No augmentation of indoleamine 2,3-dioxygenase (IDO) activity through belatacept treatment in liver transplant recipients.

Belatacept is a second-generation cytotoxic T lymphocyte antigen (CTLA)-4 immunoglobulin (Ig) fusion protein approved for immunosuppression in renal transplant recipients. It was designed intentionally to interrupt co-stimulation via CD28 by binding to its ligands B7·1 and B7·2. Experimental evidence suggests a potential additional mechanism for CTLA-4 Ig compounds through binding to B7 molecules expressed on antigen-presenting cells (APCs) and up-regulation of indoleamine 2,3-dioxygenase (IDO), an immunomodulating enzyme that catalyzes the degradation of tryptophan to kynurenine and that down-regulates T cell immunity. So far it remains unknown whether belatacept up-regulates IDO in transplant recipients. We therefore investigated whether belatacept therapy enhances IDO activity in liver transplant recipients enrolled in a multi-centre, investigator-initiated substudy of the Phase II trial of belatacept in liver transplantation (IM103-045). Tryptophan and kynurenine serum levels were measured during the first 6 weeks post-transplant in liver transplant patients randomized to receive either belatacept or tacrolimus-based immunosuppression. There was no significant difference in IDO activity, as indicated by the kynurenine/tryptophan ratio, between belatacept and tacrolimus-treated patients in per-protocol and in intent-to-treat analyses. Moreover, no evidence was found that belatacept affects IDO in human dendritic cells (DC) in vitro. These data provide evidence that belatacept is not associated with detectable IDO induction in the clinical transplant setting compared to tacrolimus-treated patients.

Regulatory T Cells Promote Natural Killer Cell Education in Mixed Chimeras.

Therapeutic administration of regulatory T cells (Tregs) leads to engraftment of conventional doses of allogeneic bone marrow (BM) in nonirradiated recipient mice conditioned with costimulation blockade and mammalian target of rapamycin inhibition. The mode of action responsible for this Treg effect is poorly understood but may encompass the control of costimulation blockade-resistant natural killer (NK) cells. We show that transient NK cell depletion at the time of BM transplantation led to BM engraftment and persistent chimerism without Treg transfer but failed to induce skin graft tolerance. In contrast, the permanent absence of anti-donor NK reactivity in mice grafted with F1 BM was associated with both chimerism and tolerance comparable to Treg therapy, implying that NK cell tolerization is a critical mechanism of Treg therapy. Indeed, NK cells of Treg-treated BM recipients reshaped their receptor repertoire in the presence of donor MHC in a manner suggesting attenuated donor reactivity. These results indicate that adoptively transferred Tregs prevent BM rejection, at least in part, by suppressing NK cells and promote tolerance by regulating the appearance of NK cells expressing activating receptors to donor class I MHC.

Murine models of transplantation tolerance through mixed chimerism: advances and roadblocks.

Organ transplantation is the treatment of choice for patients with end-stage organ failure, but chronic immunosuppression is taking its toll in terms of morbidity and poor efficacy in preventing late graft loss. Therefore, a drug-free state would be desirable where the recipient permanently accepts a donor organ while remaining otherwise fully immunologically competent. Mouse studies unveiled mixed chimerism as an effective approach to induce such donor-specific tolerance deliberately and laid the foundation for a series of clinical pilot trials. Nevertheless, its widespread clinical implementation is currently prevented by cytotoxic conditioning and limited efficacy. Therefore, the use of mouse studies remains an indispensable tool for the development of novel concepts with potential for translation and for the delineation of underlying tolerance mechanisms. Recent innovations developed in mice include the use of pro-apoptotic drugs or regulatory T cell (Treg ) transfer for promoting bone marrow engraftment in the absence of myelosuppression and new insight gained in the role of innate immunity and the interplay between deletion and regulation in maintaining tolerance in chimeras. Here, we review these and other recent advances in murine studies inducing transplantation tolerance through mixed chimerism and discuss both the advances and roadblocks of this approach.

