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David K C Cooper

Publications and source records attributed to David K C Cooper.

At least 19 recordsLinked to original sources
PubMed2007

Gene expression of porcine lymphotrophic herpesvirus-1 in miniature Swine with posttransplant lymphoproliferative disorder.

Porcine lymphotropic herpesvirus-1 (PLHV-1) is a gamma-herpesvirus related to Epstein-Barr virus (EBV) and associated with development of posttransplant lymphoproliferative disorder (PTLD) following allogeneic stem cell or spleen transplantation in miniature swine. Oligonucleotide microarrays were designed based on known open reading frames (ORFs) of PLHV-1. Expression was compared by cohybridization of cDNA from lymph nodes of PLHV-1+ swine after allogeneic spleen transplantation between either: 1) PTLD-affected and PTLD-unaffected swine; or 2) PTLD-affected swine vs. samples from the same animal prior to diagnosis. In PTLD-affected animals, consistent upregulation (nine ORFs) and downregulation (four ORFs) of PLHV-1 mRNA was observed in comparison to those without PTLD. No differences in gene expression were discovered at the time of clinical PTLD diagnosis compared to six to nine days prior to diagnosis in the same animals. This model provides insights into the pathogenesis of PTLD and, by extension, potential diagnostic and therapeutic tools for human EBV-associated PTLD.

Animals↗
PubMed2007

Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies.

INTRODUCTION: Pig islets have been proposed as an alternative to human islets for clinical use, but their use is limited by rejection. The availability of genetically modified pigs devoid of alpha1,3-galactosyltransferase might provide islets more suitable for xenotransplantation. To limit the costs involved in the logistics and health care of pigs for clinical xenotransplantation, we have studied whether younger, rather than older, pigs that are typically preferred can be used as islet donors. METHODS: We utilized pancreases from Yorkshire and White Landrace wild-type pigs and alpha1,3-galactosyltransferase gene-knockout pigs of three main different age and size groups: (i) <6 months, (ii) 6 to 12 months, and (iii) >2 yr of age, inclusive of retired breeders. We compared isolation yield and in vitro and in vivo function of islet cells obtained from these groups. RESULTS: Islets from adult pigs (>2 yr) offered not only higher islet yields, but retained the ability to preserve intact morphology during the isolation process and culture, in association with high functional properties after transplantation. Following isolation, islet cells from young (<6 m) and young-adult (6 to 12 m) pigs dissociated into small aggregates and single cells, and exhibited inferior functional properties than adult islets both in vitro and in vivo. CONCLUSIONS: These data support the conclusion that, in view of the large number of islets needed to maintain normoglycemia after xenotransplantation, organ-source pigs need to reach adult age (>2 yr) before being considered optimal islet donors, in spite of the higher costs involved.

Aging↗
PubMed2006

Hormonal therapy of the brain-dead organ donor: experimental and clinical studies.

Experiments in Cape Town in the 1980s demonstrated that acute brain death is followed by massive catecholamine release resulting in systemic hypertension, acute left ventricular failure, and multiple cardiac arrhythmias along with substantial decreases in cortisol, insulin, thyroid, and antidiuretic hormone levels, a change from aerobic to anaerobic metabolism, and increases in inflammatory cytokines. Hormonal replacement results in rapid recovery of cardiac function in both experimental animals and humans and enables significantly more organs to be transplanted. Organ Procurement and Transplantation Network/United Network for Organ Sharing multivariate studies on hormonal treatment of brain-dead donors revealed significant increases in organs transplanted and in one-year survival of kidneys and hearts.

Animals↗
PubMed2006

Allosensitization does not increase the risk of xenoreactivity to alpha1,3-galactosyltransferase gene-knockout miniature swine in patients on transplantation waiting lists.

BACKGROUND: The recent availability of alpha1,3-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.

Animals↗
PubMed2006

Elicited antibodies in baboons exposed to tissues from alpha1,3-galactosyltransferase gene-knockout pigs.

