Search

Background: A human Fcγ-Fcε fusion protein (GE2) designed to inhibit FcεRI signaling by coaggregating FcεRI with the inhibitory receptor FcγRIIB has been shown to inhibit mast cell activation and block cutaneous anaphylaxis. A critical issue remained as to whether the mechanism of GE2 inhibition is competition for IgE binding or inhibitory signaling through FcγRIIB. Objective: Our aim was to define the in vitro and in vivo mechanism of action of a mouse homolog of GE2 (mGE) and to assess the potential of human GE2 (hGE2) for therapeutic administration. Methods: The in vitro activity of mGE on mediator release and signaling pathways was characterized in IgE-sensitized bone marrow–derived mast cells (BMMCs). The in vivo activity of mGE was examined in mouse passive cutaneous and passive systemic anaphylaxis models, and the therapeutic activity of hGE2 was evaluated in Ascaris suum–sensitized cynomolgus monkeys. Results: mGE inhibited release of histamine and cytokines by BMMCs from wild-type mice but not by BMMCs from FcγRIIB-deficient mice. In mice mGE blocked IgE-dependent anaphylaxis mediated by mast cells with sustained efficacy. In BMMCs mGE decreased spleen tyrosine kinase and extracellular signal-regulated kinases 1/2 phosphorylation and induced FcγRIIB phosphorylation and the subsequent recruitment of SH2 domain–containing inositol polyphosphate 5′ phosphatase (SHIP) 1 and SH2 domain–containing protein tyrosine phosphatase (SHP) 1/2 phosphatases. When administered therapeutically, hGE2 protected sensitized monkeys from local anaphylaxis for 3 weeks. Conclusion: mGE-mediated inhibition of mast cell activation is associated with inhibitory signaling through FcγRIIB that results from activation of SHIP-1 and SHP-1/2 phosphatases. [Copyright &y& Elsevier]

Background: Tissue-engineered pulmonary arteries could overcome the drawbacks of homografts or prosthetic conduits used in the repair of many congenital cardiac defects. However, the ideal scaffold material for tissue-engineered conduits is still subject of intensive debate. In this study, we evaluated an acellularized allogeneic matrix scaffold for pulmonary artery tissue engineering with and without in-vitro reseeding with autologous endothelial cells in the pulmonary circulation in a growing sheep model. Methods: Ovine pulmonary arteries (n = 10) were acellularized by trypsin/ethylenediamine tetraacetic acid incubation. Autologous endothelial cells were harvested from carotid arteries, and the pulmonary conduits were seeded with endothelial cells. We implanted in-vitro, autologous, reendothelialized (group A, n = 5) and acellularized pulmonary conduits (group B, n = 5) in the pulmonary circulation. The animals were sacrificed 6 months after the operation. Explanted valves were examined histologically and by immunohistochemistry. Results: The conduit diameter increased in both groups (group A, 44% ± 11%; group B, 87% ± 18%; p < 0.05). In group A, however, a proportional increase in diameter was present, whereas in group B, a disproportionate increase resulting in aneurysm formation was observed. Histologically, the conduit wall integrity was destroyed in group B and preserved in group A. In group B, the extracellularmatrix degenerated with a reduced amount of collagens and proteoglycanes. Furthermore, no elastic fibers were detectable. In contrast, the extracellularmatrix in group A was close to native ovine tissue. Conclusions: Tissue-engineered pulmonary conduits (autologous endothelial cells and allogeneic matrix scaffolds) functioned well in the pulmonary circulation. They demonstrated an increase in diameter and an extracellular matrix comparable to that of native ovine tissue. [Copyright &y& Elsevier]

Abstract: Tissue engineering is an emerging field in regenerative medicine to overcome the problem of end-stage organ failure. However, complex tissues and organs need a vascular supply to guaranty graft survival and render bioartificial organ function. Here we developed methods to decellularize porcine small bowl segments and repopulate the remaining venous and arterial tubular structures within these matrices with allogeneic porcine endothelial progenitor cells. Cellular adherence and vitality was characterized by quantitative 2-[18F]-fluoro-2′-desoxy-glucose (FDG) positron emission tomography (PET) and subsequent immunohistological work up. The generated matrices showed insulin-dependent FDG uptake predominantly in the region of the former vascular structures. Stain for vitality and the specific endothelial markers CD31, VE-Cadherin and Flk-1 matched this functional finding. Providing evidence for vitality up to 3 weeks post reconstitution and typical endothelial differentiation, these results indicate that our generated matrix allows the generation of complex bioartificial tissues and organs for experimental and future clinical application. [Copyright &y& Elsevier]

