Transplantation tolerance: the big picture. Where do we stand, where should we go?

A major goal in organ transplantation has been to safely exploit the natural processes of immune tolerance in order to minimize the dose and duration of drug immunosuppression. In this commentary, I argue that we can learn from how tumours avoid rejection, to evolve a three-stage tolerance-inducing strategy for transplanted tissues.

Smoking during Pregnancy Is a Risk Factor for Executive Function Deficits in Preschool-aged Children.

Introduction: Maternal nicotine use during pregnancy has a negative impact on the child. Numerous studies have demonstrated an association between smoking during pregnancy and psychological deficits. This study looks at deficits in executive functioning in preschool-aged children. Methods: The executive functioning of preschool children was assessed by asking parents to complete the parental form of the Behavior Rating Inventory of Executive Functions - Preschool Version (BRIEF-P, German version). The results for preschool children whose mothers had smoked during pregnancy (n = 71) were compared with those of a control group. In a subsample, parental assessments of children of smokers (n = 42) and non-smokers (n = 27) were complemented by the teacher form of the BRIEF-P (German version), which allowed inter-rater agreement (parents vs. preschool teachers) to be assessed. Results: An increased incidence of executive function deficits was noted in the children of smokers, based on parental assessment. Clinically relevant deficits were particularly evident with regard to inhibition, with inhibitory deficits in children of smokers found to be almost four times higher than in the control group (p = 0.006). Inhibitory deficits were reported both by parents and by preschool teachers. Discussion: The increased percentage of executive function deficits described here, particularly the increased inhibitory deficits, confirms the current state of research on smoking during pregnancy. Poor inhibition or impulse control is a key symptom of ADHD.

Guiding postablative lymphocyte reconstitution as a route toward transplantation tolerance.

Anti-lymphocyte-depleting antibodies have increasingly been utilized in the clinic as induction therapy aiming to improve transplantation outcomes by reducing the need for long-term immunosuppression. However, maintenance immunosuppression is still required as lymphocyte reconstitution through homeostatic proliferation, partially driven by IL-7, continues to replenish tolerance-refractory immune cells capable of rejection. In murine models of MHC mismatched skin grafting, we investigated whether it is feasible to control the lymphocyte reconstitution process to delay rejection and favor tolerance processes. We found that a short course of anti-IL-7 receptor blocking antibody following T cell depletion, combined with the mammalian target of rapamycin inhibitor Rapamycin, could significantly delay graft rejection in one mouse strain, and achieve transplantation tolerance in another. The combination treatment was found to delay T cell reconstitution and, in the short term, enriched for Foxp3+ regulatory T cells (Tregs), at the expense of effector cells. Extended graft survival and tolerance were dependent on TGF-ß, indicating a role for induced Tregs. These findings point to the feasibility of building on lympholytic induction by guiding early lymphocyte reconstitution to favor endogenous regulatory mechanisms.

[The relationships of intelligence and memory assessed using the WAIS-IV and the WMS-IV]. / Zusammenhänge von Intelligenz- und Gedächtnis-diagnostik anhand von WAIS-IV und WMS-IV.

BACKGROUND: This study examines the relationships of intelligence and memory scores derived from WAIS-IV and WMS-IV. We were especially interested in the reciprocal predictive values of the test scales. METHODS: A sample of 137 healthy adults with an age range between 16 and 69 years was assessed with the WAIS-IV and the WMS-IV. The test order was balanced. Pearson correlations were conducted on the subtest and scale level. A series of 14 linear regression models was tested with memory performance as predictor for intelligence and vice versa. RESULTS AND CONCLUSION: A model including the 3 main memory scales of the WMS-IV was able to predict the global IQ best. It nevertheless explained only 46% of the variance. The memory and intelligence measures show significant relationships, but also represent distinct functions. WAIS-IV and WMS-IV complement one another.

CD73 and adenosine generation in the creation of regulatory microenvironments.

