Mechanisms of intrathymic tolerance induction to isolated rat hepatocyte allografts

Intrathymic injection of alloantigen in young adult rats is capable of mediating long-lived transplantation tolerance. In this study, we use a well-defined model of isolated hepatocyte transplantation to define the mechanisms of intrathymic induced tolerance. The recipient rats are Nagase analbuminemic rats (NAR) that are deficient in albumin, to allow for following transplant acceptance using metabolic and genetic markers. Tolerance to allogeneic hepatocyte transplants could be mediated by intrathymic injection of live allogeneic splenocytes, lethally irradiated splenocytes, or isolated hepatocytes. Intrathymic injection of live allogeneic splenocytes, but not of hepatocytes or irradiated splenocytes, resulted in donor microchimerism in peripheral lymphoid organs, with preferential expansion of CD4-positive T cells in the recipient spleens. Tolerance could be adoptively transferred from tolerant animals to naive recipients, but only from those animals that had been inoculated with intrathymic donor splenocytes. We conclude that donor microchimerism is found after intrathymic inoculation of live splenocytes, but is not required for tolerance induction and that microchimerism is not an absolute requirement for the generation of regulatory cells.     

Induction of allograft nonresponsiveness after intrathymic inoculation with donor class I allopeptides. II. Evidence for persistent chronic rejection despite high levels of donor microchimerism

We have recently demonstrated that three synthetic peptides corresponding to the donor class I RT1.Aa molecule induce long-term survival of cardiac allografts in the PVG.R8-to-PVG.1U rat strain combination (disparate for one isolated class I, RT1.A, molecule) when presented to the recipient immune system in the thymus. Long-term graft survivors had measurable levels of donor-reactive alloantibodies in their serum. In this study, we examined long-term allografts for the presence of chronic rejection and donor microchimerism to assess whether this regimen of immune modulation establishes true tolerance and whether this tolerance is dependent upon the presence of donor-recipient microchimerism. Histological examination of long-term heart grafts (>100 days) demonstrated chronic rejection, including a mild degree of myocardial infiltration by mononuclear cells, mild to moderate myocardial fibrosis, and various vascular changes ranging from focal intimal thickening to total vascular lumen blockade due to smooth muscle cell proliferation. In contrast, long-term syngeneic hearts transplanted under similar experimental conditions lacked these pathological manifestations. Donor microchimerism was analyzed using the polymerase chain reaction with a pair of oligonucleotides specific for the donor class I RT1.Aa gene and genomic DNA harvested from various tissues from graft recipients. We detected high levels of donor microchimerism in the heart, kidney, liver, skin, bone marrow, thymus, and lymph nodes of long-term graft recipients. Donor microchimerism was also detected in unmanipulated control graft recipients at rejection (7 days) and in intrathymically manipulated recipients that rejected allografts in a delayed fashion (12-82 days). These data clearly demonstrate that intrathymic inoculation of donor class I allopeptides induces long-term graft survival but does not prevent chronic rejection. Allograft rejection occurred despite high levels of donor microchimerism, providing direct evidence that donor-recipient microchimerism is not sufficient for the prevention of acute or chronic rejection in this model.     

Pretransplant injection of allograft recipients with donor blood or lymphocytes permits allograft tolerance without the presence of persistent donor microchimerism

