bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bio artificial pancreas – the immune barrier and oxygen deficiency
Authors: Barkai Uriel , Ludwig Barbara , Bornstein Stefan R. , Zimermann Baruch , Gendler Zohar , Rotem Avi 
Published: April 2014

BackgroundXenogeneic transplantation of islets of Langerhans into type I diabetes patients imposes a major challenge. This is even more exaggerated when the target is set as xenotransplantation of islets in relatively oxygen poor environment without the use of immunosuppressive therapy. To overcome these hurdles, adequate encapsulation technique and active oxygen supply should be employed.MethodsA two‐article device was developed (the βAir). The first contains an implantable bioreactor module comprising of islets of Langerhans encapsulated in alginate hydrogel and protected from the host immune system by a complex membrane. The second is an external oxygen injection device for replenishing of the islet biomass in the implantable device once a day.ResultsBoth small and large diabetic animal models (rats and pigs) were experimented. The βAir device proved: (i) ability to maintain near normal glycemic control in rats transplanted with either isogeneic or allogeneic islets for a period of over 6 months and in pigs transplanted with rat islets using marginal dose of islets (~6,500 IEQ/kg) for a period of up to 75 days; (ii) ability to maintain adequate oxygen tension required to nourish the islets graft for a period of 24 h while maintaining optimal islet's function. (iii) long term separation of transplanted graft from host immune system thus maintaining normal islets function in rat allotype, in rat‐to‐pig and in human‐to‐rat transplantation systems and (iv) high level of biocompatibility and tolerability of the device.ConclusionThe βAir device proved to offer functional solution to islets transplantation in allogeneic and xenogeneic setting. It may provide a solution to transplantation of porcine islets to diabetic patients.References[1] Barkai U, Weir GC, Colton CK et al. Enhanced oxygen supply improves islet viability in a new bioartificial pancreas. Cell Transplant. 2013; 22: 1463–1476.[2] Neufeld T, Ludwig B, Barkai U et al. The efficacy of an immunoisolating membrane system for islet xenotransplantation in minipigs. PLoS One. 2013; 8(8): e70150.
Reduction of xeno‐antigens in porcine pulmonary heart valves by decellularization and glycolytic enzymatic treatment
Authors: Hilfiker Andres , Ramm Robert , Findeisen Katja , Goecke Tobias , Haverich Axel 
Published: April 2014

BackgroundHeart valve replacement therapy with allogenic decellularized heart valve matrices allows an in vivo autologisation by repopulation of the foreign matrix with autologous cells. This method, which is superior based on experimental evidence to traditional heart valve replacements either of mechanical or glutaraldehyde fixated heart valve grafts, is current object of a multicentric international clinical trial (ESPOIR) granted by the EU. To circumvent the limited availability of allogenic heart valve matrices, we aim to the reduction of immunogenic epitops common to xenogeneic materials by high efficient decellularization paralleled by enzymatic treatment of the glycocalyx.MethodsPorcine pulmonary heart valves of landrace pigs harvested at the local slaughterhouse were subjected to detergent and proteinase based decellularization protocols widely described in the literature. In a second step decellularized matrices were exposed to α1‐3 Galactosidase or PNGase F digestion. Alterations in αGal epitope levels and glycocalyx structures were investigated by inhibitory ELISAs with anti‐αGal antibodies (M86) and IgGs of unconditioned human sera on crushed matrix, and histochemical stains on cryosections exploiting lectins as isolectin B4 (staining αGal), wheat germ agglutinin (staining GalNAc), and Datura stramonium lectin (staining GlcNAc). Equally treated GalT‐KO pig and human pulmonary artery tissues served as controls.ResultsInhibitory ELISA results show that all decellularization protocols reduced αGal epitope levels significantly, some down to <30% of native porcine tissue. Further reduction down to control levels was achieved by α1‐3 Galactosidase digestion. The absence of preformed non‐ αGal xenoantibodies in human IgG of unconditioned donors is evident by equal binding of human IgG to decellularized tissues of GalT‐KO and human origin.Histochemistry reveals that decellularization results in reduced Isolectin B4 (αGal) and WGA (GalNAc) staining, but not of DSL (GlcNAc) staining. Additional enzymatic treatment of decellularized matrix with PNGase F reduces binding of all lectins, whereas digestion with α1‐3 Galactosidase only affects Isolectin B4 staining (αGal).ConclusionThe reduction of αGal epitope levels is strongly dependent on the decellularization method. A further decrease can be achieved by enzymatic digestion of carbohydrates present on the decellularized matrix. Sugar structures of the glycocalyx are believed to be strongly immunogenic, therefore, their general removal by PNGase F digestion may produce non‐immunogenic heart valve matrices. However, the lack of preformed human non‐ αGal xenoantibodies in non conditioned sera requires the testing for immunogenicity in an in vivo system. Thus, in future decellularized and enzymatically treated GalT‐KO matrices will be tested in a humanized mouse model.
Ex‐vivo testing and preclinical heterotopic thoracic cardiac xenotransplantation of multi‐transgenic organs
Authors: Abicht Jan‐Michael , Mayr Tanja , Guethoff Sonja , Buchholz Stefan , Werner Fabian , Baehr Andrea , Klymiuk Nikolai , Wuensch Annegret , Wolf Eckhart , Belka Claus , Ayares David , Petersen Björn , Niemann Heiner , Bongoni Anjan K. , Rieben Robert , McGregor Christopher , Reichart Bruno , Brenner Paolo 
Published: April 2014

