Time-dependent changes in donor brain death related processes.

Donor brain death (BD) affects kidney function and survival after transplantation. Studies on brain dead kidney donors indicate that, besides inflammation and coagulation, cytoprotective gene expression is activated as well. Here, we evaluated in a time-course experiment progression of these renal BD-related processes. Animals were sacrificed 0.5, 1, 2 or 4 h after BD and compared to sham-operated controls. Proinflammatory genes (E-selectin, MCP-1, II-6) were massively up-regulated (p < 0.05) already 0.5 h after BD. Inducers of proinflammatory gene expression were either activated (NF-kappaB) or induced in expression (Egr-1) after 0.5 h of BD. Increased numbers of infiltrating granulocytes were seen in the interstitium from 0.5 h on. Also, expression of protective genes HO-1 and HSP70 were increased within 0.5 h. Remarkably, reactive oxygen species formation was detectable only in the later phase of BD. Among 14 measured serum cytokines, MCP-1 and KC-protein were significantly elevated from 0.5 h on. In conclusion, a fast induction of proinflammatory and stress-induced protective processes in brain dead donor kidneys was demonstrated, probably triggered by changes occurring during BD induction. Importantly, hypoxia appeared not to be one of the initial triggers, and early increased systemic levels of chemokines MCP-1 and KC may be regarded as the starting point for the inflammatory cascade in brain dead donor kidneys.

Multiparameter flow cytometric approach for simultaneous evaluation of T lymphocyte-endothelial cell interactions.

Since vascular endothelium is now recognized as an immunologically active tissue, a better understanding of the relationship between endothelial cells and T lymphocytes is critical to the field of solid organ transplantation. Investigations of endothelial cell-T cell interactions have been limited by methodology. We developed a flow cytometric method allowing for concurrent investigation of multiple cell populations within the same culture that can be applied to these complex interactions. Allogeneic CD8+ or CD4+ T cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) were added to a murine endothelial cell monolayer, in which endothelial proliferation was not inhibited by irradiation or addition of a cell cycle-blocking agent. At specific time points, the coculture was analyzed by flow cytometry. T-cell proliferation could be detected by gating on the T-cell subset and evaluating the CFSE fluorescence peaks. By directly analyzing cellular division, we minimized erroneous interpretation of the data encountered by previous studies, which utilized (3)H-thymidine incorporation as sole measure of proliferation. Further subgating on cells that divided facilitated the study of CD8+ lymphocyte activation, differentiation, and acquisition of effector function. By gating on the endothelial cell population, phenotypic changes such as upregulation of surface MHC molecules or immune-mediated apoptosis could be detected. In conclusion, we present a flow cytometric approach that could have important applications for clinical immunological monitoring in allogeneic or xenogeneic transplantation, and might provide the requisite information to better tailor immunotherapy to prevent chronic rejection.

Emergent lung retransplantation after discovery of two primary malignancies in the donor.

A 50-year-old woman underwent single lung transplantation for advanced chronic obstructive pulmonary disease. Shortly after the procedure, it was discovered that the donor suffered from both a renal cell carcinoma and a spindle-cell sarcoma of the ascending aorta, which had metastasized to the spleen. The patient was emergently listed for a retransplantation and underwent bilateral lung transplantation after a new donor became available 4 days after the initial transplantation procedure. After 24 months, the patient is without evidence of malignancy. This case illustrates the role of immediate retransplantation for patients who have inadvertently received thoracic organs from donors harboring occult malignancies.

A simple method for culturing mouse vascular endothelium.

Vascular endothelium is an important site for a wide array of immunological processes such as inflammation, atherosclerosis and allograft rejection. Culture methods of mouse vascular endothelium would provide an important in vitro correlate to immunological murine in vivo models. We describe a simple method to culture mouse vascular endothelium from thoracic aorta. Our cultured cells express typical phenotypic (CD105, CD31, CD106), morphological and ultrastructural (intercellular junctions, Weibel-Palade bodies) markers of vascular endothelium. They also possess functional receptors for uptake and processing of acetylated low-density lipoproteins. The mouse vascular endothelium within our system expresses high levels of MHC class I and MHC class II after activation with IFN-gamma. In addition, these cells express the accessory molecules CD80 and CD54, while they lack constitutive expression of CD86 and CD40, providing them the means to function as antigen presenting cells. Alloreactive CD4(+) and CD8(+) T lymphocytes demonstrate evidence of DNA synthesis after co-culture with activated vascular endothelium indicating their commitment to proliferation. In conclusion, we describe a simple culture system to isolate and grow mouse vascular endothelium, which provides a powerful tool to study biological interactions in vitro.

