New approaches to replacing failing organs.

The foremost cause of death and disability is organ failure. Failure of the heart, lungs, kidney, and liver is typically addressed by organ transplantation; however, the number of human organs available for this purpose is quite limited. For this reason, some new technologies are being advanced for the treatment of organ failure. This communication summarizes potential limitations of these technologies.

The possibility of B-cell-dependent T-cell development.

The development of T cells is thought to be independent of B cells. However, defects in cell-mediated immunity in individuals with B-cell deficiency suggest the contrary. To test whether B cells affect T-lymphocyte development, we constructed mice with a monoclonal T-cell compartment (MT) and monoclonal B- and T-cell compartments (MBTs). In these mice, the T cells expressed a DO 11.10 transgenic (DO-T) cell receptor restricted to major histocompatibility complex (MHC) class IId. While CD4+ DO-T lymphocytes are rare in transgenic H-2b MT mice, we found that in H-2b MBT mice under the influence of B cells, DO-T lymphocytes mature into large numbers of CD4+ peripheral T cells. H-2b MBT mice have more CD4+ thymocytes than H-2b MT mice. These data are consistent with the view that B cells play some role in thymocyte development.

Human xenospecific T suppressor cells inhibit T helper cell proliferation to porcine aortic endothelial cells, and NF-kappaB activity in porcine APC.

Human T suppressor cells (Ts), capable of preventing autologous T helper cells (Th) from reacting against xenogeneic pig endothelial cells and pig APC can be generated in vitro. Ts derive from a population of CD3(+)CD8(+)CD28(-) T lymphocytes and specifically recognize the MHC class I antigens of the APC used for in vitro immunization. To study the mechanism that underlies suppression, we investigated whether Ts inhibit the expression of costimulatory molecules in xenogeneic professional and semiprofessional APC. We found that Ts down-regulate Th-induced expression of CD86 in pig APC, and that this effect occurs at the level of transcription, as indicated by nuclear run-on and Northern blot assays. EMSA results revealed that inhibition of CD86 expression is mediated by inactivation of transcription factor NF-kappaB. Furthermore, transfection of pig APC with a vector expressing NF-kappaB p65 partially rescued Th-induced expression of the CD86 molecule. These results strongly support the concept that xenospecific Ts inhibit the APC function of xenogeneic cells by preventing activation of NF-kappaB.

The pathology of cardiac xenografts.

The pathology of cardiac xenografts has yielded critical insights into the mechanisms of xenograft rejection and the therapeutic procedures that might be applied to preventing or treating it. The conditions seen in rejecting cardiac xenografts include hyperacute rejection, acute vascular rejection, and cellular rejection. Hyperacute and acute vascular rejection of cardiac xenografts have features typical of humoral injury. Less is known about cellular rejection and only speculation can be offered about chronic rejection. Still, these features allow critical testing of pathogenetic mechanisms and therapies.

The immunological hurdles to cardiac xenotransplantation.

The main hurdle to clinical application of cardiac xenotransplantation is the immune response of the recipient against the graft. Although all xenografts arouse an intense immune response, the effect of that response depends very much on whether the graft consists of isolated cells or an intact organ, such as the heart. Intact organs, which are transplanted by primary vascular anastomosis, are subject to severe vascular injury owing to the reaction of immune elements with the endothelial lining of donor blood vessels. Vascular injury leads to hyperacute rejection, acute vascular rejection, and chronic rejection. The immunological basis for these types of rejection and potential therapies, which might be used to avert them, are discussed.

Phenotypic and functional maturation of dendritic cells mediated by heparan sulfate.

Primary immune responses are thought to be induced by dendritic cells. To promote such responses, dendritic cells must be activated by exogenous agonists, such as LPS, or by products of activated leukocytes, such as TNF-alpha and IL-1. How dendritic cells might be activated in the absence of exogenous stimuli, or without the immediate presence of activated leukocytes, as might occur in immunity to tumor cells or transplants, is unknown. We postulated that heparan sulfate, an acidic, biologically active polysaccharide associated with cell membranes and extracellular matrices, which is rapidly released under conditions of inflammation and tissue damage, might provide such a stimulus. Incubation of immature murine dendritic cells with heparan sulfate induced phenotypic maturation evidenced by up-regulation of I-A, CD40, CD54 (ICAM-1), CD80 (B7-1), and CD86 (B7-2). Dendritic cells exposed to heparan sulfate exhibited a markedly lowered rate of Ag uptake and increased allostimulatory capacity. Stimulation of dendritic cells with heparan sulfate induced release of TNF-alpha, IL-1beta, and IL-6, although the maturation of dendritic cells was independent of these cytokines. These results suggest that soluble heparan sulfate chains, as products of the degradation of heparan sulfate proteoglycan, might induce maturation of dendritic cells without exogenous stimuli, thus contributing to the generation and maintenance of primary immune responses.

