Graciela Elgue

Are Therapeutic Human Mesenchymal Stromal Cells Compatible with Human Blood

Multipotent mesenchymal stromal cells (MSCs) are tested in numerous clinical trials. Questions have been raised concerning fate and function of these therapeutic cells after systemic infusion. We therefore asked whether culture‐expanded human MSCs elicit an innate immune attack, termed instant blood‐mediated inflammatory reaction (IBMIR), which has previously been shown to compromise the survival and function of systemically infused islet cells and hepatocytes. We found that MSCs expressed hemostatic regulators similar to those produced by endothelial cells but displayed higher amounts of prothrombotic tissue/stromal factors on their surface, which triggered the IBMIR after blood exposure, as characterized by formation of blood activation markers. This process was dependent on the cell dose, the choice of MSC donor, and particularly the cell‐passage number. Short‐term expanded MSCs triggered only weak blood responses in vitro, whereas extended culture and coculture with activated lymphocytes increased their prothrombotic properties. After systemic infusion to patients, we found increased formation of blood activation markers, but no formation of hyperfibrinolysis marker D‐dimer or acute‐phase reactants with the currently applied dose of 1.0–3.0 × 106 cells per kilogram. Culture‐expanded MSCs trigger the IBMIR in vitro and in vivo. Induction of IBMIR is dose‐dependent and increases after prolonged ex vivo expansion. Currently applied doses of low‐passage clinical‐grade MSCs elicit only minor systemic effects, but higher cell doses and particularly higher passage cells should be handled with care. This deleterious reaction can compromise the survival, engraftment, and function of these therapeutic cells. Stem Cells2012;30:1565–1574

Mesenchymal stromal cells engage complement and complement receptor bearing innate effector cells to modulate immune responses.

Infusion of human third-party mesenchymal stromal cells (MSCs) appears to be a promising therapy for acute graft-versus-host disease (aGvHD). To date, little is known about how MSCs interact with the bodys innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46) and DAF (CD55), but were protected from complement lysis via expression of protectin (CD59). Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells.

Composite islet-endothelial cell grafts : a novel approach to counteract innate immunity in islet transplantation.

An instant blood‐mediated inflammatory reaction (IBMIR) is elicited when islets come in contact with blood after intraportal transplantation. In contrast, endothelial cells (EC) readily tolerate contact with blood. A conceivable strategy to overcome IBMIR would be to create composite islet‐EC grafts. Human islets were co‐cultured with primary human aortic endothelial cells (HAEC) for 2–7 days to obtain 50–90% coverage. HAEC‐coated islets were exposed to ABO‐identical blood and analyzed with regard to clotting time, signs of inflammation and cell infiltration. Composite islet‐HAEC graft survival was assessed after transplantation to athymic (nu/nu) nude mice. Exposed to blood, HAEC‐coated islets induced less activation of coagulation and complement compared to control islets. Also, platelet and leukocyte consumption in blood was decreased. Clots with entrapped HAEC‐coated islets showed less infiltration of CD11b+ cells. The extent of protection correlated to the level of HAEC coverage. Transplanted composite grafts stained positive for insulin and PECAM‐1 demonstrating presence of both islets and HAEC within the islet graft 7 weeks after transplantation. Composite islet‐HAEC grafts reduce all components of IBMIR. Refinement of the technique will allow introduction of composite islet‐EC grafts in clinical islet transplantation, using autologous EC expanded in vitro and kept frozen until allogeneic islets become available for that specific recipient.