Donor-recipient microchimerism has recently been suggested to play a critical role in the induction and maintenance of allograft tolerance. In this study we sought evidence for this hypothesis using the LEW-to-ACI cardiac allograft as a model system. Donor-specific tolerance to cardiac allografts was induced by intravenous or intraportal injection of graft recipients with donor peripheral blood, T cells, or B cells 7 days before transplantation. All the graft recipients injected with donor antigens accepted donor heart grafts indefinitely when compared with control recipients that rejected donor allografts in 12 days. Long-term graft survivors rejected third-party BN heart allografts in 14 days without an adverse effect on the survival of the first LEW heart allografts, demonstrating the specificity of the tolerance. Tissue lysates prepared from heart, kidney, liver, bone marrow, thymus, lymph nodes, and spleen of tolerant (>120 days) graft recipients were analyzed for the presence of donor DNA using LEW T cell receptor C beta gene-specific primers for polymerase chain reaction that detects donor DNA at > or = 1:10,000 dilution. Donor DNA was detected in 77% of tolerant graft recipients. Chimeric recipients showed variations in the levels and presence of donor DNA in different tissues. The status of donor microchimerism, with respect to its presence and tissue distribution, was dependent upon the donor cell type and route of injection used for the induction of tolerance. Intraportal injection of the graft recipients with donor peripheral blood resulted in the highest degree of chimerism, whereas intravenous injection with donor B cells did not induce detectable microchimerism in this group of recipients. These data clearly demonstrate that the presence of microchimerism is common following administration of donor cells, but that its presence is not an absolute requirement for the long-term survival of allografts.     

Defining the outcome of immunosuppression withdrawal after liver transplantation

Successful immunosuppression withdrawal should benefit the natural history of organ transplantation patients. To identify the clinical hazards of removing drug treatment and possible characteristics that predict a favorable outcome in long-term liver recipients, immunosuppression was withdrawn completely and the clinicopathological outcome documented in 18 liver recipients. Indication for transplantation, HLA matching, early rejection history, and presence of microchimerism were examined as predictors of outcome. Chimerism was determined by polymerase chain reaction-based examination for donor-specific HLA-DRB1 alleles and Y-gene-specific nucleotide sequences. At 3 years, 5 patients (28%) remained completely off immunosuppression; 12 patients (67%) experienced histological graft changes: acute rejection in 4, portal tract inflammation/hepatitis in 7, and necrosis in 1. Hepatitis B or C viral infections did not account for the nonrejection patterns. Unmasking of systemic disorders occurred. Chimerism, demonstrated in 7 patients (39%), with skin the optimal tissue, was not associated with tolerance. Parameters associated with successful drug withdrawal were transplantation for non-immune-mediated liver disorders, fewer donor-recipient HLA A, B, and DR mismatches, and a low incidence of early rejection. Immunosuppression withdrawal is a feasible option in a proportion of selected liver recipients, but identification of tolerant patients remains imprecise.     

Outcomes of recipients of both bone marrow and solid organ transplants. A review

In this review we examine the clinical outcomes of patients who have received both bone marrow transplantation (BMT) and solid organ transplantation (SOT) and discuss the possible immunologic consequences of the dual transplants. We collected cases through a comprehensive literature search (MEDLINE database, English literature only) covering the years 1990 through 1997 and correspondence with the International Bone Marrow Transplant Registry. Our study selected case reports of patients who have undergone both bone marrow and solid organ transplants; cases in which bone marrow transplantation was undertaken as an adjunct ot induce or augment donor-specific tolerance in a recipient to the transplanted organ were excluded. Clinical characteristics included patient's demographic information, underlying disorders for each transplant, source of donor organ or tissue, time between transplants, and immunosuppressive regimens used to prevent graft-versus-host disease (GVHD) or graft rejection. Clinical outcomes included patient survival, complications of transplantation, and donor-specific tolerance that was experienced in many cases. Twenty-one cases of SOT after BMT and 7 cases of BMT after SOT were reviewed. Solid organ transplantations included lung, liver, cardiac, and kidney for a variety of BMT-related complications including GVHD, hepatic veno-occlusive disease, chronic renal failure, end-stage pulmonary disease, and severe cardiomyopathy. Bone marrow transplants were performed following SOT for aplastic anemia and hematologic malignancies. Clinical outcomes for patients who received both BMT and SOT were variable and depended on transplant indication and degree of histocompatibility. Prior bone marrow transplantation may tolerize for a subsequent organ transplant from the same donor. Conversely, severe GVHD may follow BMT from human leukocyte antigen (HLA)-matched donors following SOT. The favorable survival in this high-risk group of patients may represent a literature review bias (that is, an undetermined number of unsuccessful cases may not have been reported). Nonetheless, dual transplantation is clearly feasible in selected cases.     

