Immunomodulation by a novel, dissociated Vitamin D analogue.

The biologically active metabolite of vitamin D3, 1alpha,25-dihydroxyvitamin D3, has potent immunomodulatory activity; however, its clinical use is limited because of its hypercalcaemic activity in anti-inflammatory active doses. Here, we present ZK203278, a novel, structurally different vitamin D3 analogue with profound immunomodulatory activities. It potently inhibits lymphocyte proliferation in the mixed lymphocyte reaction, and release of cytokines that are central in inflammation, such as TNFalpha and IL-12 in the low nanomolar range. Similarly, expression of cell-surface molecules involved in cell adhesion and antigen presentation, e.g. to T cells, is down-regulated on human monocytes by low nanomolar concentrations of ZK203278. Potent anti-inflammatory activity has been demonstrated also in vivo in rodent disease models. ZK203278 inhibited allergic contact dermatitis in rodents after oral administration in doses approximately two orders of magnitude below the hypercalcaemic threshold dose. Moreover, ZK203278 significantly prolonged skin allograft survival in rats in well-tolerated doses. Altogether ZK203278, in contrast to 1alpha,25-dihydroxyvitamin D3, exerts considerable immunomodulatory activity at non-hypercalcaemic dosages and may have therapeutic potential for immune disorders or transplant rejection.

Short-term anti-CD4 plus anti-TNF-alpha receptor treatment in allogeneic small bowel transplantation results in long-term survival.

BACKGROUND: Despite improved immunosuppression, intestinal transplantation is still complicated by severe rejection episodes. To further improve immunosuppressive concepts, we evaluated an anti-CD4 antibody and an anti-tumor necrosis factor (TNF)-alpha monoclonal antibody for their immunosuppressive efficacy in the standard rat model of intestinal transplantation. METHODS: Intestinal transplantation was performed in the DA to Lewis combination, and recipients were treated perioperatively with either the anti-CD4 antibody RIB5/2 (day -1, 0, postoperative days 1, 2, 4, 7, 10, 14, 17, and 21), the anti-TNF antibody etanercept (60 min before reperfusion, postoperative days 3, 6, and 9) or a combination of both. Survival, histology and expression of immunologic mediator genes on days 3 and 4 after transplantation were investigated. RESULTS: Treatment with anti-CD4 antibody alone (19.71+/-5.94) and the antibody combination (171.58+/-122.76) prolonged survival. The chemokine MIP-1alpha was significantly decreased in both anti-CD4 antibody treatment groups, possibly indicating an additional effect of the TNF-alpha blockade on the immune modulation by RIB5/2. CONCLUSIONS: Our study demonstrated long-term graft survival in short-term treatment with a combination of an anti-CD4 antibody and a TNF-alpha antibody in more than 50% of the recipients of intestinal grafts. Such a combined approach could also be useful in clinical small bowel transplantation.

Allo-specific T-cells encoding for viral IL-10 exert strong immunomodulatory effects in vitro but fail to prevent graft rejection.

Recently, we demonstrated the capacity of allo-specific gene-engineered T lymphocytes as transport vehicle for therapeutic transgenes into allografts. In this study, the influence of viral IL-10 as therapeutic transgene was addressed. Lewis rat T-cell lines specific for DA rat alloantigens were engineered to express vIL-10 by using a retroviral gene expression system. Like T regulatory 1 cells, vIL-10 transgenic T lymphocytes express the phenotype CD4(+)25(+) and secrete, in addition to vIL-10, rat IL-10 and IFN-gamma but no IL-4. First, the capacity of vIL-10 transgenic T-cell lines to modulate alloantigen-specific immune responses was evaluated in vitro. In comparison to control MLR with no transgenic cells or equal numbers of control T(EGFP)-lymphocytes, the proliferation as well as production of IFN-gamma by naive responder cells were significantly diminished. Despite this regulatory capacity in vitro, T(vIL-10)-lymphocytes were not able, either alone or in combination with suboptimal doses of Cyclosporine A, to prolong the survival of either DA rat cardiac or renal allografts in Lewis rat recipients. These data demonstrate that intra-graft IL-10 over-expression is not sufficient to prolong allograft survival in a high-responder strain combination and that the regulatory capacity of T cells in vitro does not predict their in vivo efficiency.

