Bjørg Steinkjer

Alginate microbeads are complement compatible, in contrast to polycation containing microcapsules, as revealed in a human whole blood model

Alginate microbeads and microcapsules are presently under evaluation for future cell-based therapy. Defining their inflammatory properties with regard to humans is therefore essential. A lepirudine-based human whole blood model was used as an inflammation predictor by measuring complement and leukocyte stimulation. Alginate microbeads were complement-compatible since they did not activate complement as measured by the soluble terminal complement complex (sTCC), Bb or the anaphylatoxins C3a and C5a. In addition, alginate microbeads were free of surface adherent leukocytes. In contrast, microcapsules containing poly-L-lysine (PLL) induced elevated levels of sTCC, Bb, C3a and C5a, surface active C3 convertase and leukocyte adhesion. The soluble PLL induced elevated levels of sTCC and up-regulated leukocyte CD11b expression. PMCG microcapsules containing poly(methylene-co-guanidine) complexed with sodium alginate and cellulose sulfate triggered a fast sTCC response and C3 deposition. The PMCG microcapsules were still less activating than PLL-containing microcapsules as a function of time. The amounts of anaphylatoxins C3a and C5a were diminished by the PMCG microcapsules, whereas leukocyte adherence demonstrated surface activating properties. We propose the whole blood model as an important tool for measuring bioincompatibility of microcapsules and microbeads for future applications as well as determining the mechanisms leading to inflammatory reactions.

Microencapsulation of Cells Producing Therapeutic Proteins: Optimizing Cell Growth and Secretion

Microencapsulation of genetically engineered cells may have important applications as delivery systems for therapeutic proteins. However, optimization of the microcapsules with regard to mechanical stability, cell growth, and secretion of proteins is necessary in order to evaluate the future use of this delivery technology. We have explored the growth, survival, and secretion of therapeutic proteins from 293-EBNA cells producing endostatin (293 endo cells) and JJN3 myeloma cells producing hepatocyte growth factor (HGF) that have been embedded in various types of alginate capsules. Parameters that affect capsule integrity such as homogenous and inhomogenous gel cores and addition of an outer poly-l-lysine (PLL)–alginate coating were evaluated in relation to cell functions. When cells were encapsulated, the PLL layer was found to be absolutely required for the capsule integrity. The JJN3 and 293 endo cells displayed completely different growth and distribution patterns of live and dead cells within the microcapsules, as shown by 3D pictures reconstructed from images taken with confocal laser scanning microscopy (CLSM). Encapsulated JJN3 cells showed a bell-shaped growth and HGF secretion curve over a time period of 5 months. The 293 endo cells reached a plateau phase in growth after 23 days postencapsulation; however, after around 30 days a fraction of the microcapsules started to disintegrate. Microcapsule disintegration occurred with time irrespective of capsule and cell type, showing that alginate microcapsules possessing relatively high gel strength are not strong enough to keep proliferating cells within the microcapsules for prolonged time periods. Although this study shows that the stability of an alginate-based cell factory can be increased by a PLL–alginate coating, further improvement is necessary with regard to capsule integrity as well as controlling the cell growth before this technology can be used for therapy.

Immunomagnetic isolation of NK and LAK cells.

The present study describes the immunomagnetic isolation of human natural killer (NK) and lymphokine activated killer (LAK) cells. Antibodies against CD56 and sheep anti-mouse IgG-coated magnetic monodisperse particles (Dynabeads M-450) were used for the positive isolation of CD56+ cells from unstimulated mononuclear cells (PBMC). A highly enriched population of CD56+ cells (less than or equal to 3% contaminating cells) was obtained with this method. The cellular yield of CD56+ cells was high (5.3% of the unseparated PBMC). The CD56+ cells remained unactivated after separation and preserved their functional characteristics, as measured by cytotoxic activity against the NK sensitive K562 cells. Incubating the CD56+ cells with IL-2 resulted in high LAK activity, as measured by cytotoxic activity against Daudi cells. Large numbers of functionally active CD56+ cells were obtained from IL-2 stimulated lymphocytes using anti-CD56 coated Dynabeads 450. A further enrichment of effector cells with LAK activity was accomplished by depleting the CD56+ cells for T-cells by anti-CD3 coated Dynabeads M450. The immunomagnetic isolation technique described was easy to perform, did not require expensive equipment and yielded NK and LAK cells of satisfactory purity.