Hypoxia induces high-mobility-group protein I(Y) and transcription of the cyclooxygenase-2 gene in human vascular endothelium

BACKGROUND: Animals reconstituted with allogeneic whole bone marrow (WBM) are often tolerant of donor-specific solid organ grafts. Clinical application of bone marrow transplantation in solid organ transplantation has been limited, however, principally by graft-versus-host disease. We previously demonstrated that hematopoietic stem cells (HSCs) reconstitute lethally irradiated allogeneic mice without producing graft-versus-host disease. The purpose of this study was to determine whether tolerance to solid organ grafts could be induced in mice reconstituted with HSCs. METHODS: BALB/c mice were lethally irradiated and reconstituted with allogeneic C57BL/Ka, Thy-1.1 WBM or HSCs. An isolated group was given a limited number of HSCs (250 cells) and a subpopulation of allogeneic cells known to facilitate HSC engraftment (facilitators). C57BL/Ka, Thy-1.1 neonatal heart grafts were placed in reconstituted animals either at the time of hematopoietic transplant or 35 days later. Third-party C3H grafts were placed over 2 months after hematopoietic reconstitution. Tolerance was defined as the persistence of cardiac contraction for the duration of evaluation (125-270 days). RESULTS: All surviving mice that were reconstituted with C57BL/Ka, Thy-1.1 HSCs, WBM, or HSCs and facilitators were tolerant of C57BL/Ka grafts long-term. Third-party C3H grafts placed in reconstituted animals were rejected by day 12, whereas those placed in unmanipulated mice were rejected by day 9. CONCLUSION: These data indicate that tolerance to concurrently or subsequently placed solid organ grafts can be reliably achieved with limited numbers of purified HSCs in a model where immunocompetence to third-party major histocompatibility complex antigens is delayed but intact.     

UVB-irradiation of human bone marrow: potential for donor specific tolerance

Bone marrow mononuclear cell (BMMC) transplant may serve to produce donor specific tolerance for a coincident solid organ graft, but with the risk of graft versus host disease (GVHD). We examined in vitro the immunomodulatory effect of UVB on human BMMCs as potential prophylaxis against GVHD for clinical transplantation. After 10-200 J/m2 UVB-irradiation, BMMCs were examined by proliferative response (in mixed lymphocyte reaction and following phytohemagglutinin stimulation) and by cytokine profile. We also evaluated CFU-GM, CFU-GEMM, and BFU-E progenitor viability by 2-week methyl cellulose cultures following UVB-irradiation. Parallel studies were applied to marrow that was T-cell depleted by soybean agglutination (SBA) or by SBA and sheep erythrocyte rosetting (SBA-E-). We found that (1) UVB produces a dose-dependent inhibition of the proliferative response to stimulators by human BMMCs; (2) increasing doses of UVB-irradiation and increasing levels of T-cell depletion (TCD) are both inversely related to production of lymphokines (IL2, IL3, LIF, IFN-gamma, and GMCSF) and (3) T-cell depletion, but not UVB-irradiation, decreases the production of monokines (IL1, TNF, IL6). Progenitor cell viability was decreased but preserved at 100 J/m2 of UVB. Our findings suggest that UVB compares favorably with TCD as a technique for inhibition of GVHD and therefore that UVB-modulation of bone marrow (BM) inoculum may be useful in the prevention of GVHD in clinical bone marrow transplantation accompanying a solid organ graft.     