The heterotopic thoracic pig‐to‐baboon heart transplantation has been established by our group as a safe preclinical model. Since the recipient's own heart remains in place, it is possible to evaluate immunological reactions of various types and the symptoms of the thrombotic microangiopathy under working heart conditions. However, these experiments are complex and costly. In order to limit the number of transplants, we tested our multi‐transgenic porcine donor hearts in an ex‐vivo perfusion model first.Ex‐vivo model: Both beating ventricles were perfused with heparinized freshly drawn human whole blood using a centrifugal pump and a membrane oxygenator. During 3 h of observation, cardiac function parameters were obtained continuously; specimens from the perfusate, coronary sinus blood, and myocardial biopsies were assessed. Various genetically modified porcine donor hearts were tested; we are currently evaluating the impact of human thrombomodulin on the porcine microcirculation; the efficacy of complement regulation is another work package.Pig‐to‐baboon heterotopic cardiac xenotransplantation: In our latest group, seven baboons received five double (Gal‐KO/hCD46), and two triple (Gal‐KO/hCD46/hTM resp./HLA‐E) transgenic pig hearts. Our immunosuppressive regimen included preoperative anti‐CD20‐antibody, bortezomib, dexamethasone and cyclophosphamide; postoperatively, ATG, tacrolimus, MMF, anti‐CD20‐antibody, bortezomib and dexamethasone were administered. Total lymph node irradiation (6 Gy) was applied on postoperative day five.The triple transgenic hearts survived 35 and 37 days respectively. Both hearts maintained function throughout the experiment. Both recipients succumbed to fungal infections. Humoral rejection was seen only once. The next barrier appears to be the complex event of thrombotic microangiopathy that needs to be addressed with the additional expression of human thrombomodulin strategies.
B cell activation and induced antibody responses to porcine antigen can be diminished by PD‐L1‐mediated triggering of PD‐1
Authors: Buermann Anna , Borns Katja , Römermann Dorothee , Plege‐Fleck Annegret , Schwinzer Reinhard 
Published: April 2014

