Human AB Research Articles

Mesenchymal stem cells and cardiac regeneration: a sophisticated approach depends on trophic effects—what's left over? Focus on “Activation of Toll-like receptor 3 amplifies mesenchymal stem cell trophic factors and enhances therapeutic potency”

application of stem cells for tissue, in particular cardiac, regeneration represents a promising field of investigation but has not achieved clinical utility ([Fig. 1][1]). From the latter point of view, Strauer and colleagues ([7][2]) in 2001 achieved an important milestone by their administration

Impact on N-Glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties

Prophylaxis of hemolytic disease of newborns is based on the ability of polyclonal anti-D antibodies for suppressing maternal immune response against D-positive fetal red blood cells. The immunosuppressive effect of anti-D antibody is mediated by interaction between its Fc-fragment and low-affinity IgG Fc-receptor (FcγR) on the immune cell. No clinically effective monoclonal anti-D antibody (mAb) that can replace polyclonal anti-D immunoglobulin has been developed yet. The goals of this study were comparison of structural and functional properties of human anti-D polyclonal and monoclonal Abs and assessment of the possibility to manipulate the effector properties of the mAb. N-Glycosylation and particularly the content of nonfucosylated glycans are crucial for affinity of mAb to FcγRIIIA, which plays the key role in the clearance of sensitized cells. We studied and compared glycoprofiles and FcγRIIIA-mediated hemolytic ability of human polyclonal antibodies and anti-D mAbs produced by human B-cell lines, human-rodent heterohybridomas, and a human non-lymphoid cell line PER.C6. Replacement of producing cell line and use of glycosylation modulators can convert an inert mAb into an active one. Nevertheless, rodent cell lines, as well as human non-lymphoid cells, distort natural glycosylation of human IgG and could lead to the loss of immunosuppressive properties. All of the anti-D mAbs secreted by human B-cell lines have a glycoprofile close to human serum IgG. Hence, the constant ratio of IgG glycoforms in human serum is predetermined by glycosylation at the level of the individual antibody-producing cell. The anti-D fraction of polyclonal anti-D immunoglobulin compared to the total human IgG contains more nonfucosylated glycans. Thus, only human transformed B-cells are an appropriate source for efficient anti-D mAbs that can imitate the action of polyclonal anti-D IgG.

Peptide mimotopes of malondialdehyde epitopes for clinical applications in cardiovascular disease

Autoantibodies specific for malondialdehyde-modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, MDA-LDL is a high variability antigen with limited reproducibility. To identify peptide mimotopes of MDA-LDL, phage display libraries were screened with the MDA-LDL-specific IgM monoclonal Ab LRO4, and the specificity and antigenic properties of MDA mimotopes were assessed in vitro and in vivo. We identified one 12-mer linear (P1) and one 7-mer cyclic (P2) peptide carrying a consensus sequence, which bound specifically to murine and human anti-MDA monoclonal Abs. Furthermore, MDA mimotopes were found to mimic MDA epitopes on the surface of apoptotic cells. Immunization of mice with P2 resulted in the induction of MDA-LDL-specific Abs, which strongly immunostained human atherosclerotic lesions. We detected IgG and IgM autoAbs to both MDA mimotopes in sera of healthy subjects and patients with myocardial infarction and stable angina pectoris undergoing percutaneous coronary intervention, and the titers of autoAbs correlated significantly with respective Ab titers against MDA-LDL. In conclusion, we identified specific peptides that are immunological mimotopes of MDA. These mimotopes can serve as standardized and reproducible antigens that will be useful for diagnostic and therapeutic applications in cardiovascular disease.

Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer

AbstractCancer patients often have an activated clotting system and are at increased risk for venous thrombosis. In the present study, we analyzed tissue factor (TF) expression in 4 different human pancreatic tumor cell lines for the purpose of producing derivative tumors in vivo. We found that 2 of the lines expressed TF and released TF-positive microparticles (MPs) into the culture medium. The majority of TF protein in the culture medium was associated with MPs. Only TF-positive cell lines activated coagulation in nude mice, and this activation was abolished by an anti–human TF Ab. Of the 2 TF-positive lines, only one produced detectable levels of human MP TF activity in the plasma when grown orthotopically in nude mice. Surprisingly, < 5% of human TF protein in plasma from tumor-bearing mice was associated with MPs. Mice with TF-positive tumors and elevated levels of circulating TF-positive MPs had increased thrombosis in a saphenous vein model. In contrast, we observed no difference in thrombus weight between tumor-bearing and control mice in an inferior vena cava stenosis model. The results of the present study using a xenograft mouse model suggest that tumor TF activates coagulation, whereas TF on circulating MPs may trigger venous thrombosis.

Human B-cell ontogeny in humanized NOD/SCID γcnull mice generates a diverse yet auto/poly- and HIV-1-reactive antibody repertoire

Characterization of the human antibody (Ab) repertoire in mouse models of the human immune system is essential to establish their relevance in translational studies. Single human B-cells were sorted from bone marrow and periphery of humanized NOD/SCID γcnull mice at 8–10 months post-engraftment with human cord blood-derived CD34+ stem cells. Human immunoglobulin variable heavy (VH) and kappa (Vκ) genes were amplified, cognate VH-Vκ gene-pairs assembled as single-chain variable fragment-Fc antibodies (scFvFcs) and functional studies performed. Although overall distribution of VH genes approximated the normal human Ab repertoire, analysis of the VH-third complementarity determining regions (H-CDR3) in the mature B-cell subset demonstrated an increase in length and positive charges suggesting autoimmune characteristics. Additionally, >70% of Vκ sequences utilized Vκ4-1, a germline gene associated with autoimmunity. The mature B-cell subset-derived scFvFcs displayed the highest frequency of autoreactivity and polyspecificity, suggesting defects in checkpoint control mechanisms. Furthermore, these scFvFcs demonstrated binding to recombinant HIV envelope corroborating previous observations of poly/autoreactivity in anti-HIVgp140 antibodies. These data lend support to the hypothesis that anti-HIV BnAbs may be derived from auto/polyspecific Abs that escaped immune elimination and that the hNSG mouse could provide a new experimental platform for studying the origin of anti-HIV neutralizing Ab responses.

Different Adaptations of IgG Effector Function in Human and Nonhuman Primates and Implications for Therapeutic Antibody Treatment

Safety of human therapeutic Abs is generally assessed in nonhuman primates. Whereas IgG1 shows identical FcγR interaction and effector function profile in both species, fundamental differences in the IgG2 and IgG4 Ab subclasses were found between the two species. Granulocytes, the main effector cells against IgG2- and IgG4-opsonized bacteria and parasites, do not express FcγRIIIb, but show higher levels of FcγRII in cynomolgus monkey. In humans, IgG2 and IgG4 adapted a silent Fc region with weak binding to FcγR and effector functions, whereas, in contrast, cynomolgus monkey IgG2 and IgG4 display strong effector function as well as differences in IgG4 Fab arm exchange. To balance this shift toward activation, the cynomolgus inhibitory FcγRIIb shows strongly increased affinity for IgG2. In view of these findings, in vitro and in vivo results for human IgG2 and IgG4 obtained in the cynomolgus monkey have to be cautiously interpreted, whereas effector function-related effects of human IgG1 Abs are expected to be predictable for humans.

Humoral anti-KLH responses in cancer patients treated with dendritic cell-based immunotherapy are dictated by different vaccination parameters

