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.

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.

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.

[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.

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.

Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity.

Pericyte loss and capillary regression are characteristic for incipient diabetic retinopathy. Pericyte recruitment is involved in vessel maturation, and ligand-receptor systems contributing to pericyte recruitment are survival factors for endothelial cells in pericyte-free in vitro systems. We studied pericyte recruitment in relation to the susceptibility toward hyperoxia-induced vascular remodeling using the pericyte reporter X-LacZ mouse and the mouse model of retinopathy of prematurity (ROP). Pericytes were found in close proximity to vessels, both during formation of the superficial and the deep capillary layers. When exposure of mice to the ROP was delayed by 24 h, i.e., after the deep retinal layer had formed [at postnatal (p) day 8], preretinal neovascularizations were substantially diminished at p18. Mice with a delayed ROP exposure had 50% reduced avascular zones. Formation of the deep capillary layers at p8 was associated with a combined up-regulation of angiopoietin-1 and PDGF-B, while VEGF was almost unchanged during the transition from a susceptible to a resistant capillary network. Inhibition of Tie-2 function either by soluble Tie-2 or by a sulindac analog, an inhibitor of Tie-2 phosphorylation, resensitized retinal vessels to neovascularizations due to a reduction of the deep capillary network. Inhibition of Tie-2 function had no effect on pericyte recruitment. Our data indicate that the final maturation of the retinal vasculature and its resistance to regressive signals such as hyperoxia depend on the completion of the multilayer structure, in particular the deep capillary layers, and are independent of the coverage by pericytes.

Myeloablative conditioning is well tolerated by older patients receiving T-cell-depleted grafts.

Older age has been linked to increased transplant-related mortality from graft-versus-host disease (GvHD). Depletion of T cells from stem cell grafts seems to protect from complications of GvHD particularly in older patients. After myeloablative conditioning, patients with haematological malignancies received allogeneic grafts from HLA identical siblings. For GvHD prophylaxis, PBPC grafts were treated ex vivo with anti-CD52, and therapeutic doses of cyclosporin until day +90. Survival of patients younger or older than the population age median was analysed. In all, 62 consecutive patients with a median age of 42.5 years were studied. Death was procedure related in 17% and from relapse of malignancy in five. At a median, follow-up is 662 (7-2316) days, 74% survive disease free. The rate of haematopoietic recovery and treatment-related mortality was similar in both groups. A total of 73% of 30 individuals in the younger group and 75% (P=0.8) in the older cohort survive at a median follow-up of 444 and 806 days (P=0.4). GvHD occurred in 13% and was the only adverse factor for survival (P<0.04). Myeloablative conditioning is well tolerated up to the age of 59 in patients receiving T-cell-depleted grafts. This information is useful to more precisely select patients who would benefit most from reduced intensity conditioning schedules.

Radiotherapy-based conditioning is effective immunosuppression for patients undergoing transplantation with T-cell depleted stem cell grafts for severe aplasia.

BACKGROUND: We studied the outcome of individuals with aplastic anemia conditioned with a radiation-containing regimen followed by an infusion of stem cell grafts that had been depleted of lymphocytes with CAMPATH-1H (antiCD52; humanised). METHODS: The conditioning regime consisted of fractionated (f) TBI 8 Gy followed by f total nodal irradiation (TNI) 6 Gy. In addition, patients received CY 60 mg/kg on 2 consecutive days. Cytokine-mobilized peripheral blood grafts from HLA-identical siblings were T-cell depleted with CAMPATH-1H 'in the bag'. CsA was commenced on day -1 and continued until day +90. RESULTS: Seventeen heavily transfused patients with aplastic anemia, median age 18 years (range 14-56 years), were studied. The median time from diagnosis to transplantation was 172 days (range 34-443 days). The median CD34(+) cell number infused was 3.47 x 10(6)/kg (range 1.03-18.4 x 10(6)/kg). All patients engrafted. Recovery was fast and patients reached 0.5 x 10(9)/L polymorphs by median day 11 (range 9-17 days). Toxicity from the conditioning included grade 4 hematologic toxicity in all patients. Another major toxicity was gastrointestinal mucosal damage, which exceeded grade 2 in two instances. One patient developed thrombotic thrombocytopenic purpura, which responded to substitution of CsA with tacrolimus and plasmapheresis. Another patient, who had normal blood counts, died of infection on day 241. Chimerism studies at 6 months post-transplantation confirmed the donor origin of hematopoiesis in all seven patients tested. None of the patients developed acute or chronic GvHD. There was no delayed graft failure and 94% of patients had survived disease free at a median of 1303.5 days (range 216-2615 days) from graft infusion. DISCUSSION: In this cohort of multiply transfused patients, the radiation-containing schedules described in this study led to universal engraftment with limited toxicity despite T-cell depletion. No patient developed GvHD or late graft failure. Lower doses of radiation-containing conditioning should be explored further.