Extracellular adenosine 5'-triphosphate (ATP) acts on many immune cells to promote inflammation. Conversely, the ATP metabolite adenosine is mainly an anti-inflammatory molecule. The ecto-enzymes CD39 and CD73 can dephosphorylate extracellular ATP to adenosine, thereby controlling this important pathway of immune modulation. Despite their established roles in the immune system, little is known of how CD39 and CD73 are themselves regulated. Recent data have shown that CD73 expression and adenosine generation are up-regulated by transforming growth factor-ß, depending on the cytokine content of the local microenvironment. We review here these recent findings and discuss their implications in disease.

Th17 cells induce a distinct graft rejection response that does not require IL-17A.

IL-17A-producing helper T (Th17) cells have been implicated in the pathogenesis of autoimmune disease, inflammatory bowel disease and graft rejection, however the mechanisms by which they cause tissue damage remain ill-defined. We examined what damage Th17 cell lines could inflict on allogeneic skin grafts in the absence of other adaptive lymphocytes. CD4(+) Th17 cell lines were generated from two TCR transgenic mouse strains, A1(M).RAG1(-/-) and Marilyn, each monospecific for the male antigen Dby. After prolonged in vitro culture in polarizing conditions, Th17 lines produced high levels of IL-17A with inherently variable levels of interferon gamma (IFNγ) and these cells were able to maintain IL-17A expression following adoptive transfer into lymphopenic mice. When transferred into lymphopenic recipients of male skin grafts, Th17 lines elicited a damaging reaction within the graft associated with pathological findings of epidermal hyperplasia and neutrophil infiltration. Th17 cells could be found in the grafted skins and spleens of recipients and maintained their polarized phenotype both in vivo and after ex vivo restimulation. Antibody-mediated neutralization of IL-17A or IFNγ did not interfere with Th17-induced pathology, nor did it prevent neutrophil infiltration. In conclusion, tissue damage by Th17 cells does not require IL-17A.

Successful attenuation of humoral immunity to viral capsid and transgenic protein following AAV-mediated gene transfer with a non-depleting CD4 antibody and cyclosporine.

The ability of transient immunosuppression with a combination of a non-depleting anti-CD4 (NDCD4) antibody and cyclosporine (CyA) to abrogate immune reactivity to both adeno-associated viral vector (AAV) and its transgene product was evaluated. This combination of immunosuppressants resulted in a 20-fold reduction in the resulting anti-AAV8 antibody titres, to levels in naïve mice, following intravenous administration of 2 × 10(12) AAV8 vector particles per kg to immunocompetent mice. This allowed efficient transduction upon secondary challenge with vector pseudotyped with the same capsid. Persistent tolerance did not result, however, as an anti-AAV8 antibody response was elicited upon rechallenge with AAV8 without immunosuppression. The route of vector administration, vector dose, AAV serotype or the concomitant administration of adenoviral vector appeared to have little impact on the ability of the NDCD4 antibody and CyA combination to moderate the primary humoral response to AAV capsid proteins. The combination of NDCD4 and CyA also abrogated the humoral response to the transgene product, that otherwise invariably would occur, following intramuscular injection of AAV5, leading to stable transgene expression. These observations could significantly improve the prospects of using rAAV vectors for chronic disorders by allowing for repeated vector administration and avoiding the development of antibodies to the transgene product.

[Prediction of learning disability at school by means of SOPESS]. / Vorhersage von schulischen Lernstörungen durch SOPESS.

AIM OF THE STUDY: The responsibilities of public health authorities include early detection of risks for healthy development at school. Reading /writing disorder and math disorder are among the most common developmental disorders in childhood. METHOD: The present study conveys information about the prevalence of specific developmental disorders of scholastic skills (N=372) and assesses the prognostic validity of the social-paediatric screening of developmental status for school entry (SOPESS), the relevant criteria being DERET 1-2+, DEMAT 1+, and ELFE 1-6. RESULTS: The prevalence of specific developmental disorders of scholastic skills ranges from 1.1% for dyscalculia to 3.0% for dyslexia. Adequate correlations of r= -0.42(DERET 1-2+; DEMAT 1+) and r= -0.43 (ELFE 1-6) as well as substantial negative predictive values (0.80-0.93) suggest an acceptable screening performance. CONCLUSION: Children without clinical findings in SOPESS do not display any learning disabilities at onset of the 2nd grade while children marked at risk by SOPESS seem to benefit from concurrent intervention (e.g., language promotion programmes): such disabilities emerge in only half of these children.

