Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes.

We investigated the pathogenesis of chronic allograft rejection in mouse cardiac allografts. Long-term survival occurred after administration of monoclonal antibody to CD4 or CD40-ligand (CD40L) plus donor cells. Both treatments induced permanent graft survival, but, in contrast to transplants in mice treated with CD4 monoclonal antibody, grafts in mice treated with CD40L monoclonal antibody lacked evidence of chronic rejection, including transplant arteriosclerosis. Freedom from chronic rejection in the group treated with CD40L monoclonal antibody correlated with vascular expression of the 'protective' genes heme oxygenase-1 (HO-1), Bcl-xL and A20. Moreover, arteriosclerosis was induced in allografts in immunoglobulin-deficient mice by antibody transfer only when the transfer was done before expression of protective genes. A direct role for protective gene expression in endothelial cells was demonstrated by in vitro experiments in which induction of HO-1 or Bcl-xL suppressed alloantibody-stimulated endothelial activation. Finally, induction of HO-1 in vivo protected allografts against chronic injury. These data show a role for protective genes in the prevention of chronic rejection, and indicate new approaches to protect grafts against development of transplant arteriosclerosis.

Islet cell autoantigen 69 kD (ICA69). Molecular cloning and characterization of a novel diabetes-associated autoantigen.

We have identified a novel 69-kD peptide autoantigen (ICA69) associated with insulin-dependent diabetes mellitus (IDDM) by screening a human islet lambda gt11 cDNA expression library with cytoplasmic islet cell antibody positive sera from relatives of IDDM patients who progressed to the overt disease. The deduced open reading frame of the ICA69 cDNA predicts a 483-amino acid protein. ICA69 shows no nucleotide or amino acid sequence relation to any known sequence in GenBank, except for two short regions of similarity with BSA. The ICA69 cDNA probe hybridizes with a 2-kb mRNA in poly(A+) RNA from human pancreas, brain, heart, thyroid, and kidney, but not with skeletal muscle, placenta, spleen, or ovary. Expression of ICA69 was also detected in beta cells and cell lines, as well as in tumoral tissue of islet cell origin. The native ICA69 molecule migrates to 69 kD in SDS-PAGE as detected with specific antibodies. Serum samples from relatives of IDDM patients specifically reacted with affinity-purified recombinant ICA69 on Western blotting. The structural gene for ICA69 was designated ICA1. A homologue in the mouse, designated Ica-1 was mapped to the proximal end of chromosome 6 (within 6 cM of the Met protooncogene). ICA69 adds a novel autoantigen to the family of identified islet target molecules, and by the manner of its identification and characterization large amounts of antigen are available for development of quantitative, convenient predictive assays for autoantibodies and analysis of the role of this molecule in diabetes autoimmunity, as well as its physiologic function.

Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury.

We examined the effects of upregulation of heme oxygenase-1 (HO-1) in steatotic rat liver models of ex vivo cold ischemia/reperfusion (I/R) injury. In the model of ischemia/isolated perfusion, treatment of genetically obese Zucker rats with the HO-1 inducer cobalt protoporphyrin (CoPP) or with adenoviral HO-1 (Ad-HO-1) significantly improved portal venous blood flow, increased bile production, and decreased hepatocyte injury. Unlike in untreated rats or those pretreated with the HO-1 inhibitor zinc protoporphyrin (ZnPP), upregulation of HO-1 by Western blots correlated with amelioration of histologic features of I/R injury. Adjunctive infusion of ZnPP abrogated the beneficial effects of Ad-HO-1 gene transfer, documenting the direct involvement of HO-1 in protection against I/R injury. Following cold ischemia/isotransplantation, HO-1 overexpression extended animal survival from 40% in untreated controls to about 80% after CoPP or Ad-HO-1 therapy. This effect correlated with preserved hepatic architecture, improved liver function, and depressed infiltration by T cells and macrophages. Hence, CoPP- or gene therapy-induced HO-1 prevented I/R injury in steatotic rat livers. These findings provide the rationale for refined new treatments that should increase the supply of usable donor livers and ultimately improve the overall success of liver transplantation.

Computer-assisted rational design of immunosuppressive compounds.