A Proviral Role for CpG in Cytomegalovirus Infection

TLR9-dependent signaling in plasmacytoid dendritic cells is a key contributor to innate immune defense to mouse CMV infection. We aimed to study the expression and potential contribution of TLR9 signaling in human CMV (HCMV) infection of primary fibroblasts. HCMV infection strongly induced TLR9 expression in two of three fibroblast types tested. Furthermore, the TLR9 ligand CpG-B induced a strong proviral effect when added shortly after HCMV infection, enhancing virus production and cell viability. However, not all CpG classes displayed proviral activity, and this correlated with their IFN-β-inducing ability. The proviral effect of CpG-B correlated completely with concurrent viral up-regulation of TLR9 in fibroblasts. Importantly, the timing of CpG addition was a critical parameter; in striking contrast to the proviral effect, CpG addition at the time of infection blocked viral uptake and nearly abolished HCMV production. The contrasting and time-dependent effects of CpG on HCMV infectivity reveal a complex interplay between CpG, TLR9, and HCMV infection. Additionally, the data suggest a potentially harmful role for CpG in the promotion of HCMV infection.

The induction of cytokines by polycation containing microspheres by a complement dependent mechanism.

The cytokine-inducing potential of various microspheres were evaluated in a short-time screening assay of lepirudin-anticoagulated human whole blood utilizing the Bio-Plex Human cytokine 27-plex system. The inflammatory cytokines IL-1β, TNF and IL-6; the anti-inflammatory mediators IL-1ra and IL-10; the chemokines IL-8, MIP-1α and MCP-1; and the growth factor VEGF were induced by polycation (poly-l-lysine or poly(methylene-co-guanidine)) containing microspheres. Alginate microspheres without polycations did not induce the corresponding cytokine panel, nor did soluble alginate. By inhibiting complement C3 using compstatin analog CP20, a total inhibition of complement activation as well as the inflammatory mediators was achieved, indicating that complement activation alone was responsible for the induced cytokines. A strong deposition of C3c on the poly-l-lysine containing surface, while not on the microspheres lacking polycations, also points to the formation of C3 convertase as involved in the biomaterial-induced cytokine induction. These results show that complement is responsible for the induction of cytokines by polycation containing microspheres. We point to complement as an important initiator of inflammatory responses to biomaterials and the lepirudin anticoagulated whole blood assay as an important tool to identify the most tolerable and safe materials for implantation to humans.

Evaluation of different types of alginate microcapsules as bioreactors for producing endostatin.

The use of nonautologous cell lines producing a therapeutic substance encapsulated within alginate microcapsules could be an alternative way of treating different diseases in a cost-effective way. Malignant brain tumors have been proposed to be treated locally using engineered cells secreting proteins with therapeutic potential encapsulated within alginate microcapsules. Optimization of the alginate capsule bioreactors is needed before this treatment can be a reality. Recently, we have demonstrated that alginate-poly-L-lysine microcapsules made with high-G alginate and a gelled core disintegrated as cells proliferated. In this study we examined the growth and endostatin secretion of 293-EBNA (293 endo) cells encapsulated in six different alginate microcapsules made with native high-G alginate or enzymatically tailored alginate. Stability studies using an osmotic pressure test showed that alginate-poly-L-lysine-alginate microcapsules made with enzymatically tailored alginate was mechanically stronger than alginate capsules made with native high-G alginate. Growth studies showed that the proliferation of 293 endo cells was diminished in microcapsules made with enzymatically tailored alginate and gelled in a barium solution. Secretion of endostatin was detected in lower amounts from the enzymatically tailored alginate microcapsules compared with the native alginate microcapsules. The stability of the alginate microcapsules diminished as the 293 endo cells grew inside the capsules, while empty alginate microcapsules remained stable. By using microcapsules made of fluorescenamine-labeled alginate it was clearly visualized that cells perforated the alginate microcapsules as they grew, destroying the alginate network. Soluble fluorescence-labeled alginate was taken up by the 293 endo cells, while alginate was not detected in live spheroids within fluorescence-labeled alginate microcapsules. Despite that increased stability was achieved by using enzymatically tailored alginate, the cell proliferation destroyed the alginate microcapsules with time. It is therefore necessary to use cell lines that have properties more suited for alginate encapsulation before this technology can be used for therapy.