Four-year metabolic outcome of a randomised controlled CD3-antibody trial in recent-onset type 1 diabetic patients depends on their age and baseline residual beta cell mass.

AIMS/HYPOTHESIS: The aim of the study was to examine the 48 month outcome of treating recent-onset type 1 diabetic patients for 6 days with humanised CD3-antibody, ChAglyCD3. METHODS: Eighty patients, aged 12-39 years, were recruited for a phase 2 multicentre trial and randomised to placebo (n=40) or ChAglyCD3 (n=40) treatment by a third party member; participants and care-givers were blinded. The change in insulin dose (U kg(-1)day(-1)) over 48 months was chosen as primary endpoint and compared in 31 placebo-and 33 ChAglyCD3-treated patients. Adverse events were followed in 35 and 38 patients, respectively. RESULTS: Treatment with ChAglyCD3 delayed the rise in insulin requirements of patients with recent-onset diabetes and reduced its amplitude over 48 months (+0.09 vs +0.32 U kg(-1)day(-1) in the placebo group). Using multivariate analysis this effect was correlated with higher baseline residual beta cell function and a younger age. It was associated with better outcome variables in subgroups selected according to these variables. In the ChAglyCD3 subgroup with higher initial beta cell function, 0/11 patients became C-peptide-negative over 48 months vs 4/9 in the corresponding placebo subgroup. In the subgroup aged <27 years old, antibody treatment preserved initial beta cell function for 36 months (vs >80% decline within 24 months in the placebo subgroup <27 years old), resulted in lower HbA1c concentrations and tended to reduce glycaemic variability (p=0.08). No longterm adverse events were observed. CONCLUSIONS/INTERPRETATION: A 6 day ChAglyCD3 treatment can suppress the rise in insulin requirements of recent-onset type 1 diabetic patients over 48 months, depending on their age and initial residual beta cell function. In younger patients this effect is associated with reduced deterioration of metabolic variables. These observations help to define inclusion criteria for prevention trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT00627146 FUNDING: Center grants from the Juvenile Diabetes Research Foundation (4-2001-434, 4-2005-1327) and grants from the Belgian Fund for Scientific Research-Flanders and from Brussels Free University-VUB.

[Social pediatric screening of developmental status for school entry (SOPESS): validity in the cognitive domain]. / Screening des Entwicklungsstandes bei der Einschulungsuntersuchung: Validität der kognitiven Skalen des SOPESS.

The newly developed social paediatric screening of developmental status for school entry (SOPESS) comprises assessments in visuomotor coordination, selective attention, precursors of number and set comprehension as well as visual cognition and reasoning. In various validation studies, these domains are correlated to coextensive scales of well-established psychometric instruments. In addition, a preliminary evaluation of screener performance in terms of sensitivity, specificity and predictive capability is given. The SOPESS features high specificity and results in reliable true negative findings. In the intended field of application, a mild proneness to false positive findings is tolerable, and may also be attributed to a limited reliability of the criteria instuments in the lower ranges of performance.

[Social pediatric screening of developmental status for school entry (SOPESS): validity in the domain of speech and language]. / Die Validität der Sprachskala des SOPESS unter Berücksichtigung der Erstsprache.

In addition to general cognitive and motor skills, the social-pediatric screening of developmental status for school entry (SOPESS) provides subtests for assessing speech and language in a differentiated way. In a special validation study, these domains are correlated to coextensive scales of SETK 3-5. The SOPESS features high specificity and results in reliable true negative findings. In addition, a preliminary evaluation of language skills considering migrant background is given. Children with an unsatisfactory status of language competence are treated separately in the SOPESS.

CD8+ T-Cell depletion and rapamycin synergize with combined coreceptor/stimulation blockade to induce robust limb allograft tolerance in mice.

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti-CD154 mAb, nondepleting anti-CD4 combined to either depleting or nondepleting anti-CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long-term allograft survival in both combinations. Nevertheless, robust donor-specific tolerance, defined by the acceptance of a fresh donor-type skin allograft and simultaneous rejection of third-party grafts, required initial CD8(+) T-cell depletion. Mixed donor-recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL-2, IFN-gamma and TNF-alpha in MLC with donor antigen while significant alloreactivity persisted against third- party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor-recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8(+) T-cell depletion and costimulation/coreceptor blockade.