The Australian brushtail possum (Trichosurus vulpecula) gonadotrophin alpha-subunit: analysis of cDNA sequence and pattern of expression

Substantial evidence has accumulated to suggest that in the near future implementation of the veto-cell-suppressor concept in the treatment of kidney allograft recipients might lead to the establishment of life-long specific allograft tolerance in the absence of further immunosuppressive therapy. Veto suppression prevents the generation of antigen-specific T-helper and cytotoxic T lymphocytes in vitro provided that the T-lymphocyte precursors specifically recognize antigenic peptides associated with the major histocompatibility complex molecules class II and class I, respectively, expressed on the surface of the veto-active cell. Data from a large number of experimental and clinical studies strongly indicate that veto-active cells function in vivo and are capable of preventing allograft rejection. Thus, donor-cell-mediated veto activity is the most likely explanation for the well-known graft tolerizing effect of pretransplant donor blood transfusions in kidney graft recipients. A prerequisite for a veto-active environment in vivo is the establishment of lymphoid microchimerism, in which veto-active donor and recipient cells mutually downregulate potential alloaggression.     

Sample suitability for the detection of minor white cell populations (microchimerism) by polymerase chain reaction

BACKGROUND: There is increasing use of highly sensitive testing with polymerase chain reaction (PCR) to study white cell microchimerism after transfusion and transplantation. This study investigated possible artifactual sources of allogeneic sample contamination before PCR testing. STUDY DESIGN AND METHODS: Quantitative Y-chromosome PCR was used to study microchimerism among transfused patients with sickle cell disease (SCD) and thalassemia by using residual specimens from the clinical laboratory. High levels of circulating male white cells among transfused patients with SCD but not thalassemia led to concern over the artifactual origin of male cells. To investigate, paired specimens were collected from 26 female SCD patients: one specimen underwent processing only for PCR, while the other underwent testing in the clinical laboratory before PCR as a process control. All laboratory instruments were also assessed for their ability to impart male allogeneic cells to aliquots of female blood. RESULTS: Thirty-three (31%) of 107 SCD samples, but 0 of 20 thalassemia samples, gave a high-level PCR signal. One of 26 paired samples that was not exposed to clinical laboratory equipment had low-level PCR positivity while 10 of the 26 became strongly positive after testing on a blood cell analyzer and a reticulocyte analyzer. Sixteen of 32 female samples became positive after reticulocyte analysis, while none became positive after blood cell analysis. Samples from thalassemia patients tested PCR-negative because reticulocyte counts had not been performed. CONCLUSION: Allogeneic cell contamination is common with clinical laboratory equipment. These samples may not be suitable for microchimerism studies. In addition to method controls, process controls should be employed where appropriate.     

Antiviral cytotoxic activity across a species barrier in mixed xenogeneic chimeras: functional restriction to host MHC

Reconstitution of lethally irradiated mice with a mixture of mouse and rat bone marrow cells (mouse + rat-->mouse) results in mixed xenogeneic chimerism and donor-specific tolerance. The current study demonstrates that mouse and rat T lymphocytes that have developed in xenogeneic chimeras are restricted to Ag presentation by mouse, but not rat, APC. Restriction to host Ags results in functional immunocompetence with generation of antiviral cytotoxic activity in vivo, within and across species barriers. These data demonstrate for the first time that the host thymus is sufficient to support development and positive selection of functional cross-species T lymphocytes. The superior immunocompetence, as compared with fully xenogeneic (rat-->mouse) chimeras, may prove to be of significant benefit in the clinical application of xenotransplantation to solid organ transplantation and immune reconstitution for AIDS.     

Specific tolerance induction and transplantation: a single-day protocol

Bone marrow transfusion is a well-established method for induction of mixed hematopoietic chimerism and donor-specific tolerance in animal models. This procedure, however, is inapplicable in clinical transplantation using cadaveric donors due to the interval (1 week to 7 months) between tolerance induction and organ transplantation. For clinical use, it is essential that allografts be placed at the time of bone marrow transfusion. In the present study, we performed skin transplantation within 1 hour after a nonlethal conditioning regimen. Recipient mice were treated with anti-CD3, anti-CD4, low-dose total body irradiation (3 to 6 Gy TBI) and fully mismatched or haploidentical donor bone marrow cells. Stable multilineage chimerism and specific T-cell nonresponsiveness developed. Donor skin grafts were permanently accepted. These results suggest that this single day protocol has clear potential for application in both cadaveric and living-related organ transplantation.     

