Surveillance for prion disease in cervids, Germany.

An active survey on transmissible spongiform encephalopathies was performed from 2002 to 2005 on 4,255 roe deer, 1,445 red deer, and 1,604 fallow deer in Germany. All cervids tested negative. This survey has been the largest in European wildlife and provides no evidence of prion diseases in free-living German cervids.

FTY720 prevents anti-CD4 mAb-induced tolerance but cannot reverse established tolerance in a rat kidney transplantation model.

FTY720 is highly effective in various models of transplantation and autoimmunity. In order to find drugs that act synergistically with a tolerance-inducing nondepleting anti-CD4 mAb we studied this combination in a strong DA to LEW kidney transplantation model. Rats were treated with 0.3 mg/kg of FTY720 for 14 days and anti-CD4 mAb RIB5/2, alone or in combination. After kidney transplantation serum creatinine and blood lymphocyte counts were monitored. Immunohistology, ELISPOT and TaqMan trade mark -PCR analysis of biopsies were performed. Short-term application of RIB5/2 but not FTY720 induced long-term survival of kidney transplants. Moreover, the combination of FTY720 + RIB5/2 prevented tolerance induction. In the combination group serum creatinine levels increased 1 week after cessation of therapy and all rats died from uremia within 72 days. Intragraft immunohistology, ELISPOT and real-time RT-PCR analysis at day 21 demonstrated an enhanced T-cell infiltration and activation but a diminished up-regulation of protective genes in the grafts from recipients receiving the combination therapy. In contrast, delayed application of FTY720 to RIB5/2-treated rats did not interact with RIB5/2-induced tolerance. In summary, FTY720 is powerful in preventing intragraft infiltration by naive T cells but this might also affect the early development of graft-protecting regulatory T cells and tolerance induction.

IFN-gamma regulation in anti-CD4 antibody-induced T cell unresponsiveness.

The anti-rat CD4 mAb RIB5/2 is very potent in inducing allospecific tolerance in vivo. It is interesting that the unresponsiveness is breakable by exogenous IL-2 applied during the induction phase of tolerance. The molecular mechanisms underlying anti-CD4 antibody-mediated inhibition of allospecific T cell activation and how this is antagonized by exogenous IL-2 were investigated. Anti-CD4 treatment, in vivo and in vitro, completely abrogated IL-2 production by alloreactive T cells. In contrast, anti-CD4-treated alloactivated T cells showed similar IFN-gamma mRNA expression as untreated alloactivated T cells but did not secrete any protein. Thus, the anti-CD4 antibody cannot prevent IFN-gamma mRNA expression but is interfering with posttranscriptional mechanisms that control IFN-gamma production during alloactivation of T cells. Addition of IL-2 but not IL-15 to anti-CD4-treated alloactivated T cells restored IFN-gamma protein production without leading to enhanced IFN-gamma mRNA expression. Further investigations revealed a diminished activation of translation initiation factor eIF2alpha in anti-CD4-treated T cells, which was restored by exogenous IL-2. As activated eIF2alpha is essential for the translation of IFN-gamma mRNA, the results may explain the reversibility of anti-CD4-induced unresponsiveness by exogenous IL-2. Furthermore, these results not only shed further light onto the molecular mechanisms of tolerance induction but also reveal the possible weaknesses of anti-CD4 antibody-induced unresponsiveness.

Bag-1 up-regulation in anti-CD4 mAb-treated allo-activated T cell confers resistance to activation-induced cell death (AICD).

The non-depleting anti-CD4 monoclonal antibody (mAb) RIB5/2 is a powerful inducer of tolerance to major histocompatibility complex (MHC)-incompatible allografts in rat recipients. The unresponsiveness induced is characterized by the persistence (over 300 days) of donor-reactive regulatory T cells within the graft. We applied differential-display reverse-transcription polymerase chain reaction (RT-PCR) to identify differences at the mRNA level between graft-infiltrating cells of anti-CD4 mAb-treated and non-treated control rats at day 5 after kidney transplantation. A 550-bp DNA fragment appearing only in anti-CD4 mAb-treated rats is identical with the anti-apoptotic protein Bag-1. A further investigation of Bag-1 expression during mixed lymphocyte reactions (MLR) revealed a three-four-fold up-regulation of Bag-1 mRNA expression in anti-CD4 mAb-treated allogeneic cultures. Bag-1 up-regulation is associated with higher protection against apoptosis of anti-CD4 mAb-treated cultures. Application of antisense oligonucleotides specific for Bag-1 leads to both a reduction in Bag-1 expression and sensibility against apoptosis. Thus, the expression of Bag-1 in anti-CD4 mAb-treated alloreactive T cells conferred resistance against apoptosis, which may contribute to the long-term survival of tolerance-mediating T cells in vivo.

Bag-1 up-regulation in anti-CD4 mAb treated allo-activated T cells confers resistance to apoptosis.