Donor antigen-presenting cells are important in the development of obliterative airway disease.

OBJECTIVE: Obliterative airway disease, which resembles obliterative bronchiolitis histologically, develops in murine heterotopic tracheal allografts. Chimeric tracheas were used to examine whether donor-type antigen-presenting cells are important in the development of obliterative airway disease. To separate the contributions of CD4(+) and CD8(+) direct pathways, we transplanted tracheas from knockout mice lacking major histocompatibility complex (MHC) class I or II antigens. METHODS: Chimeric tracheas were created via bone marrow transplantation in fully MHC-mismatched combinations. Tracheas from naive B6, autologously reconstituted B6, chimeric B6 bearing recipient-type C3H antigen-presenting cells, MHC class I knockout B6 (B6(I-)), MHC class II knockout B6 (B6(II-)), or C3H mice were transplanted into C3H recipients. The tracheas were harvested at days 14 and 28. RESULTS: At day 28, isografts showed no occlusion, normal respiratory epithelium, and minimal infiltrates. Naive or autologously reconstituted B6, B6(I-), and B6(II-) tracheas showed minimal occlusion at day 14 but contained intraepithelial infiltrates. By day 28, the naive or autologously reconstituted B6 tracheas had occlusion of 69.5% +/- 11.6% (mean +/- standard error of the mean), and in comparison, B6(I-) and B6(II-) tracheas had occlusions of 53.0% +/- 16.3% and 52.2% +/- 15.9%, respectively (P =. 20,.19). In chimeric B6 tracheas, minimal occlusion was seen at day 14 and remained 33.6% +/- 16.2% (P =.039) at day 28. Subtle epithelial changes and minimal infiltrates were seen. CONCLUSIONS: Obliterative airway disease appears to involve donor-type antigen-presenting cells and develops in the absence of either MHC class I or II antigens. These findings suggest that either CD8(+) or CD4(+) direct allorecognition is important in the development of obliterative airway disease.

Immune monitoring in xenotransplantation: the multiparameter flow cytometric mixed lymphocyte culture assay.

Xenotransplantation requires monitoring of complex cellular interactions in vitro. A tool to monitor cell proliferation in detail would be instrumental in understanding these cellular interactions in heterogeneous xenogeneic lymphocyte cultures and in patients after xenotransplantation. To accomplish this, we used a fluorescent cell proliferation marker, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), in combination with flow cytometry. CFSE, a green fluorescent molecule, binds covalently to intracellular macromolecules. Each cell division reduces the fluorescent intensity per cell by half and shows a characteristic multipeak pattern in flow cytometric analysis. For this study, human lymphocytes were labeled with CFSE and cultured in the presence of irradiated porcine lymphocytes. Cell proliferation was detected in CFSE-labeled lymphocytes in both a single and a multiparameter flow cytometry setting. Concurrently, tritiated ((3)H) thymidine incorporation, a common method to measure gross cell proliferation, was assessed. The kinetics of CFSE-labeled cell proliferation correlated with (3)H-thymidine incorporation in that both methods showed a lag phase for days 1-3 and a log phase for days 4-7. Multiparameter flow cytometric monitoring of mixed lymphocyte cultures allowed phenotyping and assessment of viability of proliferating populations in heterogeneous xenogeneic stimulated human lymphocyte cultures and complemented the classical (3)H-thymidine incorporation assay. The use of this technique will allow a wide array of immunologic parameters to be measured in a heterogeneous xenogeneic mixed lymphocyte culture. The information gained from these assays is essential to understanding the biological significance of xenogeneic cellular interaction and for monitoring the immune status of the xenotransplanted patient.

Replacement of graft-resident donor-type antigen presenting cells alters the tempo and pathogenesis of murine cardiac allograft rejection.