BackgroundT cell activation is regulated by the integration of positive and negative signals. A strategy to down‐regulate human immune responses to a porcine xenograft is the enhancement of negative costimulatory signals. The programmed death‐1 receptor (PD‐1) is one of the known negative regulators for T cell activation and we have previously shown that human in vitro T cell responses to porcine transfectants over‐expressing human PD‐Ligand 1 (PD‐L1) are reduced as compared to responses triggered by control cells. However, PD‐1 is also expressed on B cells and its role in B cell activation is not well understood so far. Thus, the present study focused on the effects of PD‐1/PD‐L1 targeting on the humoral immune response to xenoantigens.MethodsIn vitro activation of human B cells was induced by co‐culturing with porcine L23 cells (B cell line) genetically engineered to over‐express human PD‐L1 (L23‐PD‐L1 cells) or “mock” ‐transfected controls. In vivo immune responses to the two cell types were characterized in a rat model by immunization experiments. The anti‐pig antibody titer in the culture supernatant or the sera of rats was examined by binding to L23 cells and visualized by flow cytometry using FITC‐conjugated IgM and IgG.ResultsStimulation of human peripheral blood mononuclear cells (hPBMC) with L23 cells resulted in up‐regulation of the early activation marker CD69 on CD19+ B cells. Furthermore, we observed T cell dependent proliferation of B cells and differentiation to antibody secreting cells in vitro. This was shown by the detection of anti‐pig antibodies in the culture supernatant of hPBMCs stimulated with xenoantigen after 7 days and the titer increased till day 12, with higher amounts of IgM than IgG antibodies. When the activation of B cells was induced by L23‐PD‐L1 cells, lower antibody titers were determined in comparison to B cell activation induced by L23 control cells. Moreover, the humoral immune response was reduced when a soluble PD‐L1 molecule was applied to hPBMCs stimulated with porcine cells. To further characterize the antibody responses to L23‐PD‐L1 and control cells in vivo, we analyzed the level of anti‐pig antibodies in rats after cell transplantation under the kidney capsule. In line with the in vitro data we found lower levels of antibodies in the serum of rats immunized with L23‐PD‐L1 cells than in rats receiving L23 control cells.ConclusionThese data suggest that the antibody response to porcine xenoantigen is diminished in the presence of the inhibitory ligand PD‐L1. Application of PD‐L1 might be a strategy to prevent induction of anti‐graft antibody responses, which are particularly strong in the pig‐to‐primate combination and represent a significant immunologic barrier for clinical xenotransplantation.
Decellularization followed by PNGase F treatment efficiently removes immunogenic αGal epitopes and other N‐acetylglucosamine structures on the glycocalyx of porcine pulmonary heart valve matrices
Authors: Findeisen Katja , Ramm Robert , Hilfiker Andres 
Published: April 2014

BackgroundXenotransplantation leads to hyperacute or acute graft reaction due to interaction of preformed human antibodies, mainly αGal‐antibodies, with carbohydrate structures present on non‐human tissue. By removing these carbohydrate structures from porcine decellularized pulmonary heart valves (PHV), grafts might be generated that allow heart valve replacement therapy as with allogeneic PHV matrices.MethodsThus, after detergents based decellularization (0.5%SDS/0.5%Triton‐X100) cell free PHV matrices were enzymatically treated with α1‐3,6‐galactosidase or PNGase F. The potential impact on the glycocalyx was investigated by histochemical stains utilizing isolectin B4 (IL‐B4), wheat germ agglutinin (WGA), Datura stramonium lectin (DSL), and Ricinus communis agglutinin (RCA I) on decellularized only, and decellularized and enzymatically treated specimens. Native PHV tissue served as controls.ResultsAll used lectins stained native heart valve tissue. Decellularization resulted in reduced IL‐B4 and WGA staining, whereas cell removal had no effect on DSL and RCA I staining. Enzymatic PNGase F treatment resulted in a further decrease of IL‐B4 and WGA staining whereas initially not affected DSL stain was reduced as well. Enzymatic treatment with α1‐3,6‐galactosidase led to a reduction of IL‐B4 stain only.ConclusionDecellularization per se is able to reduce αGal epitopes as demonstrated by IL‐B4 staining. This reduction of αGal epitopes can be enhanced by α1‐3,6‐galactosidase digestion as expected. Enzymatic treatment with PNGase F that recognizes GlcNacβ(1‐N)Asn sites results in removal of carbohydrate structures as αGal and N‐acetyl‐glucosamines to a high degree as demonstrated by IL‐B4, WGA, and DSL stains.In summary, detergent based decellularization followed by PNGase F treatment resembles an efficient way to remove immunogenic epitopes from porcine pulmonary heart valve matrices, thus potentially enabling the generation of xenogeneic PHV matrices for clinical application.
Discordant cellular and organ transplantation from bench to bedside
Authors: Reichart Bruno 
Published: April 2014

It is more than 1 year since our Consortium commenced. The following abstracts provide an account of what has been achieved so far and document our understanding of xenotransplantation and some of our future strategies:

Since (microencapsulated) islets are the first in the clinic, our basic research focuses on immediate (innate) rejection reactions and means of counteracting them. Once transplanted and working, porcine and primate islets may respond differently, for example to glucose challenges.
We will gain preclinical experience regarding the safety of genetically modified islets as quickly as possible.
We are currently in dialogue with German authorities (Paul‐Ehrlich‐Institution as representative of the European Medicines Agency) to decide how best to formulate a successful clinical application within European standards.
Are there any ethical objections from within German society? A recent 3‐day meeting, held at the Munich Catholic Academy explored the issues, including opinions from the Jewish and Islamic communities. In brief, the outcome revealed no major objections as long as xenogeneic procedures are safe and effective. All talks will be published in German, with abstracts in English. There will also be an English review of the meeting possibly published in the journal Xenotransplantation.
Successful preclinical organ (heart, kidney) xenotransplantation is still difficult to achieve. Heterotopic thoracic (working) heart transplantation has been established with consistent results. It seems that hyperacute and delayed humoral rejection reactions are manageable; but thrombotic microangiopathy currently presents the main obstacle. Porcine hearts that express human thrombomodulin will be used next, together with complement antibodies and/or co‐stimulation blockade. Human thrombomodulin has provided promising results in the laboratory (cell based).
Porcine heart valves will be tested using single and multiple antigen knock out.
In conclusion, the German Xenotransplantation Consortium supported by the German Research Foundation (DFG) is on track for a successful (first?) period. There seems to be broad support from a majority of German cardiovascular surgeons, and this is spreading to those in other parts of Europe [1].Reference[1] Reichart B., Guethoff S., Mayr T. et al. Discordant cardiac xenotransplantation – broadening the horizons. Eur J Cardiothorac Surg 2013; Epub ahead of print.
After decellularization of porcine heart valves: no non‐Gal antigeneic epitopes detectable by non conditioned human sera
Authors: Ramm Robert , Hilfiker Andres 
Published: April 2014

BackgroundPatients that undergo heart valve replacement surgery with glutaraldehyde fixated porcine grafts do not face acute rejection as recipients of native porcine organs and tissues do, however, grafts calcify over time and fail after 10–20 years in adults and 3–4 years in children. Responsible for hyperacute and acute rejection of unfixated tissue are preformed antibodies that recognize xenoantigens. Major targets are carbohydrates like the αGal epitope or Neu5Gc linked to proteins as well as to lipids on cells and extracellular matrix. To overcome the limited availability of decellularized allogeneic heart valve grafts, which show the ability to remodel and to grow by ingrowth of autologous cells, a characteristics lacking in glutaraldehyde fixated heart valve grafts, we want to develop a porcine derived decellularized matrix that is well tolerated in humans.MethodsThe generation of such optimal porcine heart valves matrices is dependent on a successful elimination of bound xenoantigens. To quantify the amount of xenoantigens present on decellularized matrix, we established an inhibition ELISA by exposing human IgG to crushed matrix and measuring unbound IgG in an ELISA set up. High levels of measured IgG means low levels of xenoantigens present on the decelllularized matrix. To enhance the specificity of IgG tested in the ELISA, human sera were perfused through porcine kidneys, followed by intensive washing and elution of bound xenoantibodies. Total amount of IgG and αGal specific antibodies was monitored to control the purification steps. Inhibition ELISA was performed with matrices derived from Landrace pigs, GalT‐KO pigs and humans. Binding of total IgG as well as αGal specific antibodies was assayed.ResultsThe amount of total IgG binding correlates with the amount of αGal specific antibodies binding to decellularized porcine matrix indicating the presence of remaining αGal epitopes on the porcine matrix after decellularization. However, decellularized GalT‐KO pig and decellularized human matrix showed no binding of αGal specific antibodies and comparable amounts of total IgG binding.ConclusionOur results indicate that only αGal epitopes present on decellularized porcine heart valve matrixes are recognized by preformed xenoantibodies while no other xenoantigens are detected. Therefore, further evaluation focusing on induction of immunogeneic reactions has to be conducted in an in vivo model like the humanized mouse utilizing decellularized heart valve material derived from GalT‐KO pigs.
Generation of transgenic pigs carrying siRNA vector directed against human tissue factor expression
Authors: Ahrens Hellen , Petersen Björn , Herrmann Doris , Lucas‐Hahn Andrea , Hassel Petra , Ziegler Maren , Kues Wilfried , Niemann Heiner 
Published: April 2014