The Immunosuppressive Effect of CTLA4 Immunoglobulin Is Dependent on Regulatory T Cells at Low But Not High Doses.

B7.1/2-targeted costimulation blockade (CTLA4 immunoglobulin [CTLA4-Ig]) is available for immunosuppression after kidney transplantation, but its potentially detrimental impact on regulatory T cells (Tregs) is of concern. We investigated the effects of CTLA4-Ig monotherapy in a fully mismatched heart transplant model (BALB/c onto C57BL/6). CTLA4-Ig was injected chronically (on days 0, 4, 14, and 28 and every 4 weeks thereafter) in dosing regimens paralleling clinical use, shown per mouse: low dose (LD), 0.25 mg (≈10 mg/kg body weight); high dose (HD), 1.25 mg (≈50 mg/kg body weight); and very high dose (VHD), 6.25 mg (≈250 mg/kg body weight). Chronic CTLA4-Ig therapy showed dose-dependent efficacy, with the LD regimen prolonging graft survival and with the HD and VHD regimens leading to >95% long-term graft survival and preserved histology. CTLA4-Ig's effect was immunosuppressive rather than tolerogenic because treatment cessation after ≈3 mo led to rejection. FoxP3-positive Tregs were reduced in naïve mice to a similar degree, independent of the CTLA4-Ig dose, but recovered to normal values in heart recipients under chronic CTLA4-Ig therapy. Treg depletion (anti-CD25) resulted in an impaired outcome under LD therapy but had no detectable effect under HD therapy. Consequently, the immunosuppressive effect of partially effective LD CTLA4-Ig therapy is impaired when Tregs are removed, whereas CTLA4-Ig monotherapy at higher doses effectively maintains graft survival independent of Tregs.

Donor CD4 T cells trigger costimulation blockade-resistant donor bone marrow rejection through bystander activation requiring IL-6.

Bone marrow (BM) transplantation under costimulation blockade induces chimerism and tolerance. Cotransplantation of donor T cells (contained in substantial numbers in mobilized peripheral blood stem cells and donor lymphocyte infusions) together with donor BM paradoxically triggers rejection of donor BM through undefined mechanisms. Here, nonmyeloablatively irradiated C57BL/6 recipients simultaneously received donor BM (BALB/c) and donor T cells under costimulation blockade (anti-CD154 and CTLA4Ig). Donor CD4, but not CD8 cells, triggered natural killer-independent donor BM rejection which was associated with increased production of IL-6, interferon gamma (IFN-γ) and IL-17A. BM rejection was prevented through neutralization of IL-6, but not of IFN-γ or IL-17A. IL-6 counteracted the antiproliferative effect of anti-CD154 in vitro. Rapamycin and anti-lymphocyte function-associated antigen 1 negated this effect of IL-6 in vitro and prevented BM rejection in vivo. Simultaneous cotransplantation of (BALB/cxB6)F1, recipient or irradiated donor CD4 cells, or late transfer of donor CD4 cells did not lead to BM rejection, whereas cotransplantation of third party CD4 cells did. Transferred donor CD4 cells became activated, rapidly underwent apoptosis and triggered activation and proliferation of recipient T cells. Collectively, these results provide evidence that donor T cells recognizing the recipient as allogeneic lead to the release of IL-6, which abolishes the effect of anti-CD154, triggering donor BM rejection through bystander activation.

Use of X-ray tomography to map crystalline and amorphous phases in frozen biomaterials.