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.

Aerobic endurance exercise improves executive functions in depressed patients.

BACKGROUND: Aerobic endurance exercise has been shown to improve higher cognitive functions such as executive control in healthy subjects. We tested the hypothesis that a 30-minute individually customized endurance exercise program has the potential to enhance executive functions in patients with major depressive disorder. METHOD: In a randomized within-subject study design, 24 patients with DSM-IV major depressive disorder and 10 healthy control subjects performed 30 minutes of aerobic endurance exercise at 2 different workload levels of 40% and 60% of their predetermined individual 4-mmol/L lactic acid exercise capacity. They were then tested with 4 standardized computerized neuropsychological paradigms measuring executive control functions: the task switch paradigm, flanker task, Stroop task, and GoNogo task. Performance was measured by reaction time. Data were gathered between fall 2000 and spring 2002. RESULTS: While there were no significant exercise-dependent alterations in reaction time in the control group, for depressive patients we observed a significant decrease in mean reaction time for the congruent Stroop task condition at the 60% energy level (p = .016), for the incongruent Stroop task condition at the 40% energy level (p = .02), and for the GoNogo task at both energy levels (40%, p = .025; 60%, p = .048). The exercise procedures had no significant effect on reaction time in the task switch paradigm or the flanker task. CONCLUSION: A single 30-minute aerobic endurance exercise program performed by depressed patients has positive effects on executive control processes that appear to be specifically subserved by the anterior cingulate.

Donor-specific renal, but not cardiac, allograft tolerance promotes engraftment of the normally rejected rat skin graft.

This study examined whether a heart or kidney graft could provide protection for the more resistant skin graft. Buffalo rat recipients were given a single dose of RIB 5/2 (non-depleting anti-CD4 mAb) plus i.v. Lewis splenocytes 21 days before being given Lewis heart or kidney grafts. Lewis skin was grafted either simultaneously with, or after, long-term (>50 days) Lewis heart or kidney allograft acceptance. Immune responsiveness was analyzed by in vitro mixed lymphocyte culture (MLC), cytotoxic T lymphocytes (CTLs), and limiting dilution analysis (LDA). While i.v. alloantigen plus RIB 5/2 resulted in long-term acceptance of heart and kidney, survival of skin grafts alone was not prolonged. However, simultaneous transplantation with kidney, but not heart, resulted in long-term skin graft acceptance, while skin grafts subsequently grafted to recipients tolerant to kidney or heart were not accepted. In vitro analysis revealed a down-regulation of proliferation, cytotoxicity, and precursor T-helper cells (pThs)/precursor cytotoxic T lymphocytes (pCTLs) in Buffalo recipients accepting Lewis kidney and skin allografts. While RIB 5/2 plus Lewis splenocytes do not prolong the survival of skin grafts, Lewis skin grafted simultaneously with a kidney, but not heart, is accepted indefinitely and provides donor-specific protection for a subsequent skin graft.

Inhibition of ischemia/reperfusion injury and chronic graft deterioration by a single-donor treatment with cobalt-protoporphyrin for the induction of heme oxygenase-1.