Background: The generation of autologous tracheal implants by tissue-engineering techniques is a promising concept for otherwise untreatable patients. A functional cartilaginous backbone represents a prerequisite for any bioartificial tracheal graft. The aim of this study was to define suitable cell types and culture conditions for the generation of tracheal cartilage.Methods: We obtained tracheal, costal, and auricular cartilage from porcine donor animals (n = 10). The chondrocytes were cultured two-dimensionally in cell flasks or mixed with a liquid collagen solution forming a three-dimensional culture system. Labeling with carboxy fluorescein diacetate succinimidyl ester (CFDA SE) and biochemical reduction of formazan served to determine cell viability and proliferation. The extracellular matrix produced by the chondrocytes was characterized by Western blot.Results: The CFDA SE labeling proved viability and the MTT assays documented a proliferation of the chondrocytes over time in vitro. While the chondrocytes in the three-dimensional cell culture system produced hyaline cartilage composed of collagen II, the two-dimensional culture conditions resulted in nonspecific collagen synthesis.Conclusions: Chondrocytes grown in a three-dimensional matrix can effectively proliferate and produce cartilage and are viable for more than 2 weeks. Costal chondrocytes are suitable for tracheal cartilage tissue engineering. [Copyright &y& Elsevier]

: BackgroundThe purpose of this study was to evaluate neointimal functionality of synthetic vascular grafts repopulated by host cells after implantation.: MethodsWe obtained reseeded inflow and outflow cannulas of 2 patients undergoing orthotopic heart transplantation after left ventricular assist device implantation 9 and 10 months before. After cell isolation we examined cellular function of reseeded cells and their capability to form a functional endothelial layer applying immunohistologic markers and quantitative Western blot for endothelial nitric oxide synthase activity.: ResultsNeointima formation in inflow and outflow cannulas differs macroscopically and by histologic appearance. The neointima formation on the surface of the polyethylene terephthalate fiber (Dacron) grafts differs substantially from native aortic vessel wall with respect to cellular and extracellular matrix composition and cellular function.: ConclusionsThe neointima of Dacron prostheses is composed of cells with rudimentary physiologic endothelial function. We conclude that synthetic matrices are not suitable scaffolds for generating functional cardiovascular implants. [Copyright &y& Elsevier]

: BackgroundApproaches to in vivo repopulation of acellularized valve matrix constructs have been described recently. However, early calcification of acellularized matrices repopulated in vivo remains a major obstacle. We hypothesised that the matrix composition has a significant influence on the onset of early calcification. Therefore, we evaluated the calcification of acellularized allogenic ovine (AVMC) and xenogenic porcine (XVMC) valve matrix conduits in the pulmonary circulation in a sheep model.: MethodsPorcine (n = 3) and sheep (n = 3) pulmonary valve conduits were acellularized by trypsin/EDTA digestion and then implanted into healthy sheep in pulmonary valve position using extracorporeal bypass support. Transthoracic echocardiography (TTE) was performed at 12 and 24 weeks after the implantation. The animals were sacrificed at week 24 or earlier when severe calcification of the valve conduit became evident by TTE. The valves were examined histologically and biochemically.: ResultsAll AVMC revealed severe calcification after 12 weeks with focal endothelial cell clustering and no interstitial valve tissue reconstitution. In contrast, after 24 weeks XVMC indicated mild calcification on histologic examination (von Kossa staining) with histologic reconstitution of valve tissue and confluent endothelial surface coverage. Furthermore, immunohistologic analysis revealed reconstitution of surface endothelial cell monolayer (von Willebrand factor), and interstitial myofibroblasts (Vimentin/Desmin).: ConclusionsPorcine acellularized XVMC are resistant to early calcification during in vivo reseeding. Furthermore, XVMC are repopulated in vivo with valve-specific cell types within 24 weeks resembling native valve tissue. [Copyright &y& Elsevier]