The non-depleting anti-CD4 mAb RIB5/2 is a powerful inducer of tolerance to MHC-incompatible renal and heart allografts in rat recipients. In vitro the mAb blocks the proliferation and cytokine production of alloreactive T cells. To learn more about the mechanism of anti-CD4-mediated suppression, we applied differential display reverse transcription-PCR to identify differences at mRNA level between T cells stimulated by alloantigen in the presence or absence of anti-CD4 mAb. A sequence alignment of a 550-bp DNA fragment appearing only in anti-CD4 mAb-treated cells resulted in at least 95% homology to a mouse cDNA encoding for the anti-apoptotic protein Bag-1. Further investigation of Bag-1 expression during mixed lymphocyte reactions revealed a three- to fourfold up-regulation of Bag-1 mRNA expression in anti-CD4 mAb-treated allogeneic cultures which was confirmed at protein level. Bag-1 up-regulation was associated with an increase resistance to apoptosis of T cells from anti-CD4 mAb-treated cultures. Application of antisense oligonucleotides specific for Bag-1 reduced Bag-1 protein expression and restored susceptibility to apoptosis. In addition, up-regulation of Bag-1 mRNA could also be detected in graft-infiltrating T cells from anti-CD4 mAb-treated rats in vivo. Thus, the expression of Bag-1 in a subset of anti-CD4 mAb-treated alloreactive T cells conferred resistance against apoptosis, potentially contributing to the long-term survival of these cells.

Antigen-dependent transgene expression in kidney transplantation: a novel approach using gene-engineered T lymphocytes.

So far, gene therapy in transplantation mainly focuses on the expression of therapeutic proteins in the graft itself. Insufficient transfection and inflammatory responses that are due to the immunogenicity of multiple vector systems are often limiting factors in these approaches. The potential of genetically modified T lymphocytes was investigated as a delivery system for therapeutic transgenes to transplanted organs as a way to circumvent immunogenicity and efficiency problems in a rat transplant model. Gene-engineering of alloantigen-specific rat T cell lines was performed by using a Moloney murine leukemia virus (MoMuLV)-based enhanced green fluorescence protein (EGFP) encoding retroviral transduction system. The ex vivo gene-modified lymphocytes were adoptively transferred into syngeneic rats carrying allogeneic, syngeneic, or third party kidneys. Homing behavior, activation level, and transgene expression of the adoptively given cells were monitored. The T(EGFP) lymphocytes infiltrated the transplanted kidneys in an antigen-specific manner. High numbers of alloantigen-specific T lymphocytes accumulated exclusively in allografts but not in syngeneic or third party grafts. Flow cytometric analysis revealed that only T(EGFP) lymphocytes found in allografts had an activated phenotype that resulted in higher transgene expression. Alloantigen-specific homing, activation, and transgene expression are important prerequisites for the guarantee of localized delivery and expression of transgenic proteins by gene-engineered T lymphocytes. Antigen-specific gene-targeting strategies using ex vivo modified T lymphocytes with donor specificity are a novel approach to the delivery of therapeutic transgenes in transplantation.

Fumaric acid esters are potent immunosuppressants: inhibition of acute and chronic rejection in rat kidney transplantation models by methyl hydrogen fumarate.

The effectiveness and safety of fumaric acid esters (FAEs) for the treatment of psoriasis has been demonstrated. Their mode of action, however, is poorly understood. To determine the immunomodulatory potential of calcium methyl hydrogen fumarate (CaMHF) in transplant models, we tested the compound in rat transplantation models of acute rejection (AR) and chronic rejection (CR). Orthotopic kidney transplantation was combined with contralateral nephrectomy using the strain combinations WF to BDIX (AR) and F344 to LEW (CR). Recipients were treated orally prophylactically (day -28 to day +28, AR and CR model) or therapeutically (day +30 to day +60, CR model). CaMHF significantly prolonged the time to onset of AR in the WF/BDIX model. The half-lives of the grafts were 14 days in the CaMHF group, 7 days in the placebo-treated control group and 9 days in the untreated control group ( P<0.01). Three of ten CaMHF-treated rats showed permanent graft acceptance (defined as survival for >100 days) resulting in a mean survival time of >42.3+/-41.0 ( P<0.01) in comparison with >28.3+/-38.3 days in the placebo-treated group and 9.4+/-2.6 days in the untreated control group. In the F344/LEW model of chronic graft injury, only prophylactic CaMHF treatment significantly inhibited the development of CR ( P=0.001; mean survival times >28.4+/-2.0 weeks, >23.9+/-6.0 weeks and >21.1+/-5.4 weeks in the prophylactic CaMHF, therapeutic CaMHF, and placebo group, respectively). By 30 weeks six of ten prophylactically treated animals were still alive, but only three of nine and one of ten were alive in the therapeutically treated and placebo-treated groups, respectively ( P<0.05). These findings indicate that CaMHF treatment effectively inhibits AR and CR, and demonstrates marked in vivo immunomodulatory efficacy. Thus, FAEs should be considered as drugs showing considerable immunosuppressive efficacy. This might have implications with regard to safety issues (e.g. immune monitoring) and novel potentially suitable indications (e.g. transplantation and other immune diseases).