Today, the major problem in organ transplantation is not acute graft rejection but chronic graft deterioration. In addition to alloantigen-specific events, alloantigen independent factors like donor age, previous diseases, consequences of brain death, and perioperative events of ischemia/reperfusion injury have a major impact on long-term graft function. The induction of the stress protein heme oxygenase-1 (HO-1) protects cells from injury and apoptosis. Here, we tested the protective effects of HO-1 induction in a clinically relevant kidney transplant model. Induction of HO-1 expression following cobalt-protoporphyrin (CoPP) treatment in organ donors prolonged graft survival and long-term function remarkably following extended periods of ischemia. Positive effects were observed with both optimal and marginal grafts from old donor animals. Structural changes characteristic for chronic rejection, as well as graft infiltration by monocytes/macrophages and CD8+ T cells, were substantially reduced following HO-1 induction. Up-regulation of HO-1 expression before organ transplantation was also associated with reduced levels for tumor necrosis factor (TNF)-alpha mRNA, increased levels for interferon (IFN)-gamma, and bcl-x, and insignificant differences for CD25, interleukin (IL)-2, IL-4, IL-6, and IL-10 mRNA levels. The significant improvement of long-term graft function following induction of HO-1 expression in donor organs suggests that this strategy may be a novel clinical treatment option with particular relevance for transplantation of marginal organs.

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.

Improvements in gene therapy: averting the immune response to adenoviral vectors.

Gene therapy is an interesting approach for the correction of defective genes, the treatment of cancer and the introduction of immunomodulatory genes. Various techniques for gene transfer into cells or tissues have been developed within the last decade; these can be divided generally into viral and nonviral gene transfer systems. Nonviral techniques include the liposome- or gene gun-mediated introduction of therapeutic genes; however, the efficiency of gene transfer by these applications is still very low. In contrast, viruses have optimised their strategies for efficient infection of virtually any cell type in a mammalian organism. The genetic modification of genomes from different virus families (Adenoviridae, Retroviridae, Herpesviridae) led to the development of gene therapy vectors with a similar capacity to infect cells or tissues as that of wild type viruses. In contrast to wild type viruses, gene therapy vectors are engineered to transfer therapeutic genes into the target cells or tissues. In addition, they have lost their capacity for replication in target cells, because of the removal of essential genes, which allows replication only in specialised packaging cell lines engineered for the production of recombinant viruses. Despite considerable progress over the past decade in the generation of gene transfer systems with reduced immunogenic properties, the remaining immunogenicity of many gene therapy vectors is still the major hurdle, preventing their frequent application in clinical trials. Recombinant adenoviruses have been shown to be promising vectors for gene therapy, since they are able to transduce both quiescent and proliferating cells very efficiently. However, a major disadvantage of adenoviral vectors lies in the activation of both the innate and adaptive parts of the recipient's immune system when applied in vivo. The inflammatory responses induced by adenovirus particles can be very strong and can be fatal in patients treated with these adenoviral constructs. Therefore, many experiments have been performed in the effort to prevent these inflammatory responses mediated by adenoviral particles. The depletion of cell populations responsible for these inflammatory responses as well as the application of immunosuppressive drugs have been investigated. Moreover, the generation of less immunogenic adenoviral vectors by further genetic modification within the adenoviral genome has led to vectors with reduced immunogenic properties. Both strategies to reduce inflammatory responses against adenoviral particles are discussed in this review.

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).

Physical Exercise as an Adjunct Therapy in Sleep Apnea-An Open Trial.

BACKGROUND: The aim of this study was to determine in an open trial if physical exercise in sleep apnea patients is safe and/or influences respiratory disturbance index (RDI). METHODS: After being treated 3 months or more with nasal CPAP for moderate to severe sleep apnea syndrome, eleven patients (1 female, 10 male, mean age 52.2 years) began a six-month period of supervised physical exercise twice a week, 2 hours each time. Before and after this period a Polysomnography without CPAP was recorded, along with a bicycle exercise test with lactate profile, echocardiography, body-weight, and body-height measurement. RESULTS: No adverse effects or cardiopulmonary problems were observed. There was no significant change in body weight with physical training; no significant difference in either min SaO2 nor mean SaO2; and no significant improvement in fitness. No adverse cardiopulmonary effects or problems were observed. There was a decrease of the RDI from 32.8 to 23.6 (p < 0.05), without a significant change in the REM-sleep portion of total sleep time (TST), NREM sleep, or TST. CONCLUSIONS: A prescription for mild to moderate exercise is safe in the management of sleep apnea, and, even in the absence of a fitness improvement, there occurred a decrease in RDI without a change in sleep architecture.