Since the approval of ibrutinib for the treatment of B-cell malignancies in 2012, numerous clinical trials have been reported using covalent inhibitors to target Bruton's tyrosine kinase (BTK) for oncology indications. However, a formidable challenge for the pharmaceutical industry has been the identification of reversible, selective, potent molecules for inhibition of BTK. Herein, we report application of Tethering-fragment-based screens to identify low molecular weight fragments which were further optimized to improve on-target potency and ADME properties leading to the discovery of reversible, selective, potent BTK inhibitors suitable for pre-clinical proof-of-concept studies. [ABSTRACT FROM AUTHOR]

[Display omitted] For the past two decades, BTK a tyrosine kinase and member of the Tec family has been a drug target of significant interest due to its potential to selectively treat various B cell-mediated diseases such as CLL, MCL, RA, and MS. Owning to the challenges encountered in identifying drug candidates exhibiting the potency block B cell activation via BTK inhibition, the pharmaceutical industry has relied on the use of covalent/irreversible inhibitors to address this unmet medical need. Herein, we describe a medicinal chemistry campaign to identify structurally diverse reversible BTK inhibitors originating from HITS identified using a fragment base screen. The leads were optimized to improve the potency and in vivo ADME properties resulting in a structurally distinct chemical series used to develop and validate a novel in vivo CD69 and CD86 PD assay in rodents. [ABSTRACT FROM AUTHOR]

The interaction between IgE-Fc (Fcϵ) and its high affinity receptor FcϵRI on the surface of mast cells and basophils is a key event in allergen-induced allergic inflammation. Recently, several therapeutic strategies have been developed based on this interaction, and some include Fcϵ-containing moieties. Unlike well characterized IgG therapeutics, the stability and folding properties of IgE are not well understood. Here, we present comparative biophysical analyses of the pH stability and thermostability of Fcϵ and IgG 1-Fc (Fcγ). Fcϵ was found to be significantly less stable than Fcγ under all pH and NaCl conditions tested. Additionally, the Cϵ3Cϵ4 domains of FCϵ were shown to become intrinsically unfolded at pH values below 5.0. The interaction between Fcϵand an Fcγ-FcϵRIa fusion protein was studied between pH 4.5 and 7.4 using circular dichroism and a combination of differential scanning calorimetry and isothermal titration calorimetry. Under neutral pH conditions, the apparent affinity of Fcϵ for the dimeric fusion protein was extremely high compared with published values for the monomeric receptor (KD > 10-12 M). Titration to pH 6.0 did not significantly change the binding affinity, and titration to pH 5.5 only modestly attenuated affinity. At pH values below 5.0, the receptor binding domains of Fcϵ unfolded, and interaction of FCE with the Fcγ-FcϵRIa fusion protein was abrogated. The unusual pH sensitivity of Fcϵ may play a role in antigen-dependent regulation of receptor-bound, non-circulating IgE. [ABSTRACT FROM AUTHOR]

The transcription factors of the Myc/Max/Mad network play essential roles in the regulation of cellular behavior. Mad1 inhibits cell proliferation by recruiting an mSin3-corepressor complex that contains histone deacetylase activity. Here we demonstrate that Mad1 is a potent inhibitor of the G1 to S phase transition, a function that requires Mad1 to heterodimerize with Max and to bind to the corepressor complex. Cyclin E/CDK2, but not cyclin D and cyclin A complexes, fully restored S phase progression. In addition inhibition of colony formation and gene repression by Mad1 were also efficiently antagonized by cyclin E/CDK2. This was the result of cyclin E/CDK2 interfering with the interaction of Mad1 with HDAC1 and reducing HDAC activity. Our findings define a novel interplay between the cell cycle regulator cyclin E/CDK2 and Mad1 and its associated repressor complex and suggests an additional mechanism how cyclin E/CDK2 affects the G1 to S phase transition. [ABSTRACT FROM AUTHOR]

