Ferret lung transplant: an orthotopic model of obliterative bronchiolitis.

Obliterative bronchiolitis (OB) is the primary cause of late morbidity and mortality following lung transplantation. Current animal models do not reliably develop OB pathology. Given the similarities between ferret and human lung biology, we hypothesized an orthotopic ferret lung allograft would develop OB. Orthotopic left lower lobe transplants were successfully performed in 22 outbred domestic ferrets in the absence of immunosuppression (IS; n = 5) and presence of varying IS protocols (n = 17). CT scans were performed to evaluate the allografts. At intervals between 3-6 months the allografts were examined histologically for evidence of acute/chronic rejection. IS protects allografts from acute rejection and early graft loss. Reduction of IS dosage by 50% allowed development of controlled rejection. Allografts developed infiltrates on CT and classic histologic acute rejection and lymphocytic bronchiolitis. Cycling of IS, to induce repeated episodes of controlled rejection, promoted classic histologic hallmarks of OB including fibrosis-associated occlusion of the bronchiolar airways in all allografts of long-term survivors. In conclusion, we have developed an orthotopic lung transplant model in the ferret with documented long-term functional allograft survival. Allografts develop acute rejection and lymphocytic bronchiolitis, similar to humans. Long-term survivors develop histologic changes in the allografts that are hallmarks of OB.

Embryonic stem cell-derived T cells induce lethal graft-versus-host disease and reject allogenic skin grafts upon thymic selection.

Efficient differentiation of embryonic stem cells (ESC) into hematopoietic progenitor cells (HPCs) is crucial for the establishment of stem cell-based therapies targeting the treatment of immunological and hematological disorders. However, so far, it has not been possible to induce long-term survival of murine ESC-derived HPCs without the overexpression of HoxB4, a homeobox transcription factor that confers self-renewal properties to hematopoietic cells. Yet it has not been feasible to generate T cells from HoxB4-expressing HPCs, a problem that has been attributed to HoxB4. Here, we show that Notch1 signaling in HoxB4-transduced ESCs leads to efficient derivation of T cells that survive long term. These T cells display a normal T-cell Vß repertoire, respond to mitogen stimulation and induce lethal graft-versus-host disease. Thymic selection in fetal thymic organ cultures (FTOCs) allowed negative selection and generation of T cells tolerant to 'self' and capable of rejecting MHC-mismatched skin allografts. Our data show that ESC-derived T cells, despite high expression of HoxB4, are fully immunocompetent.

Efficacy, safety and tolerability of recombinant factor VIII (REFACTO) in patients with haemophilia A: interim data from a postmarketing surveillance study in Germany and Austria.

An open-label, multicentre, postmarketing surveillance study conducted in Germany and Austria with recombinant factor VIII (REFACTO) has enrolled 217 patients (mean age 26.3 years) from 38 haemophilia centres during the first 4.8 years. Most patients (188/217; 86.6%) had severe to moderately severe haemophilia A, of whom 153 completed sufficient diary information for the main efficacy analysis. These 153 patients experienced a median of 6.6 (interquartile range 1.4-18.6) bleeding episodes per year. Patients treated with prophylaxis experienced a median of 4.4 (1.1-9.3) bleeds per year, while patients treated on-demand experienced a median of 22.8 (11.3-29.0) bleeds per year. Overall, most physicians (41/43 [95.3%]) were 'very satisfied' or 'satisfied' with the efficacy of REFACTO in the treatment of bleeding episodes. A total of 137 non-serious adverse events have been reported in 52/217 patients (24.0%) to date. In addition, 129 serious adverse events in 87 patients (40%) were reported, including 41 cases of 'less than expected therapeutic effect' (LETE). Of these, 39 LETE cases were reported in one centre; however, patients in this centre experienced considerably fewer bleeding episodes per year than patients outside this centre. Overall, six patients (2.8%) have developed de novo inhibitors, three of which were considered high titre. Four of these patients were at high risk (0-50 exposure days [ED]) of inhibitor formation, one was at intermediate risk (51-100 ED) and one was at low risk (>100 ED). These results emphasize the benefit of postmarketing surveillance and, overall, this study confirms the efficacy, safety and tolerability of REFACTO in the treatment of patients with haemophilia A.

Thalidomide and prednisolone inhibit growth factor-induced human retinal pigment epithelium cell proliferation in vitro.