BackgroundThe acute vascular rejection (AVR) is the major obstacle to successful pig‐to‐primate xenotransplantation. Changes of the endothelium to a procoagulant state which occur after xenotransplantation entail an increased expression of Tissue Factor (TF). This initiates the extrinsic coagulation cascade and results in microvascular thrombosis and rapid loss of organ function. Organs from pigs with a decreased expression of TF could be promising to overcome AVR. Since a homozygous TF knockout had shown to be lethal in mice [1], we decided to use siRNA‐technology to knockdown TF to a basal level.MethodsDifferent siRNAs directed against TF expression were tested in initial studies for their efficacy. A vector coding for the siRNA providing the highest decrease of TF mRNA levels was kindly provided by Dr. Joachim Denner, RKI Berlin. Porcine fetal fibroblasts were co‐transfected with this plasmid and a vector (DsRed, Clontech) which confers neomycin resistance and red fluorescence. After 14 days of selection with 800 μg/ml Geneticin (G418), cells were screened for siRNA expression prior to somatic cell nuclear transfers (SCNTs). Subsequently, the reconstructed embryos were transferred to synchronised recipient sows. Liveborn offspring were analysed by real‐time PCR for TF mRNA quantification. The production of the siRNA was detected via RT‐PCR followed by real‐time PCR.ResultsAn average of 95 embryos per sow was transferred to 28 synchronized recipients. The pregnancy rate was 54%. One sow was sacrificed on day 25 of pregnancy and one fetus (#893/1) was obtained. The candidate siRNA could be detected fetal tissues. Real‐time PCR analysis revealed decreased TF mRNA expression in fibroblasts of fetus #893/1 compared to wild‐type fibroblasts. Therefore, somatic cells from fetus #893/1 was used for recloning. A total of 12 live born piglets could be obtained; five piglets died within few days after delivery. The mRNA levels and production of the siRNA are currently determined. Protein expression analysis by fluorescence cytometry and functionality assessment by coagulation assays are planned for the near future with collaborating laboratories at the Medical School Hannover.ConclusionsThe AVR is currently the bottleneck in porcine‐to‐primate organ xenotransplantation. Controlling TF‐induced coagulation might provide protective effects for porcine xenografts. Thus, TF knockdown pigs could be critical in producing the ultimate multi‐transgenic pig as organ donor for human patients.References[1] Toomey J, Kratzer K, Lasky N, Stanton J, Broze GJ. Targeted disruption of the murine tissue factor gene results in embryonic lethality. Blood. 1996; 88(5): 1583–1587.
The Pattern of Change in the Abundances of Specific Bacterioplankton Groups Is Consistent across Different Nutrient-Enriched Habitats in Crete
Authors: Fodelianakis Stilianos , Papageorgiou Nafsika , Pitta Paraskevi , Kasapidis Panagiotis , Karakassis Ioannis , Ladoukakis Emmanuel D. 
Published: April 2014

ABSTRACTA common source of disturbance for coastal aquatic habitats is nutrient enrichment through anthropogenic activities. Although the water column bacterioplankton communities in these environments have been characterized in some cases, changes in α-diversity and/or the abundances of specific taxonomic groups across enriched habitats remain unclear. Here, we investigated the bacterial community changes at three different nutrient-enriched and adjacent undisturbed habitats along the north coast of Crete, Greece: a fish farm, a closed bay within a town with low water renewal rates, and a city port where the level of nutrient enrichment and the trophic status of the habitat were different. Even though changes in α-diversity were different at each site, we observed across the sites a common change pattern accounting for most of the community variation for five of the most abundant bacterial groups: a decrease in the abundance of thePelagibacteraceaeand SAR86 and an increase in the abundance of theAlteromonadaceae,Rhodobacteraceae, andCryomorphaceaein the impacted sites. The abundances of the groups that increased and decreased in the impacted sites were significantly correlated (positively and negatively, respectively) with the total heterotrophic bacterial counts and the concentrations of dissolved organic carbon and/or dissolved nitrogen and chlorophyll α, indicating that the common change pattern was associated with nutrient enrichment. Our results provide anin situindication concerning the association of specific bacterioplankton groups with nutrient enrichment. These groups could potentially be used as indicators for nutrient enrichment if the pattern is confirmed over a broader spatial and temporal scale by future studies.