Fc-disabled anti-mouse CD40L antibodies retain efficacy in promoting transplantation tolerance.

CD40L antibodies have proven to be powerful immunosuppressive agents in nonhuman primates but unfortunately perturb blood coagulation. Neither the therapeutic nor the prothrombotic mechanism of anti-CD40L is defined sufficiently to determine whether these effects can be uncoupled. Recent evidence suggests that the Fc region of anti-CD40L antibodies interacting with Fc receptors plays an important role in stabilizing platelet aggregates. An Fc-disabled, aglycosylated anti-CD40L heavy chain variant was therefore created to determine whether it might still be useful in promoting transplantation tolerance. In a number of mouse models an engineered aglycosyl anti-CD40L recapitulated the effects of the intact anti-CD40L antibody in tolerance protocols involving transplantation of allogeneic bone marrow and skin. In contrast, another anti-CD40L variant with a conventional rat gamma2b heavy chain was less effective in ensuring long-term skin graft survival, possibly associated with its faster clearance from the circulation. These results show that short pulses of anti-CD40L antibody therapy may still be useful in tolerance protocols even when the Fc region is disabled.

A pilot study of combination anti-cytokine and anti-lymphocyte biological therapy in rheumatoid arthritis.

BACKGROUND: Immunological tolerance in humans using anti-T-cell monoclonal antibodies (mAbs) may be hampered by a pro-inflammatory microenvironment. All clinical trials of such therapies in rheumatoid arthritis (RA), however, have selected patients with active disease at baseline. Concurrent neutralization of inflammation with a TNFalpha antagonist should maximize the potential of anti-T-cell mAbs to induce tolerance in RA. AIM: To evaluate the safety of combining a TNFalpha antagonist and CD4 mAb in RA. DESIGN: An iterative pilot study focused on the safety of such combination therapy. METHODS: Eight poor prognosis, seropositive RA patients were treated with combined CD4 and TNFalpha blockade. Prolonged CD4 blockade was achieved with a humanized mAb, and TNFalpha blockade with a p55 TNF receptor fusion protein. RESULTS: There was a low incidence of classical first-dose reactions to the CD4 mAb, possibly reflecting concomitant TNFalpha blockade. An unusual anaphylactoid reaction was seen, however, and one patient developed a probable allergic reaction after several infusions. Skin rashes were common, as previously reported with CD4 mAb monotherapy. No serious infections were documented during follow-up, despite CD4+ lymphopenia in some patients. Most patients appeared to demonstrate improved RA disease control after the study. After 17-49 months after therapy, one patient was in remission, one remained off disease modifying anti-rheumatic drugs and five had stable disease, three on previously ineffective doses of methotrexate. CONCLUSION: We report, for the first time in man, immunotherapy with a combination of an anti-cytokine and an anti-T-cell reagent. We witnessed an unusual first-dose reaction but there were no significant infectious complications.

Biology-inspired synthesis of compound libraries.

Biologically active small molecules represent the basis for chemical biology applications in which small molecules are used as chemical tools to probe biological processes. In this report, we review two approaches to design and synthesize compound libraries for biological screenings, i.e., diversity-oriented synthesis (DOS) and biology-oriented synthesis (BIOS).

Anti-angiogenic properties of a sulindac analogue.

