Rapamycin and CTLA4Ig synergize to induce stable mixed chimerism without the need for CD40 blockade.

The mixed chimerism approach achieves donor-specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti-CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor-specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC-mismatched/minor antigen-matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non-MHC antigens cause graft rejection and split tolerance.

Persistent molecular microchimerism induces long-term tolerance towards a clinically relevant respiratory allergen.

BACKGROUND: Development of antigen-specific preventive strategies is a challenging goal in IgE-mediated allergy. We have recently shown in proof-of-concept experiments that allergy can be successfully prevented by induction of durable tolerance via molecular chimerism. Transplantation of syngeneic hematopoietic stem cells genetically modified to express the clinically relevant grass pollen allergen Phl p 5 into myeloablated recipients led to high levels of chimerism (i.e. macrochimerism) and completely abrogated Phl p 5-specific immunity despite repeated immunizations with Phl p 5. OBJECTIVE: It was unclear, however, whether microchimerism (drastically lower levels of chimerism) would be sufficient as well which would allow development of minimally toxic tolerance protocols. METHODS: Bone marrow cells were transduced with recombinant viruses integrating Phl p 5 to be expressed in a membrane-anchored fashion. The syngeneic modified cells were transplanted into non-myeloablated recipients that were subsequently immunized repeatedly with Phl p 5 and Bet v 1 (control). Molecular chimerism was monitored using flow cytometry and PCR. T cell, B-cell and effector-cell tolerance were assessed by allergen-specific proliferation assays, isotype levels in sera and RBL assays. RESULTS: Here we demonstrate that transplantation of Phl p 5-expressing bone marrow cells into recipients having received non-myeloablative irradiation resulted in chimerism persisting for the length of follow-up. Chimerism levels, however, declined from transient macrochimerism levels to persistent levels of microchimerism (followed for 11 months). Notably, these chimerism levels were sufficient to induce B-cell tolerance as no Phl p 5-specific IgE and other high affinity isotypes were detectable in sera of chimeric mice. Furthermore, T-cell and effector-cell tolerance were achieved. CONCLUSIONS AND CLINICAL RELEVANCE: Low levels of persistent molecular chimerism are sufficient to induce long-term tolerance in IgE-mediated allergy. These results suggest that it will be possible to develop minimally toxic conditioning regimens sufficient for low level engraftment of genetically modified bone marrow.

Ensemble modelling of nutrient loads and nutrient load partitioning in 17 European catchments.

An ensemble of nutrient models was applied in 17 European catchments to analyse the variation that appears after simulation of net nutrient loads and partitioning of nutrient loads at catchment scale. Eight models for N and five models for P were applied in three core catchments covering European-wide gradients in climate, topography, soil types and land use (Vansjø-Hobøl (Norway), Ouse (Yorkshire, UK) and Enza (Italy)). Moreover, each of the models was applied in 3-14 other EUROHARP catchments in order to inter-compare the outcome of the nutrient load partitioning at a wider European scale. The results of the nutrient load partitioning show a variation in the computed average annual nitrogen and phosphorus loss from agricultural land within the 17 catchments between 19.1-34.6 kg N ha(-1) and 0.12-1.67 kg P ha(-1). All the applied nutrient models show that the catchment specific variation (range and standard deviation) in the model results is lowest when simulating the net nutrient load and becomes increasingly higher for simulation of the gross nutrient loss from agricultural land and highest for the simulations of the gross nutrient loss from other diffuse sources in the core catchments. The average coefficient of variation for the model simulations of gross P loss from agricultural land is nearly twice as high (67%) as for the model simulations of gross N loss from agricultural land (40%). The variation involved in model simulations of net nutrient load and gross nutrient losses in European catchments was due to regional factors and the presence or absence of large lakes within the catchment.

Degradation of a short-lived glycoprotein from the lumen of the endoplasmic reticulum: the role of N-linked glycans and the unfolded protein response.

We are studying endoplasmic reticulum-associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI(332), containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI(332) was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of approximately 45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI(332) in which the single used N-glycosylation consensus site had been removed (RI(332)-Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI(332) degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI(332) to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI(332). After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI(332) and RI(332)-Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI(332) and RI(332)-Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI(332) was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives.

[Reactive acalculous cholecystitis: a bland and asymptomatic course--incidence of a classical stress disease]. / Reaktive steinfreie Cholecystitis: blande und asymptomatische Verlaufsform--zur Dunkelziffer einer klassischen Stresserkrankung.

A systematic search for reactive acalculous cholecystitis (RAC) performed from 11/85 until 11/87 rendered an incidence of 9 in 1272 patients recovering from surgery and 1 in 930 patients treated conservatively for various diseases. 8 patients presented with typical but discrete clinical symptoms whereas 2 remained asymptomatic. Abdominal sonography proved to be the most useful diagnostic procedure. The ratio of classic fulminant disease to cases presenting with lesser severity was calculated to be 1:50. All patients in this series were managed nonoperatively in respect to RAC. The pathoanatomic and pathogenetic context is reviewed and the author's impression corroborated that relevant cases may frequently go by unnoticed by clinicians.

[Acute cholecystitis in cholelithiasis: importance of early surgery (a comparative study)]. / Akute Cholezystitis bei Cholezystolithiasis: zum Stellenwert der Frühoperation--Eine vergleichende Studie.

The results of early surgery (ES) for acute calculous cholecystitis obtained in 74 patients operated on between 3/78 and 12/87 were compared with relevant data obtained in 74 sex- and age-matched patients with a history of acute cholecystitis operated on for biliary colic or jaundice during the same period. Operative procedures, incidence of jaundice and common bile duct calculi, duration of operation, number of patients requiring blood transfusions, surgical and general complications and mortality showed no significant difference. Only operative blood loss was significantly higher in the ES group, but this was of no practical relevance. ES precludes the sequelae of emergency surgery in the delayed surgery group not infrequently necessary for failure of conservative treatment of acute cholecystitis, which necessarily precedes planned delayed surgery, and thus renders a significant reduction of over-all risk. This forms the rationale for ES as treatment concept.