed in grafts, maintained by chronic immunosupression, presumably due to de novo responses to non‐Gal antigens.

Allosensitization does not increase the risk of xenoreactivity to α1,3-galactosyltransferase gene-knockout miniature swine in patients on transplantation waiting lists

Background. The recent availability of α1,3-galactosyltransferase knockout (GalT-KO) miniature swine has eliminated anti-Gal antibodies as the major barrier to xenotransplantation, potentially bringing this modality closer to clinical application. Highly-allosensitized patients, who have poor prospects of receiving a suitable cross-match negative human organ, might be the first patients to benefit from xenotransplantation of porcine organs. However, concerns exist regarding cross-reactivity of alloreactive anti-human leukocyte antigen (HLA) antibodies against xenogeneic swine leukocyte antigen (SLA) antigens. We have investigated this question using sera from such patients on GalT-KO target cells. Methods. Using flow cytometry and complement-dependent cytotoxicity (CDC) assays, we have tested a panel of 88 human serum samples from patients awaiting cadaveric renal allotransplantation for reactivity against: 1) human; 2) standard miniature swine; and 3) GalT-KO peripheral blood lymphocytes (PBL) and cultured endothelial cells. Results. Anti-swine IgM and IgG antibody binding, as well as CDC, were significantly attenuated on GalT-KO versus standard swine. No correlation was found between the degree of anti-human panel reactive antibodies (PRA) and xenoreactivity against either standard or GalT-KO miniature swine. Treatment of sera with dithiothreitol (DTT) showed that the majority of remaining lymphocytotoxicity against GalT-KO swine was mediated by preformed IgM antibodies. Patients with high alloreactivity but low anti-GalT-KO xenoreactivity were readily identified. Conclusions. Highly allosensitized patients awaiting renal transplants appear to be at no increased risk of xenosensitization over their non-sensitized cohorts, and could therefore be candidates for xenotransplantation using GalT-KO swine donors.

Alloantibody and xenoantibody cross-reactivity in transplantation

The recent availability of pigs homozygous for alpha1,3-galactosyltransferase gene knockout, and improved immunosuppressive regimens that prevent an elicited antibody response, are expected to contribute to significantly increased survival of pig organs transplanted into primates, bringing clinical trials of xenotransplantation closer. Patients highly sensitized to human leukocyte antigens, who may be precluded from obtaining a human donor organ, would be one group that might benefit from xenotransplantation. However, there have been few studies on whether there is cross-reactivity of anti-human leukocyte antigen antibodies with pig antigens. What data there are suggest that such cross-reactivity exists and that this may be detrimental to the outcome after transplantation of a pig organ. Neither is it known whether sensitization after a pig xenograft would preclude subsequent allotransplantation, although the data available suggest that this will not be the case. Further investigation on allo- and xenoantibody cross-reactivity is required.

Bone marrow transplantation from α1,3-galactosyltransferase gene-knockout pigs in baboons

Abstract:  Background:  Successful hematopoietic cell allotransplantation results in donor‐specific tolerance, but this approach has been unsuccessful in the wild‐type pig‐to‐baboon xenotransplantation model, as pig cells were lost from the circulation within 5 days. However, after cessation of immunosuppressive therapy on day 28, all baboons demonstrated non‐specific unresponsiveness on mixed leukocyte reaction (MLR) for at least 30 days. We have now investigated the transplantation of bone marrow (BM) cells from miniature swine homozygous for α1,3‐galactosyltransferase gene‐knockout (GalT‐KO).

Immunohistochemical analysis of epidermal growth factor receptor family members in stage I non-small cell lung cancer

BACKGROUND To elucidate the relationship between the expression of epidermal growth factor receptor family members (ErbB-1, neu/ErbB-2, ErbB-3, and ErbB-4) and tumor recurrence. METHODS We used immunohistochemistry to examine the expression of four epidermal growth factor receptor family members in 73 patients with stage I non-small cell lung cancer. RESULTS Using Cox univariate analysis, we determined that angiolymphatic tumor emboli and non-well-differentiated tumor cells were two significant conventional pathologic predictors of tumor recurrence, and that ErbB-1 and ErbB-3 were also significant predictors. Co-expression of ErbB-1+, -3+, or expression of three or more epidermal growth factor receptor family members had a significant effect on lung cancer recurrence. A stepwise multivariate Cox proportional hazards regression analysis provided a predictive model for tumor recurrence. CONCLUSIONS The present study shows that in patients with a non-well-differentiated tumor, overexpression of ErbB-3 is a useful marker for predicting tumor recurrence. The present study also confirmed that ErbB-1 expression increased in proportion to the loss of tumor differentiation. The correlation between ErbB-3 and distant metastasis was good.