Thalidomide and prednisolone were recently introduced as treatment modalities in age-related macular degeneration (AMD). Growth factor-induced activation of retinal pigment epithelial (RPE) cells is a crucial event in this disease. The purpose was to examine the effect of thalidomide and prednisolone on growth factor-preactivated RPE cells. Human RPE cells were stimulated with 10 ng/ml platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), or vascular endothelial growth factor (VEGF) for 24 h. Afterwards, thalidomide (50 microg/ml) or prednisolone (100 ng/ml) were added for 24 h. RPE cell proliferation was determined by [3H]-thymidine incorporation. PDGF and bFGF significantly stimulated human RPE cell proliferation (p < 0.005), the value for VEGF stimulation was not significant (p = 0.3). The effect of the growth factors was diminished after addition of thalidomide and prednisolone (p < 0.005). The current study shows that the inhibitory properties of thalidomide and prednisolone remain even after growth factor activation of the cells.

Allogeneic recombinant soluble MHC class I molecules modify urinary odor cues in rats.

The highly polymorphic genes of the major histocompatibility complex (MHC) determine, in part, the odor cues and behavior of an individual. In animal models, MHC-associated odors that regulate distinct behavior have been identified mainly in urine. However, the underlying mechanism is still not clear. Here, we show that injected recombinant soluble (rs) MHC class I molecules (DA, rsRT1.A(a) and Lewis, rsRT1.A(l)) temporarily alter urine odor of Lewis test rats (RT1.A(l)). This change in urinary signals was observed in behavioral assays using the habituation/dishabituation test and in odor signals analysed by gas chromatography (GC) and mass spectrometry (MS). Gas chromatographic analysis revealed that these altered odor signals are caused by quantitative changes of at least two nitrogen-containing urinary compounds. The results suggest that urinary olfactory cues are directly or indirectly influenced by MHC class I gene products.

Donor-derived soluble MHC antigens plus low-dose cyclosporine induce transplantation unresponsiveness independent of the thymus by down-regulating T cell-mediated alloresponses in a rat transplantation model.

BACKGROUND: In vitro, soluble MHC (sMHC) antigens modulate and induce apoptosis in alloreactive and antigen-specific T cells, demonstrating their potency to regulate T cell-mediated immune responses. However, their efficacy to regulate immunological responses in vivo remains unclear. Here, we report that repetitive intraperitoneal injection of recombinant Lewis rat-derived MHC class I antigens in Dark Agouti (DA) rats modulates alloreactivity. METHODS: RT1.A1 (Lewis derived) genes were cloned into mammalian expression vectors, and RT1.Aa (DA derived) genes were used to transfect a rat myeloma cell line. RT1.A1 molecules were injected intraperitoneally in DA recipients that subsequently underwent transplantation with Lewis-derived cardiac allografts. RESULTS: Soluble class I antigens were secreted by the transfected cells and were shown to be heterodimeric, peptide-loaded, and conformationally folded. Injection of donor-derived soluble MHC significantly reduced the ability of recipient animals to mount a cytotoxic T-cell response to donor-derived tissue. More interestingly, this treatment significantly prolonged donor-graft survival and allowed 60% of treated animals to develop graft tolerance (>120 days), when donor sMHC were combined with a single subtherapeutic dosage of cyclosporine. Thymectomy of recipient animals before transplantation did not interfere with induction of peripheral tolerance. CONCLUSIONS: Donor-derived sMHC are potential tolerogens for down-regulating the cytotoxic T-cell response of animals that undergo transplantation. Thus, these data provide for the first time a rationale for the application of directly injected sMHC in vivo to down-regulate immunological responses and aid the induction of graft tolerance.

HLA-G has a concentration-dependent effect on the generation of an allo-CTL response.