BACKGROUND: Graft-resident antigen presenting cells (APCs) are potent stimulators of the alloresponse. To test whether replacement of graft-resident donor-type APCs with those of recipient-type alters allorecognition and the pathogenesis of both acute and chronic rejection, we created chimeric hearts for transplantation into naive recipients. METHODS: To replace donor-type APCs with those of recipient-type, chimeric animals were created by bone marrow transplantation (BMT) in fully allogeneic mouse and rat strain combinations. The degree of APC replacement in chimeric organs was assessed phenotypically and functionally. Chimeric hearts were transplanted heterotopically into untreated recipients. RESULTS: Flow cytometric and immunohistochemical analysis did not detect residual bone marrow recipient-type APCs in mouse BMT chimeras. Although semi-quantitative reverse transcription polymerase chain reaction detected 0.001-0.01% residual cells, APCs isolated from chimeric organs were functionally unable to stimulate donor-type cells. When transplanted into naive recipients, chimeric mouse hearts had significantly prolonged survival but were nevertheless rejected acutely. Similar results were obtained in the ACI --> LEW rat strain combination. However, in the PVG --> DA rat model, the majority of chimeric hearts survived >100 days and all long-surviving hearts developed cardiac allograft vasculopathy. CONCLUSIONS: BMT leads to near complete replacement of organ-resident APCs. The virtual absence of donor-type APCs in chimeric hearts delays or prevents acute rejection in a strain-dependent manner. In contrast, this type of graft modification does not prevent cardiac allograft vasculopathy. This suggests that, although the CD4+ direct pathway may play a role in acute rejection, it is not essential for the development of chronic rejection in rodent cardiac allografts.

Creation of chimeric hearts: a tool for testing the "passenger leukocyte" hypothesis.

BACKGROUND: Bone marrow-derived antigen-presenting cells (APCs) are thought to be a migratory component of organ allografts that activate the rejection response, and recently they have been postulated to play a critical role in tolerance induction. Our goal was to create chimeric hearts (organs with parenchyma and APCs of differing genotype) for use in models of transplantation to test the "passenger leukocyte" theory. METHODS: Murine bone marrow transplantation were performed in two fully major histocompatibility complex (MHC) mismatched strain combinations: C3H-->B10 and CBA-->BALB/c. Recipients were lethally irradiated (10 Gy) and then received 1 x 10(7) bone marrow cells intravenously. Bone marrow transplant survivors had their organ APCs isolated by digestion with collagenase D, followed by density gradient centrifugation. The APC-enriched fraction was stained with fluorescein-labeled monoclonal antibodies specific for either donor (I-Ak) or recipient (I-Ab/d) class II MHC antigens, which are expressed by all APCs but not by parenchymal cells. Donor and recipient class II expression was determined by flow cytometry. RESULTS: Sixty-nine of 100 (69.0%) of C3H-->B10 and 52/107 (48.6%) of CBA-->BALB/c bone marrow transplant recipients survived more than 100 days, whereas all B10 (n = 12) and BALB/c (n = 10) irradiation controls died within 14 days. Mortality appeared to be caused by engraftment failure as most recipients died before day 20. Flow cytometry demonstrated complete APC replacement in hearts (n = 17) and spleens (n = 40), as APC-enriched fractions stained only for donor class II MHC antigens. CONCLUSION: Bone marrow transplantation leads to replacement of heart APCs in two murine models. Chimeric hearts are now being used to test the role of APCs in allograft rejection and in tolerance induction.

Monoclonal immunoglobulin A derived from peritoneal B cells is encoded by both germ line and somatically mutated VH genes and is reactive with commensal bacteria.

We transferred peritoneal cells from BALB/c mice into C.B17 scid/scid mice. Six to eight months after injection, only cells with the B1 phenotype were retained in the spleens and peritoneal cavities of these mice. The lamina propria of the intestine contained many peritoneal, donor-derived, immunoglobulin A (IgA)-producing cells. The mesenteric lymph nodes of these mice were found to be a major site of proliferation and generation of IgA plasmablasts. We established eight IgA-producing hybridomas from the mesenteric lymph nodes of such mice, and all the hybridomas reacted with different but partially overlapping fecal bacterial populations. Cloning and sequencing of the VH genes of these hybridomas showed that two hybridomas utilized germ line-encoded VH genes while the VH genes of the six hybridomas showed somatic mutations, some of which are indicative of an antigen-driven selection process.