Delayed acquisition of high-avidity anti-cytomegalovirus antibody is correlated with prolonged antigenemia in solid organ transplant recipients

Previous studies have demonstrated that maturation of cytomegalovirus (CMV)-specific antibodies in solid organ transplant recipients is delayed after primary CMV infection. To investigate the clinical significance of this finding, the avidity indices of anti-CMV antibody were determined in parallel with other serologic and virologic parameters in serial serum samples from 24 solid organ transplant recipients who had primary CMV infection that began during the first 3 months after transplantation. The data obtained show that a delay in antibody maturation is significantly correlated with a long persistence of positive antigenemia.     

C-13 Methanol Far-Infrared Laser: Newly Discovered Lines, Predictions, and Assignments

Autologous blood donation before elective surgery is generally believed to be a safe procedure for patients with a variety of underlying medical conditions, but the accumulation of additional data continues to define its safety in unique patient groups. Patients who have received a solid organ transplant may also undergo various elective surgical procedures after transplantation, and the question of safety of autologous blood donation for these patients is raised. In one hospital-based blood collection program, we identified 4 patients who had received solid organ transplants and subsequently made autologous blood donations for elective operations unrelated to the transplantation. Two patients had received heart transplants and 2 received liver transplants. A total of 10 autologous donations were made by these 4 patients without adverse effects or complications. A low hemoglobin concentration was the only reason for temporary deferral from autologous donation. Despite having complicated clinical situations, recipients of solid organ transplants can safely donate autologous blood and should not be automatically excluded from making such donations.     

Status of microchimerism in recipients 15 years after living related kidney transplantation

To study the relevance of microchimerism to the long-term outcome of renal allografting, we analyzed the frequency of microchimerism in kidney transplant recipients who had stable graft function for 15 years or longer. Among the 104 recipients who underwent kidney transplantation between 1971 and 1980, 27 renal allografts (26%) are still functioning. Among these 27 patients, 13 recipients whose donor was still alive and cooperative were investigated for the presence of microchimerism in the peripheral blood and for their immunological status. Microchimerism was tested using the polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) method. To test the sensitivity of PCP-SSCP, the peripheral blood obtained within 5 weeks after transplantation (four kidney transplants, three liver transplants) was also examined. Microchimerism was detectable in five patients within 5 weeks of transplantation (kidney transplantation, 3/4; liver transplantation 2/3. However, in the patients studied 15 years after transplantation, microchimerism was detected in only one recipient (1/13). In this chimeric patient, mixed lymphocyte response revealed high responsiveness against donor antigen. In contrast, some patients who did not have chimerism showed donor-specific hyporesponsiveness in mixed lymphocyte response assay and did not develop antidonor antibody, according to flow cytometric analysis. Microchimerism is an infrequent state in the long-term survivors of kidney allografting, and this state is irrelevant to donor-specific unresponsiveness.     

Preclinical large animal models for hematopoietic stem cell transplantation

The use of large animal models for marrow and stem cell transplantation has become increasingly important. Large animal models have recently been used for studying the principles of hematopoiesis and illustrate the relative slow rate of stem cell turnover compared with mice. Furthermore, large animals are used to study the effectiveness of varying conditioning regimens, as well as the influence of growth factors and various immunosuppressive agents such as corticotropin-releasing factor and mycophenolate mofetil on graft-versus-host disease and graft rejection. Large animal models have become useful in studying the combination of hematopoietic stem cell and solid organ transplants for the establishment of long-term tolerance in major histocompatibility complex-mismatched settings.     