PurposeKeyhole limpet hemocyanin (KLH) attracts biomedical interest because of its remarkable immunostimulatory properties. Currently, KLH is used as vaccine adjuvant, carrier protein for haptens and as local treatment for bladder cancer. Since a quantitative human anti-KLH assay is lacking, it has not been possible to monitor the dynamics of KLH-specific antibody (Ab) responses after in vivo KLH exposure. We designed a quantitative assay to measure KLH-specific Abs in humans and retrospectively studied the relation between vaccination parameters and the vaccine-induced anti-KLH Ab responses.Experimental designAnti-KLH Abs were purified from pooled serum of melanoma patients who have responded to KLH as a vaccine adjuvant. Standard isotype-specific calibration curves were generated to measure KLH-specific Ab responses in individual serum samples using ELISA.ResultsKLH-specific IgM, IgA, IgG and all IgG-subclasses were accurately measured at concentrations as low as 20 μg/ml. The intra- and inter-assay coefficients of variation of this ELISA were below 6.7 and 9.9 %, respectively. Analyses of 128 patients demonstrated that mature DC induced higher levels of KLH-specific IgG compared to immature DC, prior infusion with anti-CD25 abolished IgG and IgM production and patients with locoregional disease developed more robust IgG responses than advanced metastatic melanoma patients.ConclusionsWe present the first quantitative assay to measure KLH-specific Abs in human serum, which now enables monitoring both the dynamics and absolute concentrations of humoral immune responses in individuals exposed to KLH. This assay may provide a valuable biomarker for the immunogenicity and clinical effectiveness of KLH-containing vaccines and therapies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-012-1263-z) contains supplementary material, which is available to authorized users.

Generation of equine TSLP-specific antibodies and their use for detection of TSLP produced by equine keratinocytes and leukocytes

Allergic horses react to innocuous environmental substances by activation of Th2 cells and production of allergen-specific IgE antibodies. The mechanisms leading to Th2 differentiation are not well understood. In humans and mice, epithelial cell-derived thymic stromal lymphopoietin (TSLP) plays a central role in this process. Little is known about equine TSLP (eqTSLP) and its role in allergic diseases and our current knowledge is limited to the assessment of TSLP mRNA expression.In order to be able to study eqTSLP at the protein level, the aim of the present study was to produce recombinant eqTSLP protein and generate TSLP-specific antibodies.EqTSLP was cloned from a skin biopsy sample from a horse with chronic urticaria and eqTSLP protein was expressed in E.coli and in mammalian cells. Recombinant proteins were designed to include C-terminal Histag, which allowed subsequent purification and detection by Histag-specific Ab. Polyclonal and monoclonal eqTSLP-specific Ab were generated after immunization of mice with E.coli-expressed TSLP. Their specificity was tested by western blotting and ELISA. In addition, a commercially available polyclonal human TSLP-specific antibody was tested for cross-reactivity with eqTSLP.Expression of TSLP protein was confirmed by western blotting using Histag-specific Ab. E.coli-expressed TSLP appears as a band of ∼13kDa, whereas mammalian cell-expressed TSLP showed several bands at 20–25kDa, probably representing several glycosylation forms. Polyclonal and monoclonal antibodies generated in this study, as well as commercially available human TSLP-specific Ab reacted with both E.coli- and mammalian cell-expressed TSLP in western blotting and ELISA. A capture ELISA was established to quantitate TSLP in cell supernatants and validated using supernatants from primary equine keratinocytes and peripheral blood leukocytes (PBL). Increased TSLP concentrations were found after stimulation of keratinocytes with PMA+ionomycine and with Culicoides extract. Similarly, increased TSLP concentrations were detected in PBL after stimulation with ConA, Culicoides extract, or IgE cross-linking.In conclusion, recombinant TSLP proteins and TSLP-specific antibodies produced in this study will allow further studies of the role of TSLP in equine allergic diseases.

Abstract 2724: Activity of the specific anti-ADAM17 inhibitory IgG antibody (Ab), D1(A12) in an ovarian cancer model in vivo