We describe the rational design of immunosuppressive peptides without relying on information regarding their receptors or mechanisms of action. The design strategy uses a variety of topological and shape descriptors in combination with an analysis of molecular dynamics trajectories for the identification of potential drug candidates. This strategy was applied to the development of immunosuppressive peptides with enhanced potency. The lead compounds were peptides, derived from the heavy chain of HLA class I, that modulate immune responses in vitro and in vivo. In particular, a peptide derived from HLA-B2702, amino acids 75-84 (2702.75-84) prolonged skin and heart allograft survival in mice. The biological activity of the rationally designed peptides was tested in a heterotopic mouse heart allograft model. The molecule predicted to be most potent displayed an immunosuppressive activity approximately 100 times higher than the lead compound.

Management of Intracranial Incidental Findings on Brain MRI.

The wider use of MRI for imaging of the head in both research and clinical practice has led to an increasing number of intracranial incidental findings. Most of these findings have no immediate medical consequences. Nevertheless, knowledge of common intracranial incidental findings and their clinical relevance is necessary to adequately discuss the findings with the patient. Based on the author´s experiences from a large population-based study, the most common incidental MR findings in the brain will be presented, discussing their clinical relevance and giving recommendations for management according to the current literature and guidelines. Key points: • Intracranial incidental findings are common.• The majority of these findings have no immediate medical consequences.• Knowledge of common incidental findings is necessary for appropriate management. Citation Format: • Langner S, Buelow R, Fleck S et al. Management of Intracranial Incidental Findings on Brain MRI. Fortschr Röntgenstr 2016; 188: 1123 - 1133.

Isolation of nonobese diabetic mouse T-cells that recognize novel autoantigens involved in the early events of diabetes.

Insulin-dependent diabetes mellitus (IDDM) is thought to result from chronic, cell-mediated, autoimmune islet damage. Our aim was to identify the earliest T-cell autoantigen in IDDM, reasoning that this antigen could be causally involved in the initiation of the disease. Identification of the earliest beta-cell-specific autoantigen is extremely important in allowing advances in prevention and treatment of initial events in the development of inflammatory insulitis that precedes beta-cell destruction and overt diabetes. Therefore, we analyzed the proliferative responses of peripheral T-cells from young, female nonobese diabetic (NOD) mice to extracts of pancreatic beta-cell lines. We were able to demonstrate that T-cells responsive to beta-cell antigens exist in the peripheral lymphoid tissue of these mice in the absence of deliberate priming before the manifestation of histologically detectable insulitis. T-cell lines and clones isolated from the peripheral lymphatic tissues of young, unimmunized, female NOD mice were also shown to react with extracts of beta-cells. Fractionation of the beta-cell extracts showed that these T-cell clones recognized multiple beta-cell-specific autoantigens but none of the previously reported putative autoantigens (glutamic acid decarboxylase [GAD]65, GAD67, Hsp65, insulin, ICA 69, carboxypeptidase-H, and peripherin). Thus, we can conclude that these responses are specific for novel beta-cell autoantigens. Finally, NOD T-cell proliferative responses were also seen to an extract of human islets suggesting potential shared antigenic determinants between human and mouse beta-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of carbon monoxide in the donor reduces graft immunogenicity and chronic graft deterioration.

Chronic allograft dysfunction remains the major obstacle for long-term successful transplantation. To date there is no effective treatment. Overexpression of protective genes has provided increased graft function and survival. This mechanism has been implicated in the process of graft accommodation. One of these genes that has been shown to mediate protective effects decodes the enzyme heme oxygenase-1 (HO-1), and an HO-1 downstream product, carbon monoxide (CO). Using an established model of kidney chronic allograft rejection in the rat, we investigated the impact of methylene chloride (MC), a CO donor, as a therapeutic tool to reduce chronic graft deterioration. We showed that donor and long-term recipient treatment with MC improved graft function and reduced histological signs of chronic rejection. Carbon monoxide may be a promising agent to improve graft quality and long-term graft function.

Alleviation of graft-versus-host disease after conditioning with cobalt-protoporphyrin, an inducer of heme oxygenase-1.

