Myelogenous leukemia in adult inbred MHC-defined miniature swine: a model for human myeloid leukemias.

This manuscript reports on five cases of spontaneous myelogenous leukemia, similar to human disease, occurring within highly inbred, histocompatible sublines of Massachusetts General Hospital (MGH) MHC-defined miniature swine. In cases where a neoplasm was suspected based on clinical observations, samples were obtained for complete blood count, peripheral blood smear, and flow cytometric analysis. Animals confirmed to have neoplasms were euthanized and underwent necropsy. Histological samples were obtained from abnormal tissues and suspect lesions. The phenotype of the malignancies was assessed by flow cytometric analysis of processed peripheral blood mononuclear cells and affected tissues. Five cases of spontaneous myeloid leukemia were identified in adult animals older than 30 months of age. All animals presented with symptoms of weight loss, lethargy, and marked leukocytosis. At autopsy, all animals had systemic disease involvement and presented with severe hepatosplenomegaly. Three of the five myelogenous leukemias have successfully been expanded in vitro. The clustered incidence of disease in this closed herd suggests that genetic factors may be contributing to disease development. Myelogenous leukemia cell lines established from inbred sublines of MGH MHC-defined miniature swine have the potential to be utilized as a model to evaluate therapies of human leukemia.

Establishment of transplantable porcine tumor cell lines derived from MHC-inbred miniature swine.

The lack of transplantable tumors has limited assessment of graft-versus-tumor effects following hematopoietic cell transplantation in clinically relevant large-animal models. We describe the derivation and characterization of porcine tumor cell lines with initial efforts of tumor transplantation using immunocompromised mice and highly inbred sublines of Massachusetts General Hospital major histocompatibility complex (MHC)-inbred miniature swine. Autopsies were performed routinely on swine that died unexpectedly or had suspicion of malignancy based on clinical symptoms or peripheral blood analysis. Tissue samples were obtained for pathology, phenotyped by flow cytometry, and placed in culture. Based on growth, lines were selected for passage into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and miniature swine. Porcine tumor recipients were preconditioned with total body irradiation from 0 to 500 cGy or with a 30-day course of oral cyclosporine. We identified 19 cases of hematologic tumors. Nine distinct tumor cell lines were established from 8 of these cases, including 3 derived from highly inbred sublines. In vivo tumor growth and serial transfer were observed in immunocompromised mice for one tumor cell line and in miniature swine for 1 of 2 tumor cell lines expanded for this purpose. These results suggest the possibility of developing a transplantable tumor model in this large-animal system.

Posttransplant lymphoproliferative disease after allogeneic transplantation of the spleen in miniature swine.

Spleen transplantation (SpTx) was performed in miniature swine across full major histocompatibility complex barriers to study the tolerogenic effect of the spleen. This study describes the development of posttransplant lymphoproliferative disease (PTLD) after allogeneic SpTx. Recipient pigs underwent whole body irradiation (100 cGy), thymic irradiation (700 cGy), and native splenectomy (day 0), and received a 45-day course of intravenous cyclosporine (trough level 400-800 ng/ml). After SpTx, two of seven pigs developed PTLD (1 donor-type, 1 host-type). These two pigs had greater T cell depletion and higher trough levels of cyclosporine. Early changes that occurred prior to the development of clinical features of PTLD were increased porcine lymphotropic herpesvirus-1 viral loads in blood and tissues, and increased numbers of leukocytes, B cells, and total serum IgM. PTLD can occur after allogeneic SpTx in swine. This model may be useful in studies of the pathogenesis of PTLD.

Histomorphometric comparison of cardiac allograft vasculopathy in miniature swine.