Alemtuzumab (Campath-1H) in allogeneic stem cell transplantation: where do we go from here?

Alemtuzumab (Campath-1H) has been widely used for T-cell depletion following both conventional and reduced-intensity conditioning allografts. We studied the impact of alemtuzumab used in vivo and in vitro on infections, immune reconstitution, and allograft outcome. The use of alemtuzumab in vivo following reduced-intensity conditioning and unrelated donor conventional transplantation was associated with durable engraftment and significant reduction in graft-versus-host disease (GVHD) but at the cost of impaired immune reconstitution and increased infectious complications. Alemtuzumab exposure in vitro was associated with durable engraftment and reduced GVHD following conventional transplants without affecting immune recovery to the same extent. Improved results were obtained following a further reduction in the alemtuzumab dose in vitro from 20 mg to 10 mg. Subsequent pharmacokinetic studies on alemtuzumab demonstrated that the antibody persisted at a higher concentration at the time of transplant and for at least 2 months thereafter when used in vivo compared to persistence for less than 30 days when used at 20 mg in vitro. In this context; an antibody with a shorter half-life, like the original rat CD52 antibody Campath-1G, would be preferable. Otherwise, our cumulative experience with alemtuzumab suggests that better results might be achieved by tailoring the dose and mode of antibody use to match the clinical situation. Further studies are needed to optimize the dose of alemtuzumab in vivo and in vitro, determined by the type of conditioning and the graft.

Campath-1 Abs 'in the bag' for hematological malignancies: the Cape Town experience.

BACKGROUND: We examined the strategy of T-cell depletion of HLA-identical sibling grafts for the prevention of GvHD, as well as disease control and overall survival. PATIENTS AND METHODS: The myeloablative conditioning was radiation based. The source of stem cells was BM in 62, and cytokine-mobilized PBPC in 68 patients. GvHD prophylaxis was by ex vivo incubation of the stem-cell concentrates with Campath-1G (anti-CD52; n=76) or Campath-1H (n=54). RESULTS: Patients receiving PBPC grafts were older (median 38.5) than those undergoing BMT (median 31; P=0.002). More patients in the PBPC group developed chronic GvHD (p<0.01). While no post-transplant GvHD prophylaxis was given to BMT recipients, prednisone 30 mg daily was prescribed to 12 and CYA for 90 days to a further 32 patients who had received PBPC grafts. Median follow-up was 1055 (range 28-4867) days. Although there was no difference in the survival between patients who received BMT or PBPC, death was from disease recurrence in 16 and nine (p=0.03; chi(2) test) subjects, respectively. Multivariate analysis showed that outcome was particularly favorable in those who were given<20 mg Campath-1 (survival: 28/39 versus 12/29; P=0.01), and in the subgroup of 30 patients who received Campath-1H and post-transplantation CYA. DISCUSSION: In patients receiving BMT, Campath-1 Abs effectively prevent GvHD. For those treated with PBPC grafts, the combination of T-cell depletion and post-transplantation CYA is equally effective, without an obvious increase in disease recurrence.

Incidence and outcome of adenovirus disease in transplant recipients after reduced-intensity conditioning with alemtuzumab.

Adenoviruses are emerging as a major cause of infectious complications after allogeneic transplantation. We evaluated the incidence and outcome of symptomatic adenovirus infection or adenovirus disease after alemtuzumab-based reduced-intensity conditioning in 86 consecutive patients. The overall probability of adenovirus disease was 18.4% (11/86 patients). Five patients died of progressive adenovirus disease, and this was the most important infectious cause of mortality in this cohort. The probability of nonrelapse mortality was 49% in patients with adenovirus disease compared with 25.5% in those without (P=.007). The severity of lymphocytopenia and continuation of immunosuppressive therapy were the most important risk factors for progressive adenovirus disease and death. In contrast, patients who were not receiving immunosuppressive therapy or had had it reduced or withdrawn cleared the virus. We also detected a correlation between the lack of preemptive anti-cytomegalovirus (CMV) therapy for CMV reactivation and the risk of progressive adenovirus disease (P=.05). Our findings highlight the emergence of adenovirus as an important posttransplantation pathogen even after reduced-intensity conditioning and demonstrate the effect of the severity of lymphocytopenia, anti-CMV prophylaxis, and immunosuppressive therapy on the outcome of adenovirus disease.

At the crossroads of chemistry and biology.

The life sciences are molecular and the harnessing of information gleaned from genomics and proteomics will require interdisciplinary research integrating chemistry and biology. This approach is illustrated by the synthesis and biological evaluation of lipidated peptides and proteins and the delineation of a concept arguing for natural product guided combinatorial chemistry.