BACKGROUND: Recently, we demonstrated that elevated expression of heme oxygenase-1 (HO-1 or Hsp-32) resulted in the modulation of several immune effector functions. Here we evaluated whether induction of HO-1 after administration of cobalt protoporphyrin (CoPP) can prevent the development of acute graft-versus-host-disease (GVHD). METHODS: Acute GVHD was initiated by injection of unfractionated spleen cells from C57BL/6 into B6D2/F1 mice. RESULTS: Administration of CoPP resulted in increased survival: 85% of CoPP-treated animals survived for >100 days compared with only 29% of saline-treated control animals (P<0.05). In contrast, administration of ZnPP, a well-known inhibitor of HO, accelerated GVHD development. The protective effect of CoPP therapy seemed to be caused by immunomodulation of donor cells, because treatment of cell donors prevented development of acute GVHD in 80% of recipients compared with 0% in control animals. Spontaneous lymphocyte proliferation could be measured with splenocytes harvested from animals developing GVHD but not with splenocytes from recipients of CoPP-treated donor cells. CoPP-treatment had no effect on interleukin-2 or interleukin-4 synthesis but inhibited interferon-gamma production. Mice with active GVHD demonstrated a defective lympho-proliferative response to alloantigens or concanavalin A. However, spleen cells isolated from survivors (on day 100) responded normally. Flow cytometric analysis of splenic T cell populations revealed a severe reduction in recipient type (H-2b,d) cells in mice with active GVHD, whereas in protected mice the number of cells remained normal. CONCLUSION: The results from this study confirmed our previous observation that up-regulation of HO-1 activity is associated with down-regulation of several immune effector functions. This resulted in protection from acute GVHD in a parent into F1 mouse model.

Prolongation of skin allograft survival in mice following administration of ALLOTRAP.

Recently, Clayberger et al. demonstrated that ALLOTRAP, small synthetic peptides derived from a conserved region of the alpha 1 helix of certain HLA class I molecules, inhibited human CTL responses in vitro. In rats, ALLOTRAP 07 therapy combined with a subtherapeutic dose of cyclosporine led to the permanent acceptance of heart allografts. In the present study, the effect of ALLOTRAP on the survival of skin allografts in mice was studied. The tail skin of male C57B1/6 (H-2b) mice was grafted on the back of male CBA (H-2k) recipients. In untreated animals, the skin graft was rejected after 11.6 +/- 1.13 days (MST +/- SD). Cyclosporine administered orally for 5 days after transplantation prolonged graft survival to 13.1 +/- 2.13 days. ALLOTRAP 2702 prolonged graft survival to 16.57 +/- 2.15 days when administered orally for five days posttransplantation and to 18.86 +/- 0.38 when administered intraperitoneally until rejection. Thus, ALLOTRAP peptides derived from human MHC class I sequences, in addition to inhibiting human T cell responses in vitro, also prolong allograft survival in rats and mice.

A synthetic dimeric HLA class I peptide inhibits T cell activity in vitro and prolongs allogeneic heart graft survival in a mouse model.

A peptide derived from the alpha 1 domain of the human HLA class I heavy chain (amino acids 75-84; B2702.75-84) has been shown to inhibit human cytotoxic T and NK cell activity in a non-allele-restricted manner. In vivo, this peptide prolonged skin allograft survival in a murine model. Here we demonstrate prolongation of heart allograft survival in mice and extend the characterization of the immunomodulatory activity of B2702.75-84. Similar to what has been observed with retrovirus-derived peptides, the inhibitory capability of this peptide was increased when bound to a carrier protein. An increased immunomodulatory activity was also observed with the dimeric peptide B2702.84-75-75-84 or the multimeric B2702.75-84.MAP. This peptide not only inhibited cytotoxic T and NK cells but also anti-CD3-induced T cell proliferation as well as a mixed lymphocyte reaction (MLR). Flow cytometric analysis of T cells harvested from anti-CD3-stimulated spleen cell culture in the presence of B2702.84-75-75-84 showed decreased expression of activation markers (CD25, ICAM-1, Pgp-1, CD69) compared with untreated control cultures. The superior activity of B2702.84-75-75-84 could also be demonstrated in vivo. Administration of B2702.84-75-75-84 prolonged the survival of B6 (H2b) hearts in CBA (H2k) recipients to 15 +/- 2.7 (P = 0.0002 vs. control) days compared with 11.4 +/- 2.6 (P = 0.01) days in B2702.75-84 treated animals and 7.5 +/- 1.1 days in untreated controls. Administration of control peptides had no significant effect on allograft survival. In combination with a subtherapeutic dose of cyclosporine, B2702.75-84 induced long-term graft survival in 60% of recipients.

Identification of patients at high risk of graft loss by pre- and posttransplant monitoring of anti-HLA class I IgG antibodies by enzyme-linked immunosorbent assay.