BACKGROUND: This study investigates anti-nonGal antibodies (Abs) in baboons after alpha1,3-galactosyltransferase gene-knockout (GalT-KO) pig heart transplantation (Tx). METHODS: Four baboons underwent pig heart Tx under chronic immunosuppression, which was discontinued after graftectomy. During follow-up, one baboon also received a pig splenocyte infusion. Hearts and splenocytes were from GalT-KO pigs (n = 3) or pigs with low Gal expression (Gal-low, n = 2), all of swine leukocyte antigen (SLA) dd haplotype. Several weeks after graftectomy, sera were tested by flow cytometry and cytotoxicity assay on porcine peripheral blood mononuclear cells (PBMC) for elicited anti-nonGal Abs. Sera were adsorbed on a Gal immunoaffinity matrix, and tested for SLA haplotype specificity using PBMC from SLA aa, cc, and dd haplotypes. RESULTS: Before heart Tx, no baboon had anti-nonGal Abs demonstrable by binding or cytotoxicity to GalT-KO PBMC. All four baboons developed anti-nonGal Abs after Tx, demonstrable by flow cytometry, and three sera from baboons showed cytotoxicity to GalT-KO PBMC of SLA(dd) haplotype. After adsorption of anti-Gal Abs, the elicited anti-nonGal Abs showed similar binding to PBMCs from pigs of all three haplotypes (SLA(dd), SLA(aa), SLA(cc)). CONCLUSIONS: Anti-nonGal Abs developed after GalT-KO pig heart Tx into baboons. The most potent of these antibodies appeared to detect antigens shared by the three pig haplotypes tested. It remains unclear whether these antibodies are directed towards shared SLA determinants or other pig antigens, and whether antibodies with specificity for allelic SLA determinants are also present, but at lower titer.

Alleles↗
PubMed2006

Facilitating physiologic self-regeneration: a step beyond islet cell replacement.

Type 1 diabetes (T1D) is an autoimmune disease, the clinical onset of which most frequently presents in children and adolescents who are genetically predisposed. T1D is characterized by specific insulin-producing beta cell destruction. The well-differentiated and specialized islet beta cells seem to physiologically retain the ability to compensate for the cells lost by reproducing themselves, whereas undifferentiated cell sources may help in generating new ones, even while the autoimmune process takes place. Diabetes clinical onset, i.e., establishment of a detectable, chronic hyperglycemia, occurs at a critical stage when autoimmunity, having acted for a while, supersedes the regenerative effort and reduces the number of beta cells below the physiologic threshold at which the produced insulin becomes insufficient for the body's needs. Clinical solutions aimed at avoiding cumbersome daily insulin administrations by the reestablishment of physiologic insulin production, like whole pancreas or pancreatic islet allotransplantation, are limited by the scarcity of pancreas donors and by the toxic effects of the immunosuppressive drugs administered to prevent rejection. However, new accumulating evidence suggests that, once autoimmunity is abrogated, the endocrine pancreas properties may be sufficient to allow the physiological regenerative process to restore endogenous insulin production, even after the disease has become clinically manifest. Knowledge of these properties of the endocrine pancreas suggests the testing of reliable and clinically translatable protocols for obliterating autoimmunity, thus allowing the regeneration of the patient's own endocrine cells. The safe induction of an autoimmunity-free status might become a new promising therapy for T1D.

Animals↗
PubMed2006

Primitive hematopoietic cell populations reside in the spleen: Studies in the pig, baboon, and human.

OBJECTIVE: We previously observed high levels (>40%) of multilineage hematopoietic cell chimerism following spleen transplantation across full MHC barriers in immunosuppressed miniature swine. We therefore investigated the spleen as a source of hematopoietic progenitor cells (HPCs). MATERIALS AND METHODS: Specific cell-surface markers were used to identify HPCs in the spleen and bone marrow (BM) of young adult (n = 15) and fetal (n = 9) miniature swine by flow cytometry. Hoechst dye-effluxing side population (SP) cells were analyzed in adult spleen, BM, and blood for their expression of c-kit. Functional HPC activity of varying repopulation potential in vitro was investigated by the ability of spleens and BM to give rise to colony-forming units (CFUs) and cobblestone area-forming cells (CAFCs) in long-term stromal cultures. Studies were also carried out on baboon and human spleens and BM. RESULTS: Spleen c-kit+ cells co-expressed more lymphoid markers, but equal myeloid markers, when compared with BM c-kit+ cells. BM and spleen both contained significant percentages of c-kit+ SP cells. Although the frequency of early-forming CFUs in the spleen was only 0.1 to 1.3% of that in the BM, the frequency of CAFCs developing after 8 weeks in culture was comparable to that of BM. Secondary CFUs in long-term culture-initiating cell assays confirmed the presence of long-term repopulating cells at comparable frequencies in spleen and BM. Similar findings were found with regard to baboon and human spleen cells. CONCLUSION: The adult spleen is a relatively rich source of very primitive HPCs, possibly hematopoietic stem cells (HSCs), and may be of therapeutic value.