BACKGROUND AND PURPOSE: Angiopoietins (Ang) are crucial for new blood vessel formation and exert their effects by acting on the Tie2 receptor. We have recently described a sulindac analogue 2-((1E,Z)-1-benzylidene-5-bromo-2-methyl-1H-inden-3-yl)acetic acid; termed C-18 from now onwards) that inhibits Tie2 receptor activity in kinase assays in vitro. Here, we have assessed the ability of C-18 to inhibit angiogenesis-related properties of endothelial cells and tested its selectivity for the Tie2 receptor. EXPERIMENTAL APPROACH: For in vitro experiments human umbilical vein endothelial cells (HUVEC) were used. Proliferation was measured using the MTT assay; migration assays were performed in a modified Boyden chamber and tube-like structure formation was determined on matrigel. The effects of C-18 in vivo were evaluated in the chicken chorioallantoic membrane (CAM). KEY RESULTS: Pre-treatment of HUVEC with C-18 blocked Ang-1-stimulated migration, but also abolished vascular endothelial cell growth factor (VEGF)- and fibroblast growth factor 2-induced responses. Incubation with C-18 inhibited serum-induced proliferation in a concentration-dependent manner; C-18 was, however, without effect on Ang-1-induced survival. In addition, we observed that C-18 did not inhibit ligand-induced receptor phosphorylation of Tie2 or VEGFR2. On the other hand, C-18 blocked activation of members of the mitogen-activated protein kinase family and of the Ser/Thr kinase Akt induced by both VEGF and Ang-1. Furthermore, incubation of CAMs with C-18 led to a dose-dependent inhibition of vascular length. CONCLUSIONS AND IMPLICATIONS: C-18 did not act as a Tie2 inhibitor, as originally thought, but rather inhibited growth factor-stimulated signalling pathways that regulate endothelial cell migration and potently reduces neovascularization in vivo.

Synthesis of palmitoylated Ras-peptides and -proteins.

In this review, an overview is given and details are provided for the synthesis of lipidated Ras (rat-adeno-sarcoma)-peptides and -proteins. The progress made in the synthesis of the lipidated peptides from the Ras superfamily is discussed with special emphasis on the recently developed solid-phase synthesis methods, since these methods have turned out to be the preferred synthesis method for the majority of the required peptides. Solid-phase lipopeptide synthesis has given access to native and modified peptides on a scale that allows peptide-consuming studies like for ligation to proteins and concomitant X-ray crystal structure determination. The access to these peptides has also enabled biological questions concerning these peptides and proteins to be resolved. The review describes different solid-phase methods, which are individually suited for different types of lipopeptides, differing for example in lipidation pattern or amino acid side-chain functionality, and their ligation to proteins. Finally, an example is provided how these peptides can serve to resolve biological aspects of the Ras family GTPases.

Protein structure similarity clustering and natural product structure as guiding principles for chemical genomics.

The majority of all proteins are modularly built from a limited set of approximately 1,000 structural domains. The knowledge of a common protein fold topology in the ligand-sensing cores of protein domains can be exploited for the design of small-molecule libraries in the development of inhibitors and ligands. Thus, a novel strategy of clustering protein domain cores based exclusively on structure similarity considerations (protein structure similarity clustering, PSSC) has been successfully applied to the development of small-molecule inhibitors of acetylcholinesterase and the 11beta-hydroxysteroid dehydrogenases based on the structure of a naturally occurring Cdc25 inhibitor. The efficiency of making use of the scaffolds of natural products as biologically prevalidated starting points for the design of compound libraries is further highlighted by the development of benzopyran-based FXR ligands.

Mechanisms of antibody immunotherapy on clonal islet reactive T cells.

Clinical intervention trials evaluating the efficacy of antibody immunotherapy in type 1 diabetes are in progress. We tested effects on prediabetic islet antigen-specific autoreactive T cells of antithymocyte globulin (ATG) and humanized monoclonal antibodies against CD3 (ChAglyCD3) or CD25 (daclizumab) with regard to downmodulation of the target protein, proliferation, cytokine production, complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), and survival. ATG leads to depletion of autoreactive CD4+ T cells by ADCC, CDC, and apoptosis, whereas anti-CD3 and anti-CD25 inhibited T-cell autoreactivity in a nondepleting fashion. ATG treatment led to a cytokine burst of Th1- and Th2-associated cytokines. Modulation of cytokine release through humanized monoclonal antibodies was moderate and selective: anti-CD25 led to increased release of IL-2 and reduced production of TNFalpha, whereas anti-CD3 decreased release of interferon-y and IL-5 and increased secretion of IL-10. ATG and the humanized monoclonal antibodies displayed contrasting mechanisms of action.

Intein-mediated in vitro synthesis of lipidated Ras proteins.

Fully functional lipid-modified Ras proteins suitable for the study of Ras-membrane interactions and embodying exclusively native amide bonds can be synthesized in preparative amounts by means of Expressed Protein Ligation.