Identification of risk factors influencing graft survival may lead to the development of models to predict graft outcome. Such models may provide guidance for immunosuppressive therapy, measure posttransplantation outcome, and eventually improve graft survival in high-risk patients. A major risk factor influencing graft survival is allosensitization. However, due to the lack of standardization of lymphocytotoxicity assays, the detection of alloantibodies utilizing this current methodology may not correlate with posttransplant events. Recently, a novel standardized enzyme-linked immunosorbent assay (ELISA) for the detection of anti-HLA class I IgG antibodies was developed. To evaluate the predictive value of this diagnostic test, a retrospective analysis of 124 renal allograft recipients with an 18-month follow-up time was performed. A highly significant (P=0.01) correlation between pre-transplant ELISA panel reactive antibody (PRA) results and graft loss was observed. Patients with pre-transplant ELISA PRA of >10% had a three times higher risk of graft loss compared with patients who tested negative. No such correlation was observed with complement-dependent cytotoxicity results independent of the reduction of IgM antibodies with dithiothreitol. Similarly, a highly significant correlation of ELISA results with the occurrence of early graft dysfunction was observed. Almost all patients (88%) with a pretransplant ELISA PRA of >50% required posttransplant dialysis, compared with 45% of patients with a pretransplant ELISA PRA of 10-50% and 27% of patients with a pretransplant ELISA PRA of <10%. No such difference was observed with complement-dependent cytotoxicity %PRA values. Analysis of posttransplant specimens by ELISA demonstrated a strong correlation of assay results with graft rejection and graft dysfunction. In summary, these results suggest that detection of anti-HLA class I antibodies by ELISA identifies patients at high risk for graft loss. No other single risk factor of such magnitude has been identified so far.

Gene transfer of immunomodulatory peptides correlates with heme oxygenase-1 induction and enhanced allograft survival.

BACKGROUND: Decapeptides derived from human HLA class I sequences have been shown to prolong allograft survival. The mechanism of action of these peptides has been uncertain, because they act in an MHC unrestricted manner. Recently, it was found that these peptides bind heme oxygenase 1 (HO-1). In the present study, we sought to determine whether local delivery of these peptides through gene transfer could extend allograft survival, and to explore the underlying mechanisms. METHODS: C57BL/6 neonatal hearts were transplanted to CBA/J recipients and the peptide, or plasmid DNA encoding the peptide, was injected directly into the allograft at the time of the transplant. RESULTS: Direct injection of 1 microg of the B2702 peptide into the allograft did not prolong survival (13.3+/-0.8 vs. 13.4+/-0.8 days for untreated controls), but injection of 400 microg of peptide did extend survival (22.0+/-0.6). Injection of plasmid DNA encoding the B2702 peptide was superior to peptide delivery, extending graft survival to 30.8+/-1.5 days. Similar results were obtained using another plasmid encoding the rationally designed peptide BC1 (28.5+/-1.7), whereas no significant prolongation was observed using a plasmid encoding the control peptide B2705 (16.5+/-1.0). To explore the hypothesis that these peptides exert their immunosuppressive effect by altering HO-1 activity, animals were treated with iron protoporphyrin, an inducer of HO-1 activity, or tin protoporphyrin, an inhibitor of HO-1. Treatment with iron protoporphyrin alone extended graft survival (24.5+/-1.6) and did not alter the benefit in survival seen with BC1 gene transfer (28.0+/-0.8). In contrast, treatment with tin protoporphyrin abolished the benefit of BC1 gene transfer (17.0+/-0.6). CONCLUSIONS: These results demonstrate that plasmid mediated gene transfer is an effective means for delivering immunosuppressive peptides to extend allograft survival. The experiments suggest that these peptides may act by increasing HO-1 activity and support a role for HO-1 in immune regulation and allograft survival.

Prolongation of porcine islet xenograft survival in mice after therapy with immunosuppressive peptides.