Activated human platelets induce factor XIIa-mediated contact activation

Earlier studies have shown that isolated platelets in buffer systems can promote activation of FXII or amplify contact activation, in the presence of a negatively charge substance or material. Still proof is lacking that FXII is activated by platelets in a more physiological environment. In this study we investigate if activated platelets can induce FXII-mediated contact activation and whether this activation affects clot formation in human blood. Human platelets were activated with a thrombin receptor-activating peptide, SFLLRN-amide, in platelet-rich plasma or in whole blood. FXIIa and FXIa in complex with preferentially antithrombin (AT) and to some extent C1-inhibitor (C1INH) were generated in response to TRAP stimulation. This contact activation was independent of surface-mediated contact activation, tissue factor pathway or thrombin. In clotting whole blood FXIIa-AT and FXIa-AT complexes were specifically formed, demonstrating that AT is a potent inhibitor of FXIIa and FXIa generated by platelet activation. Contact activation proteins were analyzed by flow cytometry and FXII, FXI, high-molecular weight kininogen, and prekallikrein were detected on activated platelets. Using chromogenic assays, enzymatic activity of platelet-associated FXIIa, FXIa, and kallikrein were demonstrated. Inhibition of FXIIa in non-anticoagulated blood also prolonged the clotting time. We conclude that platelet activation triggers FXII-mediated contact activation on the surface and in the vicinity of activated platelets. This leads specifically to generation of FXIIa-AT and FXIa-AT complexes, and contributes to clot formation. Activated platelets may thereby constitute an intravascular locus for contact activation, which may explain the recently reported importance of FXII in thrombus formation.

Paraneoplastic antigen Ma2 autoantibodies as specific blood biomarkers for detection of early recurrence of small intestine neuroendocrine tumors.

Background Small intestine neuroendocrine tumors (SI-NETs) belong to a rare group of cancers. Most patients have developed metastatic disease at the time of diagnosis, for which there is currently no cure. The delay in diagnosis is a major issue in the clinical management of the patients and new markers are urgently needed. We have previously identified paraneoplastic antigen Ma2 (PNMA2) as a novel SI-NET tissue biomarker. Therefore, we evaluated whether Ma2 autoantibodies detection in the blood stream is useful for the clinical diagnosis and recurrence of SI-NETs. Methodology/Principal Findings A novel indirect ELISA was set up to detect Ma2 autoantibodies in blood samples of patients with SI-NET at different stages of disease. The analysis was extended to include typical and atypical lung carcinoids (TLC and ALC), to evaluate whether Ma2 autoantibodies in the blood stream become a general biomarker for NETs. In total, 124 blood samples of SI-NET patients at different stages of disease were included in the study. The novel Ma2 autoantibody ELISA showed high sensitivity, specificity and accuracy with ROC curve analysis underlying an area between 0.734 and 0.816. Ma2 autoantibodies in the blood from SI-NET patients were verified by western blot and sequential immunoprecipitation. Serum antibodies of patients stain Ma2 in the tumor tissue and neurons. We observed that SI-NET patients expressing Ma2 autoantibody levels below the cutoff had a longer progression and recurrence-free survival compared to those with higher titer. We also detected higher levels of Ma2 autoantibodies in blood samples from TLC and ALC patients than from healthy controls, as previously shown in small cell lung carcinoma samples. Conclusion Here we show that high Ma2 autoantibody titer in the blood of SI-NET patients is a sensitive and specific biomarker, superior to chromogranin A (CgA) for the risk of recurrence after radical operation of these tumors.

Distinctive regulation of contact activation by antithrombin and C1-inhibitor on activated platelets and material surfaces.

Activated human plate lets trigger FXII-mediated contact activation, which leads to the generation of FXIIa-antithrombin (AT) and FXIa-AT complexes. This suggests that contact activation takes place at different sites, on activated platelets and material surfaces, during therapeutic procedures involving biomaterials in contact with blood and is differentially regulated. Here we show that activation in platelet-poor plasma, platelet-rich plasma (PRP), and whole blood induced by glass, kaolin, and polyphosphate elicited high levels of FXIIa-C1-inhibitor (C1INH), low levels of FXIa-C1INH and KK-C1INH, and almost no AT complexes. Platelet activation, in both PRP and blood, led to the formation of FXIIa-AT, FXIa-AT, and kallikrein (KK)-AT but almost no C1INH complexes. In severe trauma patients, FXIIa-AT and FXIa-AT were correlated with the release of thrombospondin-1 (TSP-1) from activated platelets. In contrast, FXIIa-C1INH complexes were detected when the FXIIa-AT levels were low. No correlations were found between FXIIa-C1INH and FXIIa-AT or TSP-1. Inhibition of FXIIa on material surfaces was also shown to affect the function of aggregating platelets. In conclusion, formation of FXIIa-AT and FXIIa-C1INH complexes can help to distinguish between contact activation triggered by biomaterial surfaces and by activated platelets. Platelet aggregation studies also demonstrated that platelet function is influenced by material surface-mediated contact activation and that generation of FXIIa-AT complexes may serve as a new biomarker for thrombotic reactions during therapeutic procedures employing biomaterial devices.