Abstract ADAM17 (TNF-α converting enzyme, TACE) is a membrane-bound metalloproteinase responsible for ectodomain shedding of TNF-α, EGFR ligands and other pathologically significant proteins. ADAM17 overexpression is reported in many cancers, with roles in cancer cell proliferation, migration and drug resistance. Small molecule inhibitors of ADAM17 lack specificity, so we developed a specific human ADAM17 inhibitory IgG Ab, D1(A12), which inhibits the proteolysis of ADAM17 substrates (TNF-α, amphiregulin, etc.,) in cancer cells in vitro (C.J. Tape, et al., Proc Natl Acad Sci USA 108: 5578-83, 2011). We have now assessed the suitability of the D1(A12) Ab for therapeutic use, by investigating its pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumour efficacy in mice. The IGROV1-Luc xenograft model of intraperitoneal (i.p.) disseminated ovarian carcinoma in nude mice was used, as IGROV1-Luc cells secrete TNF-α and other ADAM17 products and knockdown of TNF-α expression inhibits tumour growth (H. Kulbe, et al., Cancer Res., 67: 585- 592, 2007). We investigated the PK of D1(A12) Ab using a single 10 mg/kg dose i.p., first in non-tumour-bearing (NTB) mice and then in mice bearing IGROV1-Luc tumours, measuring the concentration of D1(A12) IgG in plasma and ascitic fluid by ELISA. In NTB mice, plasma Cmax was 512 nM, half life 8.6 days. The concentrations in tumour-bearing mice were expected to be lower due to a larger volume of distribution due to the ascitic fluid, and in tumour-bearing mice the Cmax was 425 nM in plasma and 391 nM in ascitic fluid. The PK data suggest that weekly dosing with 10 mg/kg D1(A12) maintains therapeutically active concentrations of the IgG, so this regimen was used for an efficacy study comparing D1(A12) with vehicle. D1(A12) showed clear PD effects: Ascitic fluid concentrations of 3 ADAM17 products were reduced when compared to vehicle: AREG (55 +/− 19 vs 302 +/− 37 pg/ml, p &amp;lt; 0.001), soluble hTNFR1α (479 +/− 100 vs 2108 +/− 204 pg/ml, p &amp;lt; 0.001) and TGF-β (0.35 +/− 0.67 vs 5.2 +/− 2.2 pg/ml, p = 0.002), indicative of inhibition of ADAM17 activity in vivo. However, D1(A12) did not significantly reduce the concentration of TNF-α in ascitic fluid (109 +/− 21 vs 134 +/−32 pg/ml, p = 0.06), suggesting that in this system ADAM17 is not the only metalloproteinase with TACE activity. Tumour burden was quantified using bioluminescence generated from the luciferase expressing cells, measured using an IVIS xenogen 200 imager. There was decreased tumour growth in the D1(A12)-treated group (compared to vehicle), although this did not reach statistical significance (p=0.09). In conclusion we have shown that the anti-ADAM17 antibody has PK properties suitable for therapeutic studies and that it can inhibit shedding of ADAM17-dependent growth factors in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2724. doi:1538-7445.AM2012-2724

Abstract 1559: A new immunotherapy for pancreatic cancer stem cells by tumor lysate vaccine, expressing α-gal epitopes

Abstract Pancreatic cancer (PC) cells, expressing the markers CD44, CD24, and epithelial-specific antigen (ESA) were identified as PC stem cells (CSCs). Treatments targeting the CSCs may be more effective in resulting in PC cure, but traditional treatments, including chemo/radiation therapy can easily miss CSCs, because they are highly resistant to these therapies. Human natural Ab, anti-Gal is an IgG known to be present in large amounts in normal subjects and cancer patients, comprising ∼1% of serum circulating IgG. Anti-Gal specifically interacts with α-gal epitopes (Galα1, 3Galα1, 4GlcNAc-R), synthesized by α1, 3 galactosyltransferase (≥1,3GT) on cell surface glycolipids and glycoproteins. We hypothesized that biosynthesis of α-gal epitopes on the carbohydrate of CSC markers, including CD44, CD24 could effectively induce Ab production against CSCs and immune-mediate eradication of PC CSCs. To address this strategy, a human PC cell line, PANC1, which expresses MUC1 was employed and transfected with α1,3GT gene (α-gal PANC1). To generate tumor lysate, 2 X 106 of either parental or α-gal PANC1 were injected S.C. into NOD/SCID mice, and the grown tumors were enucleated and homogenized. High anti-Gal α1,3GT KO mice, which displayed anti-Gal titers similar to those found in humans, were generated by immunization of pig tissue. These mice were vaccinated intraperitoneally (ip) by 10mg of either parental (parental group) or α-gal PANC1 tumor lysate (α-gal group). We isolated PANC1 cells with CD44+CD24+ phenotype by magnetic beads and assessed Ab production toward both CSCs (binding PANC1 with beads) and differentiated cancer cells (unbinding PANC1 with beads). In FACS analysis, a marked Ab production against both CSCs and differentiated cancer cells were observed in α-gal group. To demonstrate in vivo tumor destruction, an animal model was performed. Splenocytes from vaccinated KO mice were prepared, then these cells were transferred ip (30X106 cells/ip X 3 times) into NOD/SCID mice. Transferred mice were challenged with S.C. injection with either 1 X 107 of live PANC1 or 5 X 106 of CD44+CD24+ PANC1. PANC1 tumors in mice transferred from parental group reached the size of 150 mm2 at 45 days after injection. For tumors in mice transferred from α-gal group, regrowth of tumor was completely prevented. Survival of α-gal transferred mice was significantly prolonged [α-gal vs. parental: 82.5±21.9 vs. 48.0±6.7 days, p&amp;lt;0.0001]. CSC tumors in mice transferred from parental group reached the size of 100 mm2 at 49 days after injection. For CSC tumors in mice from α-gal group, no tumor formation was observed. Survival was significantly prolonged [α-gal vs. parental: 85.0±20.8 vs. 49.3±14.3 days, p=0.0002]. We conclude that immunotherapy with tumor lysate vaccine, expressing α-gal epitopes effectively targets both differentiated and PC CSCs and may provide PC cure by destruction of micro-metastasis and minimal residual cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1559. doi:1538-7445.AM2012-1559

Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions

Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified. Here, we demonstrate that the dengue virus (DENV)-specific serum Ab response in humans consists of a large fraction of cross-reactive, poorly neutralizing Abs and a small fraction of serotype-specific, potently inhibitory Abs. Although many mouse-generated, strongly neutralizing monoclonal antibodies (mAbs) recognize epitopes that are present on recombinant DENV envelope (E) proteins, unexpectedly, the majority of neutralizing Abs in human immune sera bound to intact virions but not to the ectodomain of purified soluble E proteins. These conclusions with polyclonal Abs were confirmed with newly generated human mAbs derived from DENV-immune individuals. Two of three strongly neutralizing human mAbs bound to E protein epitopes that were preserved on the virion but not on recombinant E (rE) protein. We propose that humans produce Abs that neutralize DENV infection by binding a complex, quaternary structure epitope that is expressed only when E proteins are assembled on a virus particle. Mapping studies indicate that this epitope has a footprint that spans adjacent E protein dimers and includes residues at the hinge between domains I and II of E protein. These results have significant implications for the DENV Ab and vaccine field.

Enhancement of the Antitumor Effect on Combination Therapy of an Anticancer Drug and Its Antibody against Carcinoembryonic Antigen

Background: Carcinoembryonic antigen (CEA) is frequently overexpressed in various types of human cancers and is associated with cell adhesion. There are three possible mechanisms of cancer therapy that employ anti-CEA antibody (Ab): Ab-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or the prevention of CEA interaction with the extracellular matrix and/or intercellular adhesion molecules resulting in anoikis. In this study, the effect of C2-74, a human anti-CEA monoclonal Ab was evaluated. Methods: ADCC, CDC and anoikis assays in combination with C2-74 and an anticancer drug (5-fluorouracil or cisplatin) were investigated using tumor cell lines (MKN-45, MKN-74 and KATO III). In the anoikis assay, other human anti-CEA Abs and mouse anti-CEA-related cell adhesion molecule 6 Abs were also investigated using HLC-1 cells. Results: Additive cytotoxicity was observed when the anticancer drug and C2-74 on tumor cells were combined in the CDC assays, whereas in the anoikis assay, no such additive effect was observed. Anti-CEA-related cell adhesion molecule 6 Abs, but not anti-CEA Abs, accelerated anoikis in HLC-1 cells. Conclusion: A mechanism for the additive antitumor effect when an anticancer drug and C2-74 are combined is indicated mainly by CDC activity but is irrelevant to anoikis in tumor cells.