The outcome of both cryopreservation and cryosurgical freezing applications is influenced by the concentration and type of the cryoprotective agent (CPA) or the cryodestructive agent (i.e., the chemical adjuvants referred to here as CDA) added prior to freezing. It also depends on the amount and type of crystalline, amorphous and/or eutectic phases formed during freezing which can differentially affect viability. This work describes the use of X-ray computer tomography (CT) for non-invasive, indirect determination of the phase, solute concentration and temperature within biomaterials (CPA, CDA loaded solutions and tissues) by X-ray attenuation before and after freezing. Specifically, this work focuses on establishing the feasibility of CT (100-420 kV acceleration voltage) to accurately measure the concentration of glycerol or salt as model CPA and CDAs in unfrozen solutions and tissues at 20 degrees C, or the phase in frozen solutions and tissue systems at -78.5 and -196 degrees C. The solutions are composed of water with physiological concentrations of NaCl (0.88% wt/wt) and DMEM (Dulbecco's Modified Eagle's Medium) with added glycerol (0-8 M). The tissue system is chosen as 3 mm thick porcine liver slices as well as 2 cm diameter cores which were either imaged fresh (3-4 h cold ischemia) or after loading with DMEM based glycerol solutions (0-8 M) for times ranging from hours to 7 days at 4 degrees C. The X-ray attenuation is reported in Hounsfield units (HU), a clinical measurement which normalizes X-ray attenuation values by the difference between those of water and air. NaCl solutions from 0 to 23.3% wt/wt (i.e. water to eutectic concentration) were found to linearly correspond to HU in a range from 0 to 155. At -196 degrees C the variation was from -80 to 95 HU while at -78.5 degrees C all readings were roughly 10 HU lower. At 20 degrees C NaCl and DMEM solutions with 0-8 M glycerol loading show a linear variation from 0 to 145 HU. After freezing to -78.5 degrees C the variation of the NaCl and DMEM solutions is more than twice as large between -90 and +190 HU and was distinctly non-linear above 6 M. After freezing to -196 degrees C the variation of the NaCl and DMEM solutions increased even further to -80 to +225 HU and was distinctly non-linear above 4 M, which after modeling the phase change and crystallization process is shown to correlate with an amorphous phase. In all tissue systems the HU readings were similar to solutions but higher by roughly 30 HU, as well as showing some deviations at 0 M after storage, probably due to tissue swelling. The standard deviations in all measurements were roughly 5 HU or below in all samples. In addition, two practical examples for CT use were demonstrated including: (1) glycerol loading and freezing of tissue cores and, (2) a mock cryosurgical procedure. In the loading experiment CT was able to measure the permeation of the glycerol into the sample at 20 degrees C, as well as the evolution of distinct amorphous vs. crystalline phases after freezing to -196 degrees C. In the mock cryosurgery example, the iceball edge was clearly visualized, and attempts to determine the temperature within the iceball are discussed. An added benefit of this work is that the density of these frozen samples, an essential property in measurement and modeling of thermal processes, was obtained in comparison to ice.

The region coding for the helix termination motif and the adjacent intron 6 of the human type I hair keratin gene hHa2 contains three natural, closely spaced polymorphic sites.

Mutations in distinct sites of epidermal keratins, in particular in the helix initiation and termination regions, cause human genodermatoses due to faulty intermediate filament formation. Extension of this observation to human hereditary hair and nail diseases includes population analyses of human hair keratin genes for natural sequence variations in the corresponding sites. Here we report on a large-scale genotyping of the short helix termination region (HTR) of the human type I cortical hair keratins hHa1, a3-I, and a3-II, and the cuticular hair keratin hHa2. We describe two polymorphic loci, P1 and P2, exclusively in the cuticular hHa2 gene, both creating dimorphic protein variants. P1 is due to a C to T mutation in a CpG element leading to a threonine to methionine substitution; P2 concerns a serine codon AGT that also occurs as an asparagine coding variant AAC. A third polymorphism, P3, is linked with a C to T point mutation located at the very beginning of intron 6. The three polymorphic sites are clustered in a 39-nucleotide sequence of the hHa2 gene. Both allelic frequency calculations in individuals of different races and pedigree studies indicate that the two-allelic hHa2 variants resulting from P1 and P2 occur ubiquitously in a ratio of about 1:1 (P1) and 2:1 (P2) respectively in our survey, and are clearly inherited as Mendelian traits. A genotype carrying both mutations simultaneously on one allele could not be detected in our sampling, and there was no association of a distinct allelic hHa2 variant with the known ethnic form variations of hairs. Sequence comparisons of the HTR of hHa2 with those of other type I hair keratins including the hHa2-ortholog from chimpanzee provide evidence that the P1- and P2-linked mutations must have occurred very early in human evolution and that the two P2-associated codon variants may be the result of two independent point mutations in an ancestral AGC serine codon. These data describe natural polymorphisms in the HTR of a member of the keratin multigene family.