BACKGROUND: Recently, peptides derived from the heavy chain of HLA-B2702 have been shown to modulate immune responses. In this study, we examined the use of these peptides for immunosuppression in a pig to mouse islet xenograft model. METHODS: Purified porcine islets were transplanted in autoimmune (non-obese diabetic) and non-autoimmune (streptozotocin-injected CBA or C57/Bl6) diabetic mice. Various dosing regimens of HLA-derived peptides with and without antilymphocyte therapy were administered to recipient mice. Graft rejection was determined by daily serum glucose determinations, and, at selected time points, grafts were removed to demonstrate function and provide immunohistochemical examination. RESULTS: HLA-derived peptides were demonstrated to prolong graft survival in both pretransplant and posttransplant treatment regimens. This effect was increased with concomitant antilymphocyte therapy. CONCLUSIONS: Further elucidation of the mechanism of action of these immunomodulatory peptides may help in the development of novel immunosuppressive protocols.

Synthetic MHC class I peptide prolongs cardiac survival and attenuates transplant arteriosclerosis in the Lewis-->Fischer 344 model of chronic allograft rejection.

BACKGROUND: A synthetic peptide corresponding to residues 75-84 of the alpha1 domain of HLA-B7 molecule (HLA-B7.75-84 [Allotrap]) inhibits cytotoxic T cell function in vitro and, when combined with subtherapeutic doses of cyclosporine (CsA), prolongs allogeneic cardiac survival. We now report the effects of HLA-B7.75-84 in the Lewis --> Fischer 344 rat model of chronic cardiac allograft rejection. METHODS: Animals were treated with CsA (5 mg/kg/day s.c.) alone or with CsA plus alternate-day HLA-B7.75-84 (20 mg/kg/day i.p.) for 30 days. Allografts harvested at day 100 were evaluated by histology and immunohistology. RESULTS: HLA-B7.75-84 plus CsA prolonged allograft survival (75% of allografts survived >90 days) compared with CsA alone (27% of allografts survived >90 days) (P<0.05). Histologic examination of control allografts showed dense cellular infiltrates and moderate transplant arteriosclerosis (>75% of arteries showed 10-20% occlusion). Infiltrating leukocytes consisted of macrophages (>75% cells), T cells (10-20%), and rare natural killer cells (<5%). Cell activation was shown by expression of major histocompatibility complex class II antigens (>75%), interleukin (IL) 2 receptor (5-10%), and staining for IL-2 (approximately 5% of intragraft mononuclear cells), interferon-gamma (5-10%) and tumor necrosis factor-alpha (approximately 20%). Leukocytes and vessels also showed labeling for the fibrogenic cytokine, transforming growth factor-beta, and all vessels showed dense deposition of IgG (IgG2a, IgG2b) and C3. The addition of HLA-B7.75-84 decreased overall cellularity (P<0.01) without affecting the composition of the infiltrate, but completely prevented transplant arteriosclerosis, diminished myocardial injury, and abrogated expression of IL-2R, IL-2, interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta. HLA-B7.75-84 also blunted the humoral response, which resulted in a predominance of vascular deposition of the non-complement-fixing IgG2c isotype and a concomitant decrease in C3. CONCLUSIONS: Therapy with synthetic class I major histocompatibility complex peptide (HLA-B7.75-84) attenuates key histologic features of graft arteriosclerosis, in association with inhibition of multiple cytokines and growth factors and modulation of host alloantibody responses in vivo, which is of interest since Allotrap is currently undergoing clinical trials.

Targeting of antihapten antibodies to activated T cells via an IL-2-hapten conjugate prolongs cardiac graft survival.

Antihapten antibodies binding to ligand-hapten conjugates are able to mediate complement mediated lysis in vitro. Based on this observation we propose a new in vivo immunotherapy using molecules that combine a low molecular weight hapten binding to antibodies preexisting in serum and a cell specific ligand. The ligand-hapten conjugates are potential cytotoxic drugs which may (1) be specific for a given target cell, (2) be nonimmunogenic, (3) be of low molecular weight, (4) form soluble complexes with preexisting antibodies resulting in prolonged half life of the drug, and (5) induce a potent antibody mediated rejection of target cells. These novel compounds could be useful for the elimination of certain cell subsets involved in allograft rejection, cancers, infectious diseases, etc., without some of the pitfalls of conventional immunotherapies. The feasibility of this approach was demonstrated in an animal model using a compound consisting of one interleukin 2 and one fluorescein molecule (IL-2-FITC). BALB/c mice (H2d) previously immunized and expressing anti-FITC antibodies were transplanted with a fully mismatched C57BL/6 (H2b) heterotopic heart allograft. Untreated controls rejected their graft by day 9 (MSD = 9 +/- 0.7). Mice with preexisting anti-FITC antibodies treated with IL-2-FITC maintained their grafts for 38.7 +/- 7.1 days (P < 0.02). No prolongation of graft survival was observed in immunized animals that were treated with IL-2 alone (MSD = 10 +/- 1.4). Nonimmunized animals treated with IL-2-FITC rejected their grafts on day 9.4 +/- 1.1. This demonstrates that IL-2-FITC therapy specifically prolonged graft survival in animals with circulating anti-FITC antibodies. The data suggest that a ligand/hapten pair can redirect preexisting antihapten antibodies toward target cells in vivo. Such compounds may be developed for human use as alternatives to polyclonal or monoclonal antibody therapy.