A novel hypothesis for the etiology of Graves’ disease: TSAb may be thyroid stimulating animal IgG-like hormone and TBAb may be the precursor of TSAb

There are doubtful points about the theory that autoimmunity with auto-antibody (Ab) to TSH receptor (R) causes hyperthyroidism in Graves’ disease (GD). A main doubtful point is no curative effect of corticosteroid on Graves’ hyperthyroidism in spite of curative effect of corticosteroid for all autoimmune diseases. Recently we demonstrated the immunological similarity of TSAb and TBAb-IgG to animal IgGs, except for human (h)IgG, by neutralization and purification of TSAb and TBAb-IgG using (1) heterophilic Ab to animal IgG in GD sera and (2) experimentally generated anti-animal IgG Abs [such as dog (d), bovine (b), porcine (p), and rabbit (rb)]. Furthermore, greater immunological similarity of Fab- and F(ab′)2-portion of TSAb- and TBAb-IgG to bovine Fab, compared to hFab, was demonstrated using goat anti-bovine F(ab′)2 Ab.Existence of b and p TSH-like portions in the LATS-IgG molecule (probably Fab portion) was suggested by a previous report of neutralization of LATS activity by anti-b- or anti-p-TSH Ab. We suggested the existence of a mammalian animal-TSH-like structure, excepting hTSH, in the TSAb-IgG molecule (probably Fab portion), by discovery of anti-mammalian TSH Ab (such as d, b, p, guinea-pig, rat, whale, except h) in sera of GD. Lately, similar TSHR binding of H- and L-chain of human stimulating monoclonal TSHR Ab (M22)-Fab with TSH-α and-β subunit was reported. This evidence suggests that Fab portion of TSAb has a structure like mammalian TSH, but not hTSH.IgG-λ type of d, horse, b, p, goat, ovine is 95% and IgG-κ type is 5%, while human κ and λ chain is 60:40. Previous report that LATS (TSAb)-IgG composed of predominant λ type is supporting evidence that TRAb-IgG has immunological similarity with these animal IgGs compared to hIgG.We speculate that TSAb-IgG may be referred as a mermaid consisted in face (Fab) and trunk-leg (Fc). Face may be a kind of hormone with animal TSH-like structure and trunk-leg has animal IgG-like structure (in spite of no antibody function).There are many reports for co-existence of TSAb and TBAb-IgG in sera of GD. We reported conversion from TBAb (non-thyroid stimulating type IgG) to TSAb by co-incubation of anti-hIgG Ab (containing anti-animal IgG Ab as a cross-reaction) with TBAb-bound porcine thyroid cells. Thus, we suggest that TBAb may be the precursor form of TSAb.

Interaction of the Profilaggrin N-Terminal Domain with Loricrin in Human Cultured Keratinocytes and Epidermis

The relationship between the two coexpressed differentiation markers, profilaggrin and loricrin, is not clear right now. In this study, we explored the interaction of profilaggrin N-terminal domain (PND) with loricrin in keratinocytes and epidermis. Confocal immunofluorescence microscopic analysis of human epidermis showed that PND colocalized with loricrin. Loricrin nucleofected into HaCaT cells colocalized with PND in the nucleus and cytoplasm. The PND localizes to both the nucleus and cytoplasm of epidermal granular layer cells. Nucleofected PND also colocalized with keratin 10 (K10) in the nucleus and cytoplasm. Immunoelectron microscopic analysis of human epidermis confirmed the findings in nucleofected keratinocytes. Yeast two-hybrid assays showed that the B domain of human and mouse PND interacted with loricrin. The glutathione S-transferase (GST) pull-down analysis using recombinant GST-PND revealed that PND interacted with loricrin and K10. Knockdown of PND in an organotypic skin culture model caused loss of filaggrin expression and a reduction in both the size and number of keratohyalin granules, as well as markedly reduced expression of loricrin. Considering that expression of PND is closely linked to keratinocyte terminal differentiation, we conclude that PND interacts with loricrin and K10 in vivo and that these interactions are likely to be relevant for cornified envelope assembly and subsequent epidermal barrier formation.