A molecular epidemiological probe for pig microchimerism

We have cloned and characterized a single-copy DNA sequence from the porcine alpha-1,3-galactosyltransferase gene that corresponds to a 547-base pair intron separating exons 3 and 4 of the protein coding domain. Polymerase chain reaction amplification of this sequence from flanking oligonucleotides generates a species-specific DNA probe (pgt34) capable of recognizing 50 pg chimeric template DNA at a pig to human cellular ratio of 1/10,000. Homologous DNA sequence is not identified in the macaque, baboon, or human genome by Southern hybridization. Analysis of a discordant model of pig to baboon xenotransplantation demonstrates peripheral blood microchimerism in the presence of a functioning pig kidney xenograft and persistence of microchimerism in lymphatic tissue after graft removal. This probe should be useful for tracking the fate of porcine cells in patients undergoing xenotransplantation of whole organs or free tissues such as pancreatic islet cells and should facilitate studies of microchimerism in experimental models of pig to monkey xenotransplantation.     

Identification of hashish samples with inductively coupled high-frequency plasma emission spectrometry and neutron activation analysis and data handling with neuronal networks. 1. Methods for the quantitative determination of characteristic trace elements]

The adherence of six clinical Candida albicans isolates to buccal epithelial cells obtained from AIDS patients, solid organ transplant recipients and healthy individuals was compared. It was shown that Candida albicans bound in significantly greater numbers to epithelial cells obtained from AIDS patients than to those from healthy individuals or transplant patients, and that the adherence capacity varied among the strains tested.     

Transient contralateral hypoglossal nerve palsy following third molar surgery under day-case general anaesthesia: a case report and review of the literature

We have noticed that bone marrow transplanted in a vascularized limb graft providing a continuous supply of donor BMC may prolong the survival time of skin graft from the same donor. The question arises whether the raised microchimerism plays a role in the prolonged survival of skin allograft. The aim of the study was to follow the development of microchimerism after allogeneic vascularized bone marrow transplantation (VBMTx) concomitantly with the rejection processes of transplanted skin. The BN rats served as donors and LEW rats as recipients of VBMTx and free skin flap allograft. Hind limb was transplanted followed by a full-thickness skin graft on the dorsum. Cellular microchimerism was investigated in recipients of VBMTx and skin grafts in blood, spleen, mesenteric lymph node and bone marrow with monoclonal antibody OX27 directed against MHC class I polymorthic RTI on BN cells and quantitatively analysed in FACStar. In VBMTx group free skin flap survived 70 days after weaning of CsA. Intravenous infusion of BMC in suspension equivalent to that grafted in hind limb did not prolong skin graft survival after cessation of CsA therapy. Donor-derived cells could be detected in VBMTx recipients as long 70 days after wearing of CsA but not in recipients of i.v. suspension BMC grafting.     

Monoclonal antibody against CD45RB for the therapy of rejection and autoimmune diseases

Rejection continues to be the single largest impediment to successful organ transplantation. Current therapy, which must be taken for a lifetime is nonspecific and has significant side effects including infection and cancer. There is a need to develop improved means of immunosuppression. The current goal of transplantation immunology is to induce a prolonged state of nonreactivity to the allograft but preserving an otherwise intact immune system (tolerance). We have recently reported that a monoclonal antibody against CD45RB is a potent immunosuppressive agent, and that it induces donor specific tolerance in the mouse. In this contribution we briefly review our understanding of the molecular basis for the activity of this therapy and update results in various transplant and autoimmune disease animal models. The clinical relevance and future development of this novel therapy is also discussed.     

Glycine therapy of schizophrenia

Hemolysis due to donor-derived antibodies produced by "passenger" B lymphocytes, called passenger lymphocyte syndrome, has been described in ABO-unmatched solid organ and bone marrow transplant recipients. Delayed hemolytic transfusion reactions occur within a similar time frame and have similar clinical and serologic findings. To our knowledge, we report the first case of hemolysis due to the simultaneous occurrence of passenger lymphocyte syndrome (donor-derived anti-A) and a delayed hemolytic transfusion reaction (recipient-produced anti-E) in a liver transplant patient.