Immunosuppression by D-isomers of HLA class I heavy chain (amino acid 75 to 84)-derived peptides is independent of binding to HSC70.

BACKGROUND: Peptides derived from the class I heavy chain were shown to modulate immune responses in vitro and in vivo. A peptide derived from HLA-B2702 (2702.75-84) inhibited differentiation of cytotoxic T cells as well as T cell and natural killer cell-mediated cytotoxicity in vitro. Peptide-mediated immunomodulation seemed to be independent of the MHC proteins expressed by responder and stimulator cells. In vivo studies in rodents demonstrated prolongation of heart and skin allograft survival after peptide therapy. Here, the correlation between the peptide's biological activity and its amino acid sequence was analyzed using peptides derived from amino acid 75-84 of several mouse, rat, and human MHC class I proteins as well as peptides with single amino acid substitutions in the 2702.75-84 sequence. METHODS: Peptides consisting of both L- and D-amino acids were tested for inhibition of murine and human T cell-mediated and lymphokine-activated killer cell-mediated cytotoxicity, binding to hsc70, and prolongation of heart allograft survival in vivo. RESULTS: Replacement of glutamic acid residue (E) at position 75 with valine (V) resulted in a peptide [2702.75-84(E>V)] with increased in vitro and in vivo activity but unchanged affinity for hsc70. Surprisingly, both L- and D-isomers of 2702.75-84 and 2702.75-84(E>V) inhibited cytotoxic cells in vitro and prolonged heart allograft survival in vivo. However, as expected, the peptides consisting of D-amino acids did not bind to hsc70. CONCLUSION: Assuming that both D- and L-isomers modulate immune responses by similar mechanisms, these results suggest that the peptides' effect is independent of binding to hsc70.

Prolongation of heart xenograft survival in a hamster-to-rat model after therapy with a rationally designed immunosuppressive peptide.

BACKGROUND: Modification of the aminoacid sequence of peptides derived from the HLA class I heavy chain in combination with computer rational design resulted in the development of a peptide, RDP1258, with enhanced immunosuppressive activity. METHODS: We evaluated the activity of this peptide, analyzing infiltrate by immunohistology and cytokine transcripts by reverse transcriptase-polymerase chain reaction method, in a hamster-to-rat xenograft model where recipients were treated with cobra venom factor (CVF) and peptide. RESULTS: Although CVF or peptide alone had no effect, a combination of CVF/peptide RDP1258 resulted in a significant prolongation of graft survival (7.9+/-1 vs. 4.5+/-0 and 3.5+/-0 days, P<0.001). This effect was associated with an increased expression of heme oxygenase 1 (HO-1) in spleen, a significant reduced graft infiltrate, and a decrease of tumor necrosis factor-alpha mRNA transcripts (P<0.05) compared with CVF-treated recipients (1.6+/-0.07 vs. 3.3+/-0.3%, P=0.001) on day 3 after transplantation. CONCLUSION: These observations are consistent with the observation that up-regulation of HO-1 results in inhibition of immune effector functions and suggest that the peptide acts, at least partially, through HO-1 regulation.

Immunosuppressive effects of an HLA class I-derived peptide in a rat cardiac allograft model.