BACKGROUND: Whether the pathologic characteristics of vascular lesions manifested in recipients with cardiac allograft vasculopathy (CAV) differ with the severity of the histocompatibility barrier crossed at transplantation or with the type or amount of immunosuppression used to prolong graft survival is unclear. We used miniature swine to determine whether a wide variance in heart transplantation protocols, both in histoincompatibility and immunosuppression, affects the histomorphometry of CAV. METHODS: We compared explanted hearts from major histocompatibility complex Class I-disparate recipients who were treated for 12 days with cyclosporine (Group 1) with minor-antigen-disparate hearts transplanted into mixed chimeric recipients previously engrafted with donor hematopoietic progenitor cells (Group 2). We analyzed coronary intimal lesions using computerized morphometry, immunohistochemistry, and TUNEL assay. Myocardial cytokine-gene expression was determined using RNAse protection assays and reverse-transcriptase polymerase chain reaction. RESULTS: The prevalence of CAV in Group 2 was significantly less than that observed in Group 1, but the severity of the lesions in both groups was similar. The vascular lesions that developed in both groups demonstrated the presence of alpha-smooth-muscle-actin-positive spindle cells expanding the intima, with few inflammatory cells. We noted an absence of proliferating cell nuclear antigen activity and TUNEL-positive cells in both groups. We observed prominent myocardial interferon-gamma gene expression only in Group 1. CONCLUSION: Despite differences in myocardial interferon-gamma gene expression, the histology and severity of the vascular lesions in CAV did not vary significantly with different histoincompatibilities or treatment protocols. These results suggest that the origin of CAV cannot be determined by histology alone.

Modulation of the in vivo primate anti-Gal response through administration of anti-idiotypic antibodies.

Polyclonal anti-idiotypic antibodies (AIA) were generated against human Gal alpha 1,3Gal antibodies (anti-Gal) isolated from a single donor. Specificity of the AIA was demonstrated by selective binding to anti-Gal antibodies (Ab) and absence of reactivity to non-Gal Ab. The idiotopes identified by AIA were present on anti-Gal Ab from all of the human samples evaluated (n=59) as well as on pooled samples, demonstrating that a restricted number of dominant idiotopes characterized the human anti-Gal Ab response. Furthermore, the AIA had cross-species reactivity with baboon serum samples (n=19), suggesting that the overall shape of the anti-Gal Ab combining site is conserved throughout the Old World primates and providing additional evidence of the limited heterogeneity of the anti-Gal Ab repertoire. In order to evaluate the potential effect of AIA in the modulation of the anti-Gal response in vivo, a baboon was injected with repeated doses of the purified AIA. Following AIA treatment, new Ab were generated that reduced Ab-mediated cytotoxicity to porcine cells. Furthermore, administration of the AIA to a baboon prolonged the survival of intravenously infused pig hematopoietic cells when compared with their survival in a control baboon that did not receive prior AIA treatment but underwent a similar conditioning regimen.

New intra-renal graft genes associated with tolerance or rejection.

BACKGROUND: Recipient type mononuclear cells infiltrating kidney allografts have different phenotypes and functions according to the fate of the graft. We hypothesized that different genetic programs were involved in rejected or accepted tissues and thus, transcripts that correlated with the clinical status could be identified by a differential expression strategy. This strategy was applied to miniature swine class II matched, class I disparate kidney grafts, which are accepted in recipient animals treated for 12 days with Cyclosporin A (CsA). METHODS: The mRNA differential display RT-PCR technique (DDRT-PCR) was used to detect clinical status-specific transcripts. cDNA templates for this analysis were derived from biopsies of accepted (CsA treated) and rejected (untreated) kidney grafts 8 days post-transplantation. RESULTS: A first screening procedure identified 23 PCR products differentially amplified in either tolerant or rejector samples. Nucleotide sequence of these partial transcripts showed that 11 out of 23 (48%) sequences had unknown open reading frames while 12 had substantial homology to known sequences. To validate the approach, rejection-associated (RA) cDNA 1 (RA-1) was characterized further. The results indicated that RA-1 is the porcine equivalent of secreted protein acidic and rich in cysteine (SPARC). Expression studies demonstrated that upregulation of SPARC gene transcription preceded other indicators of kidney dysfunction and correlated with the extent of graft infiltration. CONCLUSION: DDRT-PCR appears to be a powerful technique to identify genes differentially expressed in grafted tissues that correlate with tolerance or rejection. One of the gene transcripts identified through this method, SPARC, may be a reliable marker of tissue injury consequent to cellular infiltration and rejection.