The role of interleukin-2 in regulating the sensitivity of natural killer cells for Fas-mediated apoptosis.

Abstract The Fas/Fas-ligand (FasL) system seems to play a key role in regulating immunoresponses. Highly purified CD56+CD3− natural killer (NK) cells were found to be resistant to the apoptosis-inducing Fas mAb CH11 in the absence or in the presence of interleukin-2 (IL-2) for up to 3 days. However, NK cells activated with IL-2 for 3 days became apoptotic following combined treatment with CH11 and actinomycin D, suggesting the presence of an intact apoptotic machinery. In contrast, NK cells cultivated in IL-2 for 6 days became sensitive to CH11-induced apoptosis without addition of actinomycin D. At this time, a pronounced up-regulation of the Fas protein on the NK cell membrane was detected. By using reverse transcription/polymerase chain reaction it was found that the anti-apoptotic gene FLIP was strongly expressed in NK cells for up to 6 days of IL-2 stimulation. After day 6, a time-dependent decrease in the expression of FLIP was observed concomitantly with increased sensitivity for Fas-mediated apoptosis. The amount of apoptotic and necrotic NK cells in the presence of IL-2 increased in a time-dependent manner, reaching 40% at day 6 of culture. The amount of apoptotic and necrotic NK cells was reduced in the presence of Fas-Fc protein. In addition, IL-2 stimulated the NK cells to release soluble FasL in a time-dependent manner, whereas membrane FasL did not seem to increase in a similar manner. These results indicate that Fas/FasL interactions are involved in the down-regulation of IL-2-activated human NK cells.

Functional Toll-like receptors in primary first-trimester trophoblasts.

Toll-like receptors (TLRs) are an important part of the bodys danger response system and crucial for initiating inflammation in response to cellular stress, tissue damage, and infections. Proper placental development is sensitive to inflammatory activation, and a role for TLRs in trophoblast immune activation has been suggested, but no overall examination has been performed in primary trophoblasts of early pregnancy. This study aimed to broadly examine cell surface and endosomal TLR gene expression and activation in first-trimester trophoblasts. Gene expression of all ten TLRs was examined by quantitative RT-PCR (RT-qPCR) in primary first-trimester trophoblasts (n = 6) and the trophoblast cell line BeWo, and cytokine responses to TLR ligands were detected by quantitative multiplex immunoassay. Primary first-trimester trophoblasts broadly expressed all ten TLR mRNAs; TLR1, TLR2, TLR3, TLR4, and TLR6 mRNA were expressed by all primary trophoblast populations, while TLR5, TLR7, TLR8, TLR9, and TLR10 mRNA expression was more restricted. Functional response to ligand activation of cell surface TLR2/1, TLR4, and TLR5 increased IL-6 and/or IL-8 release (P < 0.01) from primary trophoblasts. For endosomal TLRs, TLR3 and TLR9 ligand exposure increased receptor-specific production of IL-8 (P < 0.01) and IFN-γ-induced protein 10 (IP-10; P < 0.001) or vascular endothelial growth factor A (VEGFA; P < 0.01). In contrast, BeWo cells expressed lower TLR mRNA levels and did not respond to TLR activation. In conclusion, primary first-trimester trophoblasts broadly express functional TLRs, with inter-individual variation, suggesting that trophoblast TLR2, TLR3, TLR4, TLR5, and TLR9 might play a role in early placental inflammation.