The role of antibodies in dysfunction of pig-to-baboon pulmonary transplants.

OBJECTIVE: Pulmonary transplantation has become the preferred treatment for end-stage lung disease, but application of the procedure is limited because of a paucity of donors. One way to solve donor limitations is to use animal organs as a donor source or xenotransplantation. The current barrier to pulmonary xenotransplantation is the rapid failure of the pulmonary xenograft. Although antibodies are known to play a role in heart and kidney xenograft rejection, their involvement in lung dysfunction is less defined. This project was designed to define the role of antibodies in pulmonary graft rejection in a pig-to-baboon model. METHODS: Orthotopic transgenic swine left lung transplants were performed in baboons depleted of antibodies by one of three techniques before transplantation: (1) ex vivo swine kidney perfusion, (2) total immunoglobulin-depleting column perfusion, and (3) ex vivo swine lung perfusion. Results were compared with those of transgenic swine lung transplants in unmodified baboons. RESULTS: All three techniques of antibody removal resulted in depletion of xenoreactive antibodies. Only pretransplantation lung perfusion improved pulmonary xenograft function compared with lung transplantation in unmodified baboons. CONCLUSIONS: The pathogenesis of pulmonary injury in a swine-to-primate transplant model is different from that in renal and cardiac xenografts. Depletion of antibodies alone does not have a beneficial effect and may actually be detrimental.

Altered intragraft immune responses and improved renal function in MHC class II-deficient mouse kidney allografts.

BACKGROUND: During renal allograft rejection, expression of MHC class II antigens is up-regulated on the parenchymal cells of the kidney. This up-regulation of MHC class II proteins may stimulate the intragraft alloimmune response by promoting their recognition by recipient CD4+ T cells. In previous studies, absence of donor MHC class II antigens did not affect skin graft survival, but resulted in prolonged survival of cardiac allografts. METHODS: To further explore the role of MHC class II antigens in kidney graft rejection, we performed vascularized kidney transplants using donor kidneys from A(beta)b-deficient mice that lack MHC class II expression. RESULTS: At 4 weeks after transplant, GFR was substantially depressed in control allografts (2.18+/-0.46 ml/min/kg) compared to nonrejecting isografts (7.98+/-1.62 ml/min/kg; P<0.01), but significantly higher in class II- allografts (4.38+/-0.60 ml/min/kg; P<0.05). Despite the improvement in renal function, class II- allograft demonstrated histologic features of acute rejection, not unlike control allografts. However, morphometric analysis at 1 week after transplantation demonstrated significantly fewer CD4+ T cells infiltrating class II- allografts (12.8+/-1.2 cells/mm2) compared to controls (25.5+/-2.6 cells/mm2; P=0.0007). Finally, the intragraft profile of cytokines was altered in class II- allografts, with significantly reduced expression of Th2 cytokine mRNA compared to controls. CONCLUSIONS: These results support a role of MHC class II antigens in the kidney regulating immune cells within the graft. Further, effector pathways triggered by class II antigens promote renal injury during rejection.

Modulation of macrophage and B cell function by glycosaminoglycans.

There is increasing evidence that the behavior of antigen-presenting cells may be regulated, in part, by the surrounding microenvironment. Components of the microenvironment of solid tissues that might influence antigen-presenting cell functions include glycosaminoglycans. We previously showed that heparan sulfate glycosaminoglycans activate macrophages, leading to profound alterations in T cell responses. Here we demonstrate the functional changes that occur in murine antigen-presenting cells induced by heparan sulfate and other glycosaminoglycans, and postulate how these functional changes influence the nature of local immune responses. Heparan sulfate triggered up-regulation of ICAM-1 and I-A, caused the release by antigen-presenting cells of interleukin (IL)-1, IL-6, tumor necrosis factor, IL-12, transforming growth factor beta, and prostaglandin E2 (PGE2), and (in macrophages) induced cytotoxic capability. Heparin induced IL-12 and interferon-gamma production but did not promote the release of other cytokines. Chondroitin sulfate and dermatan sulfate, although not stimulating the production of cytokines or of PGE2, elicited the production by macrophages of nitric oxide. These findings support a model in which the glycosaminoglycan composition of a given tissue, which may be altered by inflammatory processes, helps to regulate the behavior of antigen-presenting cells, which in turn determines the characteristics of the immune response that ensues.

Screening by tRNA primer extension analysis of porcine kidney mRNA libraries defines a novel endogenous porcine retroviral long terminal repeat.