Human leucocyte antigen (HLA) -G is expressed on trophoblast cells during pregnancy, suggesting a role in protection of the semiallogeneic fetus. Published data suggest that HLA-G protects a cell against natural killer cell lysis. It has been hypothesized that HLA-G may also protect the fetus by preventing allo-cytotoxic T lymphocyte (CTL) responses. To test this hypothesis, we assayed the effects of various concentrations of purified HLA-G on CTL response in a mixed lymphocyte culture (MLC) system. We found that concentrations > or =0.1 microg/ml of HLA-G suppressed the allo-CTL response by 30-100% over the control, but, paradoxically, concentrations of 0.01-0.05 microg/ml of HLA-G augmented the allo-CTL response by 25-50% over the control. Concentrations < or = 0.001 microg/ml HLA-G had no effect. Addition of HLA-G to preprimed allo-CTL effector cells did not affect their killing ability. Allo-CTL suppressive doses of HLA-G induced a T helper type 2 (Th2) cytokine response, whereas allo-CTL-enhancing doses of HLA-G induced a Th1-type cytokine response. HLA-G purified from first-trimester placenta does not affect allo-proliferative responses nor does it alter the percentage of CD4+ or CD8+ T cells in MLCs. These findings support a potential role for HLA-G-mediated suppression of allo-CTL formation in normal pregnancies. In addition, the effects observed at lower concentrations of HLA-G may have interesting implications for the condition of pre-eclampsia in which concentrations of this HLA class I molecule are reduced.

Growth factor combinations modulate human retinal pigment epithelial cell proliferation.

PURPOSE: Retinal pigment epithelial (RPE) cell proliferation is an important step in the pathogenesis of ocular diseases such as proliferative retinopathy. Growth factors play a major role in modulating the development and progression of these diseases. The aim of this study was to examine the effects of single growth factors as compared to growth factor combinations on the stimulation of RPE cell proliferation. METHODS: Human RPE cells were treated with 10 ng/ml IGF-1, PDGF, bFGF, EGF, VEGF, and TGFbeta(2), or with the combination of each of the growth factors. RPE cell proliferation was determined by [(3)H]-thymidine incorporation after growth factor stimulation for 24 hr. RESULTS: RPE cell proliferation was significantly increased when PDGF and bFGF were combined as compared to the single growth factor (p<0.005). There was a decrease in DNA synthesis for combined growth factors as compared to the single growth factor for IGF-1 plus TGFbeta(2) (p<0.05), IGF-1 plus VEGF (p<0.005), PDGF plus TGFbeta(2) (p<0.005), VEGF plus TGFbeta(2) (p<0.005 as compared to VEGF), and bFGF plus TGFbeta( 2) (p = 0.01 as compared to bFGF). The remaining growth factor combinations showed no significant synergistic effect on human RPE cell proliferation. CONCLUSIONS: These data lead to the implication that DNA synthesis in human RPE cell culture is synergistically affected by certain growth factor combinations, but not by all of them. This observation may be important for a better understanding of growth factor interactions occuring in proliferative diseases in the eye.

Alloreactivity and apoptosis in graft rejection and transplantation tolerance.

Weissmann wrote as early as 1889 that higher organisms contain within themselves the germs of death [1]. However, the term, programmed cell death, or apoptosis as it is now known, was defined much later [2]. Thus, it was long recognized that damaged and old cells are eliminated within the body, but the underlying mechanisms are only now beginning to emerge. Apoptosis appears central to the process of negative selection of developing T-cells in the thymus. In regard to organ transplantation, apoptosis contributes to graft rejection and the establishment of graft tolerance. Thus, understanding the regulatory mechanisms of apoptosis may help establish a new protocol for the induction of transplantation tolerance.

Co-transplantation of donor-derived hepatocytes induces long-term tolerance to cardiac allografts in a rat model.

BACKGROUND: Liver allografts transplanted between MHC-disparate mice, rats, and swine are spontaneously accepted in most strain combinations without requirement for immunosuppression. The underlying mechanism has, however, remained elusive. Here, we demonstrate that co-transplantation of donor-derived hepatocytes protect Lewis (RT1.A1) cardiac allografts from acute and chronic rejection in DA (RT1.Aa) recipients indefinitely. METHODS: Livers of donor Lewis rats were harvested and the hepatocytes separated from hepatic leukocytes by collagenase digestion and gradient separation. DA recipient animals were transplanted Lewis cardiac allografts and simultaneously intraportally infused either Lewis-derived hepatocytes or hepatic leukocytes. Recipient animals were either not further treated or received a single dose of 15 mg/kg cyclosporine. RESULTS: Donor hepatocytes alone significantly protected syngeneic cardiac allografts from rejection, whereas hepatic leukocytes failed to influence graft survival. In combination with cyclosporine, recipient cardiac allografts were indefinitely protected from rejection. Graft-infiltrating cells in tolerant animals presented as clusters of CD4+ T cells and stained mostly positive for interleukin-4, whereas graft-infiltrating cells in rejected allografts were predominantly positive for interferon-gamma. Adoptive transfer of splenocytes derived from tolerant animals protected Lewis cardiac allografts from rejection in DA recipients without immunosuppression. In contrast, hepatic leukocytes protected only 50% of the allografts from rejection. CONCLUSION: We propose that donor hepatocytes induce permanent engraftment of syngeneic allografts by establishing a Th2 type alloresponse that is transferable to new graft recipients. The results of this study demonstrate that liver parenchymal cells significantly mediate spontaneously liver-induced tolerance.