Alginate microbeads are coagulation compatible, while alginate microcapsules activate coagulation secondary to complement or directly through FXII

Alginate microspheres are presently under evaluation for future cell-based therapy. Their ability to induce harmful host reactions needs to be identified for developing the most suitable devices and efficient prevention strategies. We used a lepirudin based human whole blood model to investigate the coagulation potentials of alginate-based microspheres: alginate microbeads (Ca/Ba Beads), alginate poly-l-lysine microcapsules (APA and AP microcapsules) and sodium alginate-sodium cellulose sulfate-poly(methylene-co-cyanoguanidine) microcapsules (PMCG microcapsules). Coagulation activation measured by prothrombin fragments 1+2 (PTF1.2) was rapidly and markedly induced by the PMCG microcapsules, delayed and lower induced by the APA and AP microcapsules, and not induced by the Ca/Ba Beads. Monocytes tissue factor (TF) expression was similarly activated by the microcapsules, whereas not by the Ca/Ba Beads. PMCG microcapsules-induced PTF1.2 was abolished by FXII inhibition (corn trypsin inhibitor), thus pointing to activation through the contact pathway. PTF1.2 induced by the AP and APA microcapsules was inhibited by anti-TF antibody, pointing to a TF driven coagulation. The TF induced coagulation was inhibited by the complement inhibitors compstatin (C3 inhibition) and eculizumab (C5 inhibition), revealing a complement-coagulation cross-talk. This is the first study on the coagulation potentials of alginate microspheres, and identifies differences in activation potential, pathways and possible intervention points. STATEMENT OF SIGNIFICANCE Alginate microcapsules are prospective candidate materials for cell encapsulation therapy. The material surface must be free of host cell adhesion to ensure free diffusion of nutrition and oxygen to the encapsulated cells. Coagulation activation is one gateway to cellular overgrowth through deposition of fibrin. Herein we used a physiologically relevant whole blood model to investigate the coagulation potential of alginate microcapsules and microbeads. The coagulation potentials and the pathways of activation were depending on the surface properties of the materials. Activation of the complement system could also be involved, thus emphasizing a complement-coagulation cross-talk. Our findings points to complement and coagulation inhibition as intervention point for preventing host reactions, and enhance functional cell-encapsulation devices.

PP042. Cell surface toll-like receptors in primary first trimester trophoblasts

INTRODUCTION The first trimester of pregnancy is characterised by a mild pro-inflammatory environment, however excessive inflammation threatens placental development and function. Toll-like receptors (TLRs) are crucial in initiating inflammation. TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 are expressed on the cell surface, and respond to microbial infection and cell damage and stress signals. Recent findings of TLRs in trophoblasts indicate a role in inflammation during pregnancy, but further studies are warranted. OBJECTIVES To investigate gene expression and function of cell surface TLRs in first trimester trophoblasts, to extend knowledge on the role of trophoblast TLRs during placental development. METHODS Primary trophoblasts were isolated from first trimester placentas (n=6) by enzyme degradation and density gradient centrifugation. Gene expression of TLR1, TLR2, TLR4, TLR5, TLR6 and TLR10 was quantified by RT-qPCR in primary first trimester trophoblasts and the trophoblast cell line BeWo. Trophoblasts were stimulated with cell surface TLR ligands and pro-inflammatory cytokine release was analysed by multiplex immunoassay. RESULTS Primary first trimester trophoblasts expressed all cell surface TLR mRNAs, and activation of TLR2/1, TLR4 and TLR5 induced IL-6 and/or IL-8. CONCLUSION The broad expression of functional cell surface TLRs in primary first trimester trophoblasts suggests a central role for trophoblasts in placental inflammation and immune activation.