BACKGROUND: We have taken advantage of the common requirement of all eukaryotic retroelements for a specific tRNA primer to initiate DNA synthesis and applied a previously described in vitro screening methodology to the analysis of in vivo porcine tissues for transcriptionally active retroviral sequences. METHODS: A series of 18-base pair (bp) 3' tRNA oligomers complementary to established primer binding sites for a variety of vertebrate retroviruses, retrotransposons, and retroposons were applied to primer extension analysis of kidney poly(A) mRNA. Primer extension products are predicted to represent "strong stop" signals characteristic of the initial stages of retroviral transcription. RESULTS: Several extension products were cloned, sequenced, and analyzed as probes for screening the porcine genome for potentially active retroviral sequences. We used this strategy to identify and clone a 655-bp 5' long terminal repeat of a porcine retrovirus with significant homology to the simian sarcoma virus. This transcriptionally active virus has an 82-bp U5 region, a conserved AATAAA polyadenylation sequence, a 39-bp repeat reminiscent of other retroviral enhancers, and a unique glycine primer binding site. CONCLUSION: Our results suggest that tRNA primer cloning can effectively identify novel retroviral elements.

Human complement regulatory proteins protect swine lungs from xenogeneic injury.

BACKGROUND: Pulmonary xenotransplantation is not possible because of hyperacute lung injury, the pathogenesis of which is unknown. This study evaluates complement-dependent pathways of pulmonary injury during heterologous perfusion of swine lungs. METHODS: Lungs from unmodified swine and swine expressing human decay-accelerating factor and human CD59 (hDAF/hCD59 swine) were perfused with either human plasma or baboon blood. Pulmonary vascular resistance and static pulmonary compliance were measured serially, and swine lung tissue were examined by light microscopy. Complement activation was assessed by serial measurements of baboon plasma C3a-desArg concentrations. RESULTS: Perfusion of unmodified swine lungs with human plasma and baboon blood resulted in hyperacute lung injury within minutes of perfusion. However, function was preserved in swine lungs expressing human decay-accelerating factor and human CD59. In both study groups, xenogeneic perfusion with baboon blood resulted in at least a sevenfold increase in plasma C3a-desArg levels suggesting transient activation of complement. CONCLUSIONS: Lungs from swine expressing human decay-accelerating factor and human CD59 were resistant to injury during perfusion with human plasma and baboon blood, indicating that complement mediated some of the features of xenogeneic acute lung injury.

Partial sequence of human platelet heparitinase and evidence of its ability to polymerize.

Heparitinase cleaves heparan sulfate, a glycosaminoglycan associated with all nucleated mammalian cells and extracellular matrices. Despite the important physiologic role heparitinase is postulated to play in such processes as tumor metastasis and inflammation, the identity of the enzyme remains a matter of controversy and there is a question of whether heparitinase is CTAP III. We report a 900,000-fold purification of heparitinase from human platelets. A multi-step procedure utilizing chromatography on heparin, DEAE, hydroxyapatite and size exclusion matrices was employed and yielded a single protein as judged by Coomassie staining of protein separated by SDS-PAGE. The purified protein had an apparent molecular mass of 35 kDa by size exclusion chromatography and 55 kDa by SDS-PAGE. During purification, heparitinase activity co-eluted from the hydroxyapatite and size exclusion columns with the 35-55 kDa protein, confirming that the purified protein was indeed heparitinase. The 35-55 kDa protein reacted strongly with concanavalin A, a lectin known to bind to heparitinase, further confirming that the protein was heparitinase. Platelet heparitinase formed dimers and tetramers upon storage in a purified form, possibly accounting for the various molecular weights previously reported for the enzyme. A partial amino acid sequence of the protein revealed that heparitinase has not been previously sequenced.

The role of adhesion molecules in human leukocyte attachment to porcine vascular endothelium: implications for xenotransplantation.

Many obstacles still prevent successful xenotransplantation of porcine donor organs. When hyperacute rejection is averted, transplanted pig organs are subject to acute vascular and cellular rejection. In autologous systems, leukocyte recruitment into inflamed tissues involves selectins, integrins, and Ig family members. To determine whether these mechanisms allow human leukocytes to effectively enter porcine grafts, the pathways by which human leukocytes adhere to TNF-alpha-stimulated porcine aortic endothelium were examined under static and physiologic flow conditions. L-selectin and E-selectin had overlapping functions in neutrophil capture and rolling, whereas Ab blockade of E-selectin and the beta2 integrins inhibited firm arrest of rolling neutrophils. Combined blockade of selectins and beta2 integrins resulted in negligible human neutrophil attachment to pig endothelium. Lymphocyte attachment to porcine endothelium was primarily L-selectin mediated, whereas beta2 integrin and VCAM-1/very late Ag-4 (VLA-4) interactions promoted static adhesion. Concurrent beta2 integrin, VLA-4, VCAM-1, and L-selectin blockade completely inhibited lymphocyte attachment. Thus, interactions between leukocyte-endothelial cell adhesion receptor pairs remained remarkably intact across the human-porcine species barrier. Moreover, disrupting the adhesion cascade may impair the ability of human leukocytes to infiltrate a transplanted porcine organ during rejection.