[Communication and integration of medical documents for the teleconference use. An outcome of the BERMED project]. / Kommunikation und Integration medizinischer Dokumente für Telekonferenz-Anwendungen: Ein Ergebnis aus dem Projekt BERMED.

The collaboration between physicians is supported in the BERMED project by implementing the remote access to distributed patient data and the realisation of computer-based medical conferencing. This requires the integration of the multimedia data in form of a meta-patient record for our medical application systems. These applications are supported by a distributed information management promoting access to different information systems, imaging modalities and digital archival storage systems. The features of image processing are concerned with quantification, segmentation and 3-D-visualisation to obtain additional information. This paper gives an overview of the actual state of the teleconferencing system in radiology.

Denitrosation of N-nitrosomorpholine by liver microsomes; possible role of cytochrome P-450.

Liver microsomes from male mice and rats were incubated with N-nitrosomorpholine (MoNA) and an NADPH-regenerating system. The formation of nitrite was measured after induction or inhibition of the microsomal monooxygenase system. Pretreatment of the animals with phenobarbital (PB) enhanced nitrite formation by about 200%, while 3-methylcholanthrene (3-MC)-induction showed no exceptional effects. Various specific inhibitors of the monooxygenase function including carbon monoxide decreased nitrite formation. In conjunction with results obtained by spectra studies it is suggested that N-nitrosomorpholine is denitrosated by a reduction process in which cytochrome (cyt.) P-450 seems to be involved. Nitricoxide formed is partly converted to nitrite under these conditions.

Binding of nitrosamines to cytochrome P-450 of liver microsomes.

The interactions of 5 carcinogenic and 1 non-carcinogenic nitrosamines with hepatic microsomal cytochrome (cyt.) P-450 were investigated, using both optical difference and electron paramagnetic resonance (EPR) spectroscopic methods. Liver microsomes from phenobarbital (PB)-pretreated mice and 3-methylcholanthrene (3-MC)-pretreated rats were used, in order to have an increased specific content of cyt. P-450 and cyt. P-448 respectively. The optical and EPR spectral data obtained in the oxidised state suggest that nitrosamines are able to bind both as substrates and as ligands to the hemoprotein cyt. P-450, depending on the concentration of nitrosamine, its chemical identity and the cytochrome species present. After reduction with dithionite or NADPH in the optical difference spectrum a Soret band developed between 444 and 453 nm to an extent, which is dependent on the particular nitrosamine present. This initial nitrosamine-induced spectrum might represent a ferrous nitric oxide (NO)-cyt. P-450 complex. It appears unstable and is converted kinetically into a spectrum lacking a Soret band, but with a predominant absorbance minimum at about 425 nm. A visible band is located at 585 nm. In the EPR spectrum a sharp 3-line signal around g = 2.01 appears concomitantly. Both spectral parameters are typical of a NO-cyt. P-420 complex. These results, in conjunction with metabolic studies, indicate that nitrosamines are denitrosated by a reductive process in which cyt. P-450 appears to be involved. The resulting NO-cyt. P-450 complex denatures to a NO-cyt. P-420 complex when the dioxygen level is not sufficiently high to complete successfully.