Adult↗
PubMed2006

Induction of diabetes in cynomolgus monkeys with high-dose streptozotocin: adverse effects and early responses.

OBJECTIVES: Streptozotocin (STZ) has been widely used to induce diabetes in nonhuman primates, although it has been found difficult to achieve complete diabetes without serious adverse effects. We have investigated different types and dosages of STZ to find a way to safely induce complete diabetes in cynomolgus monkeys. METHODS: After adequate hydration, 10 monkeys received STZ. Five monkeys received conventional STZ (Sigma) at a dosage of 1250 mg/m ("high dose"; n = 4) or 60 mg/kg ("low dose"; n = 1; Group 1). Five monkeys received Zanosar STZ (Sicor Pharmaceuticals, Irvine, CA) at 150 mg/kg (high dose; n = 5; Group 2). RESULTS: High-dose Group 1 monkeys became completely diabetic (n = 4), but a protein-losing nephropathy was observed in 3 of the 4 monkeys. The monkey that received 60 mg/kg STZ failed to become fully diabetic (C-peptide, > 1.86 ng/mL). Group 2 (high-dose Zanosar-treated) monkeys became completely diabetic but with no apparent adverse effects. A triphasic blood glucose response to STZ was documented in all the high-dose STZ-treated monkeys. Low-dose STZ failed to result in a triphasic response. CONCLUSIONS: (1) High-dose Zanosar STZ induced diabetes safely in cynomolgus monkeys without adverse effects. (2) A triphasic blood glucose response suggested the complete induction of diabetes.

Animals↗
PubMed2006

Buccal mucosal cell immunohistochemistry: a simple method of determining the ABH phenotype of baboons, monkeys, and pigs.

BACKGROUND: Baboons and monkeys fail to express ABH antigens on red blood cells (RBCs), and the A or H antigens are expressed only weakly on the surface of pig RBCs. Baboons and monkeys have been previously blood typed by detection of ABH antigens in the saliva after administration of pilocarpine. A reliable method to ABH type pigs is by immunohistochemical staining of renal distal tubules in kidney biopsies. We describe a simple and efficient method to blood type baboons, monkeys, and pigs. METHODS: Baboons (n = 14) and cynomolgus monkeys (n = 8) were blood typed by staining of buccal mucosal smears and by determining the presence of serum anti-A or B antibodies following human type O adsorption. Pigs (n = 11) were tested for ABH type by immunohistochemistry for the presence of A, B, and H antigens using monoclonal antibodies on (i) renal biopsies, (ii) RBCs, and (iii) buccal mucosal smears, without pilocarpine administration, in addition, (iv) after adsorption on human type O RBCs to remove anti-human antibodies, the pig sera were typed by hemagglutination assay for the presence of anti-A or B antibodies using human A and B RBCs. RESULTS AND CONCLUSIONS: There was complete consistency among the results obtained using all of the above methods, except that no determination could be made from staining of RBCs in one pig. Staining of buccal mucosal cells proved to be the preferred method in all three species because: (i) expression of A or H antigen is weak on pig RBCs, making an accurate blood type determination difficult, and A, B, and H expression is non-existent on baboon and monkey RBCs, (ii) neither venepuncture nor organ biopsy is necessary, (iii) time-consuming adsorption of anti-human antibodies from the sera of the test animal is not required, and (iv) it proved a quick method of evaluation.