Oral feeding of an immunodominant MHC donor-derived synthetic class I peptide prolongs graft survival of heterotopic cardiac allografts in a high-responder rat strain combination.

The efficacy of two synthetic major histocompatibility complex (MHC)-derived DA (RT1.Aa) 25-mer peptides (residues 56-80 and 96-120) to modulate alloreactivity was tested in Lewis (RT1.A1) responder animals. The DA peptide 56-80, but not peptide 96-120, induced delayed-type hypersensitivity (DTH). DTH was significantly reduced by oral feeding of peptide 56-80, P = 0.004. In addition, oral feeding of this peptide in combination with a short course of cyclosporin A (CsA) prolonged graft survival of 60% of heterotope transplanted DA cardiac allografts in Lewis recipient rats. Long-term survivors developed low levels of allo-antibodies against donor tissue as compared to rejecting animals and increased levels of interleukin-4 (IL-4) within the allograft. Similarly, IL-4-secreting splenocytes were identified by flow cytometry in these animals, indicating a Th2-type cytokine pattern. However, graft survival was particularly limited to cardiac allografts because donor-type skin grafts were acutely rejected in tolerant animals. It is interesting that residue alignment of peptide 56-80 to the motif of the RT1.A1 molecule showed a preferred class I motif within this sequence, suggesting indirect presentation of this peptide to recipient T cells. Thus, peptide 56-80 appears to represent a dominant epitope that can be exploited for establishing tolerance in this transplantation strain combination.

Synthetic HLA-A2 derived peptides are recognized and presented in renal graft recipients.

Indirect presentation of allogeneic MHC antigen is an important pathway by which allografts are rejected and tolerance maintained by regulatory CD4(+) T cells. In this study HLA-A2 derived synthetic peptides were used to determine whether T cells of non-HLA-A2 renal graft recipients, which had been HLA-A2 mismatched to their organ donors, recognize some of the HLA-A2-derived peptides. Among the HLA-A2 mismatched patients, 60% recognized residues 56--69, 65--79, and 75--89. Peripheral blood lymphocytes derived from healthy individuals showed low reactivity towards allopeptides, indicating that sensitization towards HLA-A2 induced response towards HLA-A2 derived peptides. The response to the peptides was blocked by antibodies to HLA-DR, -DQ, and CD4. Depletion of antigen presenting cells abrogated response towards the allopeptides, confirming that the observed proliferation was mediated by the indirect pathway. Interestingly, although none of the HLA-A2 mismatched patients had any signs for either acute or chronic rejection, considerable response to allo-derived HLA-A2 was observed.

How does the major histocompatibility complex influence behavior?

The major histocompatibility complex (MHC) encodes highly polymorphic cell surface glycoproteins that form the basis of immunological individuality. It has been postulated that MHC besides its role in immune defence also determines behavior through formation of specific odor cues. In addition several studies have implicated MHC disassortative odor and mating preferences in mice, rats, and humans. Further leading studies have suggested that mice and humans prefer to mate with MHC dissimilar individuals. Although the latter remains controversial, MHC dependent mating preferences provide a potentially important selective factor driving the polymorphisms of the MHC genes. Here we review some of the available data and hypotheses on the influence of the MHC on behavior and discuss its molecular and chemical basis.

The role of natural killer cell mediated caspases activation in a graft-versus-host disease model of semiallogeneic small bowel transplantation.