B7.75-84, a 10-amino-acid peptide derived from the HLA-B7 molecule, prolongs rat heterotopic cardiac allograft survival time (GST) when used with cyclosporine in the Lewis-to-ACI strain combination. We evaluated the ability of B7.75-84 to prolong GST in other strain combinations without cyclosporine and studied the effect of B7.75-84 on the immune response in the Wistar-Furth (WF)-to-ACI strain combination. GST was markedly prolonged in most low-responder (ACI) recipients but only slightly prolonged in the high-responder (Lewis) recipient. Cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) limiting dilution assays (LDA) were performed 10 days after cardiac allografts from WF donors were placed in ACI recipients treated with B7.75-84. HTL-LDA assays at 10 days posttransplant showed a slight decrease in HTL precursor frequency and a decrease in their IL-2 production in B7.75-84 treated recipients with prolonged GST in response to donor antigen as well as third-party (Lewis) antigen. CTL-LDA assays at day 10 showed no difference in CTL precursor frequency among treated recipients but did show a significant decrease in CTL killing activity against donor cells in recipients with prolonged GST. No significant difference in CTL killing activity was seen against third- party cells. Antibody analysis was performed at day 8 in treated recipients. Serum from B7.75-84-treated recipients with prolonged graft survival generally showed no detectable IgG antibody response against donor MHC class I antigen. All B7.75-84 treated recipients showed a strong IgM response against donor antigen regardless of allograft outcome. Our results suggest that the immunosuppressive effect of B7.75-84 in rats is greater using a low-responder RT1 haplotype. Furthermore, B7.75-84 induces a nonspecific decrease in HTL function while producing a donor-specific decrease in CTL function and a diminished antidonor MHC class I IgG response.

Anti-Gal antibodies in humans and 1, 3alpha-galactosyltransferase knock-out mice.

BACKGROUND: Due to the absence of alphaGAL epitopes, humans and galactosyltransferase knock-out (GALT/ KO) mice express high levels of anti-Gal antibodies. We describe the properties of mouse anti-GAL antibodies. METHODS: Anti-GAL IgG antibodies were quantified by affinity purification. Antibody affinities and avidities were determined in direct binding and competition assays. Antibody-mediated rejection was investigated using hyperimmunized GALT/KO mice as recipients of GAL+ heart allografts. RESULTS: In young GALT/KO mice the levels of anti-GAL antibodies were low. Immunization of GALT/KO mice resulted in increased anti-GAL antibody expression. In mouse serum 0.6% of IgG was specific for alphaGAL compared to 0.5% in human serum. The avidity of purified mouse and human anti-GAL IgG was 30 and 6 nM, the affinity 15 and 50 microM, respectively. The isotype distribution in mouse and human anti-GAL IgG appeared to be similar to the isotype distribution in normal sera. The affinity of mouse and human anti-GAL IgM was 150 and 750 microM, respectively. Immunized GALT/KO recipients of GAL+ heart transplants rejected their grafts within 2 hr although nonimmunized GALT/KO mice retained their grafts for up to 6 days. Immunohistological examination of the rejected GAL+ hearts revealed massive deposition of IgM and IgG on endothelial cells of the graft with a concomitant deposition of complement. CONCLUSIONS: Our studies demonstrate that anti-GAL antibodies from immunized GALT/KO mice bind alphaGAL with an avidity/affinity similar to human anti-GAL antibodies and are able to induce hyperacute rejection of GAL+ heart allografts.

Soluble HLA antigens and ELISA--a new technology for crossmatch testing.

A soluble HLA ELISA for the detection of donor specific anti-HLA class I IgG antibodies was developed and compared with microlymphocytotoxicity. Donor sHLA was prepared from donor blood or purified blood lymphocytes and captured onto monoclonal antibody coated ELISA plates. After incubation of captured HLA with test serum, bound IgG antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Serum samples from patients on waiting lists to receive kidney transplants were tested by lymphocytotoxicity (AHG protocol) and/or sHLA ELISA in four different laboratories using HLA preparations from eight organ donors. Concordant crossmatch results were obtained for 854 (99%) of 864 ELISA crossmatches. In contrast, concordant results were obtained for 234 (91%) of 256 lymphocytotoxicity crossmatches. Interlaboratory reproducibility of ELISA results was 99%. In contrast, interlaboratory reproducibility of lymphocytotoxicity assay results was 78%. Endpoint titrations of serum specimens containing anti-HLA antibodies demonstrated equivalent sensitivity of ELISA and AHG lymphocytotoxicity crossmatch and similar sensitivity of ELISA and flow cytometry crossmatch. Specimens tested positive by lymphocytotoxicity without DTT treatment but negative with DTT treatment were tested negative by ELISA. Comparison of lymphocytotoxicity and ELISA crossmatch results showed an agreement of 94%. This demonstrates that detection of anti-donor HLA class I antibodies by ELISA is a reliable alternative to microlymphocytotoxicity testing.