ABO Blood-Group System↗
PubMed2006

Allosensitized humans are at no greater risk of humoral rejection of GT-KO pig organs than other humans.

BACKGROUND: The availability of pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GT-KO) has enabled study of the incidence and cytotoxicity of primate antibodies directed to antigens other than Galalpha1,3Gal (Gal), termed non-Gal antigens. METHODS: Sera from 27 healthy humans and 31 patients awaiting renal allotransplantation, who were either unsensitized [panel reactive antibodies (PRA) < 10%] or allosensitized (PRA > 70%), were tested by flow cytometry for binding of immunoglobulin M (IgM) and IgG to peripheral blood mononuclear cells (PBMC) from both wild-type (WT) and GT-KO pigs. Complement-dependent cytotoxicity to WT and GT-KO PBMC was also measured. RESULTS: IgM and IgG from all 27 (100%) healthy human sera bound to WT PBMC, while 78% and 63% of these sera had IgM and IgG that bound to GT-KO PBMC, respectively. Mean binding to WT PBMC was significantly greater than GT-KO PBMC. Whereas 100% of sera were cytotoxic to WT PBMC, only 61% were cytotoxic to GT-KO PBMC, and the extent of lysis was significantly less. Neither mean binding of IgM and IgG nor cytotoxicity of unsensitized and allosensitized sera to WT and GT-KO PBMC was significantly different to that of healthy sera. CONCLUSIONS: More than half of the healthy humans tested had cytotoxic antibodies to GT-KO PBMC, but allosensitized patients will be at no greater risk of rejecting a pig xenograft by a humoral mechanism.

Animals↗
PubMed2006

Antibodies directed to pig non-Gal antigens in naïve and sensitized baboons.

BACKGROUND: As pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GT-KO) are available, primate antibodies to pig non-Gal antigens can be studied. METHODS: Sera from 56 baboons were tested for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from both wild-type (WT) and GT-KO pigs by flow cytometry. Complement-dependent cytotoxicity was measured in 39 sera. Antibody and cytotoxicity responses were measured in two baboons exposed to a GT-KO pig heart, one not immunosuppressed and one that received only cobra venom factor. RESULTS: IgM and IgG bound to 95% and 79% of WT PBMC, and 32% and 9% GT-KO PBMC, respectively (WT vs. GT-KO, P<0.01). Whereas 97% of sera were cytotoxic to WT PBMC, only 64% were cytotoxic to GT-KO PBMC, and the level of cytotoxicity was less (mean 60% vs. 25% lysis, P<0.05). In the two baboons exposed to GT-KO hearts, anti-non-Gal antibodies increased markedly, peaking after 2 (IgM) and 3 (IgG) weeks, associated with an increase in lysis of GT-KO PBMC. CONCLUSIONS: Two-thirds of baboon sera demonstrated cytotoxicity to GT-KO PBMC. After GT-KO organ transplantation, if an elicited antibody response develops, it is likely to cause rapid graft rejection.

Animals↗
PubMed2006

ABO-incompatible allotransplantation as a basis for clinical xenotransplantation.

The 8th Congress of the International Xenotransplantation Association (IXA), held in Goteborg in September, 2005, immediately followed the 2nd International Symposium on ABO-Incompatibility in Transplantation, both congresses organized by Michael Breimer and Lennart Rydberg. The Proceedings of the Symposium on ABO-Incompatibility in Transplantation have been published (Xenotransplantation 2006; 13 (2)). The present paper provides an overview of a workshop held at the 8th Congress of the IXA, and highlights the immunological concepts emerging from ABO-incompatible allotransplants and discusses them in relation to xenotransplantation. Using specified immunomodulatory protocols, ABO-incompatible solid organ allotransplantation has become a clinical reality for a small number of patients over the last two decades. ABO-incompatible adult kidney and infant heart transplants have similar patient and graft survivals as their ABO-compatible counterparts. In contrast, ABO-incompatibility is present in up to 30% of all patients receiving hematopoietic stem cell transplants in the absence of specific immunomodulation, without affecting overall survival. Consequently, ABO-incompatible solid organ transplants and hematopoietic stem cell transplants may serve as in vivo models to elucidate the immunological mechanisms of accommodation and/or tolerance in the clinical setting. Because of similarities in the immunological hurdles that need to be overcome, knowledge obtained from ABO-incompatible allotransplantation might further promote advances in the field of xenotransplantation. The similarities and differences between ABO-incompatible allotransplantation and xenotransplantation are discussed.