Adenovirus-mediated expression of the anticoagulant hirudin in human islets: a tool to make the islets biocompatible to blood.

Human islets induce an injurious clotting reaction at the time of transplantation. A potential strategy to counteract this reaction would be to allow the islets to express hirudin, a protein with direct anticoagulative activity. Human islets were transduced with an adenoviral vector encoding hirudin, an empty corresponding vector, or left untreated. Islet culture supernatants were analyzed for hirudin using an ELISA, a chromogenic substrate assay based on the thrombin-binding properties of hirudin and in a whole blood viscosimetry assay. Immunohistochemical evaluation and determination of hirudin content revealed an abundant expression of hirudin after transduction. Hirudin content in transduced islets was in the range of the insulin content levels. A delay in human whole blood clotting time could be observed after addition of supernatants taken from islet cultures expressing hirudin. However, transduced islets showed an impaired glucose-stimulated insulin release, but could readily be retrieved 6 weeks after transplantation to athymic mice. A marked expression and secretion of hirudin with functional capacity can be induced in human islets using an adenoviral vector. The impairment in glucose-stimulated insulin release in hirudin-secreting islets, compared to controls, indicates that the additional protein synthesis affects the functional capacity of the islets.

Porcine Islets of Langerhans Isolated From Normal and hDAF Transgenic Pigs Elicit the Same Acute Inflammatory Reaction During Exposure to Human Blood; Inhibition of the Response With Soluble Complement Receptor 1 and Heparin

WE HAVE previously reported that porcine islets exposed to fresh human blood in vitro elicit an immediate inflammatory reaction, resulting in disruption of islet integrity. Complement inhibition prevents hyperacute rejection of vascularized discordant xenografts. We investigated whether inhibition of the complement and coagulation systems in human blood affected the outcome of porcine islet damage. We also tested whether islets from a single founder line (A74) of hDAF transgenic (TG) pigs are protected from this reaction.

Complement regulatory proteins on human and porcine nontransgenic and hDAF transgenic islet cells: expression and role in susceptibility to lysis by human serum

Complement regulatory proteins on human and porcine nontransgenic and hDAFtransgenic islet cells: expression and role in susceptibility to lysis byhuman serum.

Control of IBMIR Induced by Fresh and Cryopreserved Hepatocytes by Low Molecular Weight Dextran Sulfate versus Heparin

Rapid destruction of hepatocytes after hepatocyte transplantation has hampered the application of this procedure clinically. The instant blood-mediated inflammatory reaction (IBMIR) is a plausible underlying cause for this cell loss. The present study was designed to evaluate the capacity of low molecular weight dextran sulfate (LMW-DS) to control these initial reactions from the innate immune system. Fresh and cryopreserved hepatocytes were tested in an in vitro whole-blood model using ABO-compatible blood. The ability to elicit IBMIR and the capacity of LMW-DS (100 μg/ml) to attenuate the degree of activation of the cascade systems were monitored. The effect was also compared to conventional anticoagulant therapy using unfractionated heparin (1 IU/ml). Both fresh and freeze–thawed hepatocytes elicited IBMIR to the same extent. LMW-DS reduced the platelet loss and maintained the cell counts at the same degree as unfractionated heparin, but controlled the coagulation and complement systems significantly more efficiently than heparin. LMW-DS also attenuated the IBMIR elicited by freeze–thawed cells. Therefore, LMW-DS inhibits the cascade systems and maintains the cell counts in blood triggered by both fresh and cryopreserved hepatocytes in direct contact with ABO-matched blood. LMW-DS at a previously used and clinically applicable concentration (100 μg/ml) inhibits IBMIR in vitro and is therefore a potential IBMIR inhibitor in hepatocyte transplantation.