Investigation of cell abilities to growth, proliferation and (de)-differentiation in a three-dimensional distribution is an important issue in biotechnological research. Here, we report the development of a new bioreactor for three-dimensional cell culture, which allows for co-cultivation of various cell types with different culture conditions in spatial separation. Preliminary results of neonatal rat cardiomyocyte cultivation are shown.Isolated neonatal rat cardiomyocytes were cultured in spatial separated bioreactor compartments in recirculating medium on a biodegradable fibrin matrix for 2 weeks. Glucose, lactate, and lactate dehydrogenase (LDH), pO2, pCO2, and pH levels were monitored in the recirculated medium, daily. Morphological characterization of matrix and cells was assessed by hematoxylin and eosin staining, and MF-20 co-immunostaining with 4′,6-diamidino-2-phenylindole (DAPI). Cell viability was determined by LIVE/DEAD staining before cultivation and on day 3, 7, and 14.The optimized seeding density in the matrix was 2.0×107 cells retaining cellular proportions over the cell culture period. The bioreactor allows the maintenance of physiologic culture conditions with aerobic cell metabolism (low release of lactate, LDH), a high oxygen tension (pO2–183.7±18.4 mmHg) and physiological pH values (7.4±0.02) and a constant level of pCO2 (43.1±2.9) throughout the experimental course. The cell viability was sufficient after 2 weeks with 82±6.7% living cells. No significant differences were found between spatial separated bioreactor compartments.Our novel multifunctional bioreactor allows for a three-dimensional culture of cells with spatial separation of the co-cultured cell groups. In preliminary experiments, it provided favorable conditions for the three-dimensional cultivation of cardiomyocytes. [Copyright &y& Elsevier]

Objectives: To investigate the cytotoxic effect of prostaglandin E1 (PGE1), a standard combination of papaverine/phentolamine, and a triple mixture of these agents on human cavernosal endothelial cells using a cell culture model. The endothelial layer of the corpus cavernosum plays an important role in signal transduction of penile erection and is directly exposed to vasoactive agents after intracavernous injection for erectile dysfunction.Methods: Primary endothelial cells were obtained from the corpus cavernosum of 13 potent patients undergoing penile surgery. Cultured cells were exposed for 30 minutes to physiologic dilutions of 20 μg PGE1, 30 mg papaverine/1 mg phentolamine, or the same dosages of the triple mixture of these agents, each dissolved in 5 to 50 mL sodium chloride. Lactate dehydrogenase release as a cytotoxicity marker was measured 6 hours after drug exposure, and the total cell metabolic activity was quantified after 48 hours with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Additionally, the amount of viable cells was identified with a dual fluorescent staining procedure.Results: The initial release of lactate dehydrogenase was elevated up to 3.2-fold in the concentrated papaverine/phentolamine and triple mixture group compared with PGE1 and the control. After 48 hours, the papaverine-containing formulations led to a significant dose-dependent decrease in the viable cell count and metabolic activity of the cultures that was not noticed with PGE1.Conclusions: These in vitro data strongly suggest an unfavorable effect of vasoactive agents containing papaverine on cavernosal endothelial cells. Before fibrotic changes of the smooth muscle stroma, the functionally important endothelium of the corpus cavernosum might suffer significantly from intracavernous injection therapy. Therefore, papaverine should no longer be used for this indication. [Copyright &y& Elsevier]

Objective: For cardiovascular tissue engineering, acellularized scaffolds of porcine matrices have been successfully used. However, the possibility of porcine endogenous retrovirus (PERV) transmission remains debatable. In this study, we investigated whether acellularized porcine vascular scaffolds cause cross-species transmission of PERV in a xenogenic model.Methods: Porcine pulmonary arteries were acellularized and implanted into sheep in orthotopic position (n=6). Cardiopulmonary bypass support was used for all operations. Blood samples were collected regularly up to 6 months after the operation, and cellular components were tested for PERV infection by PCR and RT-PCR. Grafts were explanted 6 and 12 months after implantation. Tissue samples were characterized by histology and electron microscopy and tested for PERV sequences.Results: All animals survived the procedure and follow up until explantation of the grafts. PERV DNA was detectable in acellularized scaffolds of porcine matrices. Acellular porcine pulmonary arteries scaffolds were repopulated in vivo by autologous cells of the host, leading to a vessel consisting of all cellular components of the vessel wall. No PERV sequences were detectable neither in all tested peripheral blood samples nor in tissue samples of in vivo recellularized grafts up to 6 months after implantation. Electron microscopy revealed no signs of graft infection by retrovirus.Conclusions: Guided tissue regeneration of acellularized vascular porcine matrix scaffolds leads to structured vessels up to one year without risk of PERV transmisson. [Copyright &y& Elsevier]