ABO Blood-Group System↗
PubMed2006

Extended coagulation profiles of healthy baboons and of baboons rejecting GT-KO pig heart grafts.

INTRODUCTION: Derangements of coagulation, e.g. thrombotic microangiopathy (TM) and disseminated intravascular coagulation (DIC), limit the success of pig-to-baboon organ transplantation. Studies have investigated the coagulation profile in baboons post-xenotransplantation (XTx), but an extended coagulation profile of healthy baboons pre-XTx has not been reported. METHODS: Blood was drawn from nine healthy male baboons (approximate age 5 yr, mean weight 15 kg) that had not undergone any prior surgical or therapeutic procedures. An extended coagulation profile, consisting of markers of thrombin activation, fibrinolysis, endothelial activation, the protein C pathway, and overall reactive state, was investigated by a reference coagulation laboratory, using tests for human plasma. The mean value +/- SD was calculated for 18 parameters (analytes); values outside of the mean +/- 2 SD were excluded. Three baboons subsequently underwent transplantation with hearts from GT-KO pigs, and received either no therapy (B24603), CVF (B25003), or CVF + leflunomide (B24903), and their extended coagulation parameters were followed. RESULTS: For 14 of the 18 analytes, the human reference range reflected the coagulation status of healthy baboons. Exceptions included thrombin/antithrombin complex and fibrinopeptide A, which were elevated compared with the human reference range, while plasminogen activity was lower. The human assay failed to detect baboon plasminogen activator inhibitor-1. Immediately after GT-KO pig heart transplantation, the untreated B24603 demonstrated a coagulation profile consistent with its postoperative clinical status; DIC was not apparent, and the heart was electively excised within 2.5 h. In B25003 and B24903, that rejected their grafts on days 8 and 12, respectively, the coagulation profile showed evidence of DIC, particularly in B24903, which was clinically coagulopathic by this time. CONCLUSIONS: (i) The human reference range of extended coagulation parameters forms a basis for studies in baboons, with a few exceptions. (ii) Antibody-mediated rejection of GT-KO pig hearts in baboons can be associated with laboratory and clinical evidence of DIC.

Animals↗
PubMed2006

Measurement of anti-CD154 monoclonal antibody in primate sera by competitive inhibition ELISA.

BACKGROUND: Anti-human CD154 monoclonal antibody (mAb)-based regimens have been demonstrated to prevent T cell-dependent elicited antibody response in baboon recipients of pig hematopoietic progenitor cells, organs and islets. Monitoring of anti-CD154 mAb in serum is important to ensure maintenance of adequate levels and for adjusting dosage of the anti-CD154 mAb. We describe a method for measuring the level in primate sera. METHODS: The anti-CD154 mAb level in primate serum was measured with a competitive inhibition enzyme linked immunosorbent assay in which the extent of inhibition of binding by anti-CD154 mAb conjugated to horseradish peroxidase (anti-CD154-HRP) to soluble CD154 was used to determine the serum level. Briefly, a 96-well maxisorb plate coated with soluble human CD154, and blocked with bovine serum albumin, was loaded with graded doses of anti-CD154 mAb or primate sera containing anti-CD154 mAb. Both were mixed with a known dosage of anti-CD154-HRP before loading. Bound anti-CD154-HRP was detected by color developed using 3,3',5,5' tetramethyl-benzidine as substrate. Absorbance was measured in a Synergy HT Multi-Detection Microplate Reader at a wavelength of 450 nm. Data analysis was carried out using BioTek's KC4 Data Analysis Software. The standard curve was generated from the wells loaded with the mixture of anti-CD154 mAb and anti-CD154-HRP. RESULTS AND CONCLUSIONS: The assay has been used successfully to measure anti-CD154 mAb levels in the serum of both baboons and monkeys.

Animals↗