Multiple Receptors Trigger Human NK Cell-Mediated Cytotoxicity against Porcine Chondrocytes

Xenotransplantation of genetically engineered porcine chondrocytes may provide a therapeutic solution for the repair of cartilage defects of various types. However, the mechanisms underlying the humoral and cellular responses that lead to rejection of xenogeneic cartilage are not well understood. In this study, we investigated the interaction between human NK cells and isolated porcine costal chondrocytes (PCC). Our data show that freshly isolated NK cells adhere weakly to PCC. Consequently, PCC were highly resistant to cytolysis mediated by freshly isolated NK cells. However, the presence of human natural Abs in the coculture was often sufficient to trigger cytotoxicity against PCC. Furthermore, IL-2 stimulation of NK cells or activation of PCC with the proinflammatory cytokines TNF-α or IL-1α resulted in increased adhesion, which was paralleled by increased NK cell-mediated lysis of PCC. NK cell adhesion to PCC could be blocked by Abs against human LFA-1 and porcine VCAM-1. NKG2D and NKp44 were involved in triggering cytotoxicity against PCC, which expressed ligands for these activating NK cell receptors. Our data further suggest that NKp30 and NKp46 may contribute to the activation of NK cells by PCC under certain conditions. Finally, comparative studies confirmed that PCC are more resistant than porcine aortic endothelial cells to human NK cell-mediated lysis. Thus, the data demonstrate that human NK cells can kill pig chondrocytes and may therefore contribute to rejection of xenogeneic cartilage. In addition, we identify potential targets for intervention to prevent the NK cell response against pig xenografts.

Replicative aging and differentiation potential of human adipose tissue-derived mesenchymal stromal cells expanded in pooled human or fetal bovine serum

Mesenchymal stromal cells (MSC) are promising candidates for innovative cell therapeutic applications. For clinical-scale manufacturing, different supplements have been evaluated as alternatives for the commonly used fetal bovine serum (FBS). We have reported previously that pooled human AB serum (HS) accelerates the proliferation of adipose tissue-derived MSC (ASC) while maintaining key functions of MSC biology such as differentiation, immune suppression and growth factor secretion. ASC expanded in FBS-supplemented culture media undergo replicative aging that is associated with a progressive loss of differentiation capacity but without indications of cellular transformation. The effects of HS media on ASC long-term culture, however, remain poorly characterized. Long-term cultures of ASC in FBS and HS media were analyzed with respect to proliferation, marker expression, differentiation and immune suppression. Despite signs of an accelerated proliferation, extended life span and clonogenic capacity of ASC cultivated in HS-supplemented media, HS and FBS cultures revealed no significant differences with respect to differentiation potential and expression of senescence markers. Anchorage-independent growth, which is indicative of tumorigenic properties, was not observed in either culture conditions. Similarly, immune suppressive activities were maintained. Donor variation regarding differentiation potential and marker expression became apparent in this study independent of the culture supplement or culture duration. We have demonstrated that the use of pooled allogeneic HS maintains the characteristics of ASC even after long-term expansion, further demonstrating that the use of HS is an alternative to FBS.

Production of large numbers of plasmacytoid dendritic cells with functional activities from CD34+ hematopoietic progenitor cells: Use of interleukin-3

Plasmacytoid dendritic cells (pDC), a subset of dendritic cells characterized by a rapid and massive type-I interferon secretion through the Toll-like receptor pathway in response to viral infection, play important roles in the pathogenesis of several diseases, such as chronic viral infections (e.g., hepatitis C virus, human immunodeficiency virus), autoimmunity (e.g., psoriasis, systemic lupus erythematosus), and cancer. As pDC represent a rare cell type in the peripheral blood, the goal of this study was to develop a new method to efficiently generate large numbers of cells from a limited number of CD34(+) cord blood progenitors to provide a tool to resolve important questions about how pDC mediate tolerance, autoimmunity, and cancer. Human CD34(+) hematopoietic progenitor cells isolated from cord blood were cultured with a combination of Flt3-ligand (Flt3L), thrombopoietin (TPO), and one of the following cytokine: interleukin (IL)-3, interferon-β(IFN-β), or prostaglandin E2(PGE(2)). Cells obtained in the different culture conditions were analyzed for their phenotype and functional characteristics. The addition of IL-3 cooperates with Flt3L and TPO in the induction of pDC from CD34(+) hematopoietic progenitor cells. Indeed, Flt3L/TPO alone or supplemented with prostaglandin E2 or interferon-β produced smaller amounts of pDC from hematopoietic progenitor cells. In addition, pDC generated in Flt3L/TPO/IL-3 cultures exhibited morphological, immunohistochemical, and functional features of peripheral blood pDC. We showed that IL-3, in association with Flt3L and TPO, provides an advantageous tool for large-scale generation of pDC. This culture condition generated, starting from 2 × 10(5) CD34(+) cells, up to 2.6 × 10(6) pDC presenting features of blood pDC.