Regulation of platelet heparanase during inflammation: role of pH and proteinases.

Heparan sulfate is rapidly degraded by an endoglycosidase (heparanase) secreted by activated platelets. Since the cleavage and release of heparan sulfate would profoundly alter the local physiology of the endothelium, platelet heparanase activity should be tightly regulated. Consistent with this hypothesis, platelet heparanase was found to degrade endothelial cell heparan sulfate at pH 6.0 but not at pH 7.4, even though 25% of maximum activity was detected at pH 7.4. Loss of heparanase activity occurred rapidly (t1/2 is approximately equal to 20 min) and reversibly at physiologic pH but did not occur at acidic pH (<7.0). Inactivation of heparanase at pH 7.4 did not affect heparin binding and was reversed by 0.5 M NaCl or by heparan sulfate but not by chondroitin sulfate, suggesting inactive heparanase could be tethered on cell surfaces and the function regulated by heparan sulfate. Heparanase was gradually inactivated by trypsin and urokinase (t1/2 = 5 h) but resisted cleavage by leukocyte cathepsin G, leukocyte elastase, plasmin, and thrombin. These findings are consistent with a model in which platelet heparanase is active at the low pH of inflammation but inactive under physiologic conditions preventing inadvertent cleavage of heparan sulfate and loss of physiologic functions of endothelial cells.

Total respiratory support from swine lungs in primate recipients.

UNLABELLED: The use of nonhuman lung donors, such as swine, has the potential to provide an unlimited supply of organs. However, hyperacute rejection has prevented pulmonary xenotransplantation. OBJECTIVE: Our aim was to test the hypothesis that immunodepletion by pretransplantation swine lung perfusion will prevent hyperacute swine-to-primate pulmonary xenograft rejection and allow for a functional swine pulmonary xenograft. METHODS: Seven baboons underwent left pneumonectomy followed by orthotopic transplantation of the swine left lung. Four baboons received immunodepletion by perfusion with swine lungs before transplantation, and three received no treatment before transplantation. RESULTS: After transplantation, pulmonary xenografts from immunodepleted baboons had a low pulmonary vascular resistance and a high pulmonary blood flow compared with control animals, which had a high pulmonary vascular resistance and a low pulmonary blood flow. After 60 minutes of reperfusion, three of four immunodepleted animals also tolerated complete occlusion of the right pulmonary artery, with the baboon relying completely on the swine pulmonary xenograft for respiratory function for 11 hours. Pathologic analysis of peripheral lung biopsy specimens taken from control lungs displayed alveolar disruption and hemorrhage within small vessels, whereas swine lungs transplanted into immunodepleted baboons displayed little histologic evidence of injury. Furthermore, pulmonary xenografts transplanted into immunodepleted baboons demonstrated excellent respiratory function and adequate hemodynamics during occlusion of the right pulmonary artery. CONCLUSION: Hyperacute pulmonary xenograft rejection can be prevented by pretransplantation swine lung perfusion. Swine pulmonary xenografts can provide complete respiratory support in primates when rejection is prevented.

Specificity and function of "natural" antibodies in immunodeficient subjects: clues to B cell lineage and development.

The origin of natural antibodies has long been a subject of controversy. Polyreactive natural antibodies recognize multiple ligands and are thought to arise from B1 B cells. Natural antibodies against carbohydrate antigens such as Gal alpha 1-3Gal or against blood groups A and B are thought to be "elicited" by gut bacteria, but their origin is uncertain. To explore the origin of naturally occurring anticarbohydrate antibodies, the specificity and function of the xenoreactive antibodies and isohemagglutinins were investigated in immunodeficient subjects. Subjects with defects in T cell-dependent antibody synthesis had normal levels of xenoreactive natural antibodies, most of which, like xenoreactive antibodies from normal individuals, were specific for Gal alpha 1-3Gal. On the other hand, some subjects with hyper-IgM syndrome who were able to synthesize abundant quantities of xenoreactive antibodies and polyreactive antibodies were devoid of anti-Gal alpha 1-3Gal antibodies. These results suggest that the lineages of B cells giving rise to anti-Gal alpha 1-3Gal antibodies and isohemagglutinins are distinct from B1 B cells or at least exist at a more "advanced" stage of development than those B1 B cells that give rise to polyreactive antibodies. The findings also suggest that B cells which synthesize anti-Gal alpha 1-3Gal antibodies and isohemagglutinins may be distinct from B2 B cells or exist at a more "primitive" stage of development than B2 B cells that synthesize elicited antibodies in normal individuals.