In the clinical setting of solid organ transplantation the event of graft-versus-host disease (GvHD) is rare and not easily predictable. Even intestinal and multivisceral transplants harbour a huge amount of immunocompetent cells and they do not exert a significantly higher risk to trigger serious GvH reactions. A series of our own experimental studies has been conducted to delineate the role of the host's innate immune system in the context of GvHD following parental to F1 hybrid semiallogeneic small bowel transplantation (SBTx). These results clearly demonstrated the immunological significance of the recipient's status of natural killer (NK) cell activity to counteract donor-derived lymphocytes and related cytotoxicity. NK cells and macrophages are both endowed with Ca2+-dependent receptors of the C-type lectin family which interact with a diversity of high-affinity oligosaccharide ligands expressed on potential target cells. One of these proteins of the C-type lectin family, termed NKR-P1, has been cloned and sequenced. Activation of NKR-P1 stimulates activation-induced cell death (AICD) of bound target cells. As intracellular mediators of apoptotic cell death a new family of cysteine proteases, the caspases, have been defined. These proteases appear to be involved in the initiation of apoptosis in response to a number of stimuli. This study was conducted to investigate the impact on the activity level of host NK cells and on target cell lysis of donor-derived lymphocytes after heterotopic semiallogeneic (parental [DA;RT1.aaav1] to F1 [DA x LEW;RT1.(1)]) small bowel transplantation using a rat model. The host's NK activity was either specifically activated (by use of polyinosinic:polycytodilic acid [poly-I:C]) or suppressed (by depletion of host NK cells after intraperitoneal administration of the NKR-P1 monoclonal antibody 3.2.3). The impact of NK-activity on the incidence of GvHD and the recipients' survival was correlated with the frequency of apoptotic cell death and related expression of caspases 1 (ICE) and 3 (CPP-32) from donor and recipient small bowel tissues. Our results confirm that depletion of NK cells in F1 host rats prior to parental small bowel transplantation significantly decreased the mean survival to 11.4 days versus 16.2 days of nondepleted F1 rats (p < 0.01). Conversely, activation of host NK activity with poly-I:C abrogated GvHD in all 12 recipient rats and led to long-term survival in seven of 12 animals. Long-term survival was associated with a substantially higher frequency of apoptotic cell death in donor and recipient small bowel and mesenteric lymph nodes. On day 10 after transplantation, Northern blot analysis of these tissues revealed profound upregulation of mRNA-specific gene expression for caspase 1 and 3 as potential mediators of programmed cell death of activated lymphocytes. Our findings emphasize the importance of NK cell associated innate immunity in the context of GvHD after semiallogeneic small bowel transplantation. Killing of alloreactive donor-derived lymphocytes was mediated by the NKR-P1 protein on NK cells and could be suppressed after pretreatment of F1 hosts with anti-NKR-P1 mAb 3.2.3. Moreover, NK cell-mediated apoptosis induced upregulation of caspases 1 and 3, thus elucidating the involvement of this protein in the context of caspase-mediated target cell killing.

Different in vivo tolerogenicity of MHC class I peptides.

The efficacy of MHC class I-derived peptides to induce tolerance was tested in a cardiac transplantation model. Two 25-mer peptides from the polymorphic region of the DA class I molecule (RT1.Aa) were synthesized by F-moc chemistry and injected intrathymically or intraperitoneally into LEW (RT1.1) responder animals. Intrathymic treatment of the recipient animals with peptide 1 (residues 56-80) accompanied by intraperitoneal treatment with peptide 4 (residues 96-120) led to indefinite survival of allogeneic DA cardiac allografts (n = 7; > 100 days). The tolerogenicity of both peptides differed according to the site of inoculation, as donor-specific tolerance was only observed after administration of peptide 1 into the thymus and injection of peptide 2 into the abdominal cavity of LEW recipients, but not vice versa. Donor-specific tolerance was confirmed in vivo by grafting of full-thickness skin and in vitro by appropriate proliferation and cytotoxicity assays using donor and third-party rats. Donor-specific tolerance was associated with up-regulation of interleukin-4, transforming growth factor beta, and interleukin-10 gene expression within cardiac allografts, thus suggesting intrathymic clonal deletion and external suppression with expansion of T-helper 2-type lymphocytes as the underlying mechanisms of tolerance induction.

Soluble HLA class I molecules: biological significance and clinical implications.

Soluble class I human leukocyte antigens (sHLAs) have been detected in serum, sweat, lymphatic fluid, urine and cerebrospinal fluid. Their biological function has, however, remained a puzzle. The physiological concentration of sHLA varies more than tenfold depending on the phenotype of the individual, and is significantly upregulated in various diseases and during inflammation. This suggests that sHLAs might serve as a marker of pathological changes. Recent experiments have shown that, in vitro, sHLAs can modulate T-cell reactivity and induce cell-activated apoptosis, implicating sHLAs in the induction and maintenance of peripheral tolerance. Therefore, sHLAs have the therapeutic potential to induce tolerance to transplants.