EN

EN

A synthetic double-stranded RNA, poly I:C, induces a rapid apoptosis of human CD34+ cells

Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and melanoma differentiation-associated antigen 5 (RIG-I/MDA-5) helicases are known to sense double-stranded RNA (dsRNA) virus and initiate antiviral responses, such as production of type-I interferons (IFNs). Recognition of dsRNA by TLR3 or RIG-I/MDA-5 is cell-type-dependent and recent studies have shown a direct link between TLRs and hematopoiesis. We hypothesized that viral dsRNA recognized by either TLR3 or RIG-I/MDA-5, affects the growth of human hematopoietic stem/progenitor cells. Here we show that polyinosinic polycytidylic acid (poly I:C)-mediated very rapid apoptosis occurs within 1 hour in CD34(+) cells in a dose-dependent manner. Polyadenylic-polyuridylic acid, another synthetic dsRNA that signals only through TLR3, had no effect. Poly I:C-LMW/LyoVec, a complex between low molecular-weight poly I:C and the transfection reagent LyoVec, which signals only through RIG-I/MDA-5, induces apoptosis of CD34(+) cells. A strong and sustained upregulation of messenger RNA and protein levels of Noxa, a proapoptotic BH3-only protein that can be induced by RIG-I/MDA-5 pathway, is found in CD34(+) cells treated by poly I:C. Although poly I:C upregulates type-I IFNs in CD34(+) cells, neither exogenous IFN-α nor IFN-β induces rapid apoptosis in CD34(+) cells and neutralization or blocking of type-I IFN receptor does not rescue CD34(+) cells, whereas Z-VAD, a pan-caspase inhibitor, rescues the cells from apoptosis. These results suggest that RIG-I/MDA-5, but not TLR3, signaling triggers poly I:C-induced rapid apoptosis of human CD34(+) cells, which will provide an insight into the mechanisms of dsRNA virus-mediated hematopoietic disorders.

Human AB serum for generation of mesenchymal stem cells from human chorionic villi: comparison with other source and other media including platelet lysate

We have investigated foetal mesenchymal stem cells (MSCs) obtained from first-trimester chorionic villi (CV) and second-trimester amniotic fluid (AF), comparing them to adult bone marrow-derived MSCs. We report on cell population growth in human allogeneic serum (HS) and platelet lysate (PL), immunophenotype, cytokine expression profile and immunoregulatory activity, of these foetal MSCs on stimulated peripheral blood mononuclear and lymphocyte subpopulations. Chorionic villi cells grow rapidly in HS, with 20 populations doublings (PDs) after 59 days (six passages), and also in animal serum, with 27 PDs after 65 days (seven passages). PL allowed for expansion in 60% of the samples tested, although it was lower than in HS. HS supported an average of 40 PDs of expansion in 20% of AF cells after 90 days, whereas animal serum supported 28.5 PDs in 66 days. CV and AF cells inhibited proliferation of stimulated T lymphocytes, suppressing population growth of both CD4+ and CD8+ T subpopulations and sometimes also, CD19+ cells. Our results indicate that CV would be an optimal source of MSCs with high expansion potential in a HS propagation system and immunoregulatory capacity of T and B lymphocytes. More than 90% of CV samples achieved large-scale expansion in HS, which is encouraging for potential clinical applications of these cells.