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  • Enhancing the reproducibility of serological methods used to evaluate immunogenicity of pandemic H1N1 influenza vaccines-an effective EU regulatory approach. 22446639

    Haemagglutination-inhibition (HI) and virus neutralisation (VN) assays are routinely applied to evaluate influenza vaccine immunogenicity for regulatory approval. Despite their frequent use both assays are currently only poorly standardised causing considerable inter-laboratory variation of serological results that is particularly evident for pandemic influenza vaccines. The present study was conducted in association with the European Medicines Agency (EMA) to directly compare assay variability between vaccine manufacturer's and European regulatory agency's laboratories in an influenza pandemic scenario. To this end, a defined subset of H1N1 pdm clinical trial sera from all manufacturers that had applied at EMA for approval of pandemic H1N1 vaccines were re-tested by the National Institute for Biological Standards and Control (for HI) and the Paul Ehrlich Institute (for VN). Comparative analysis of test results determined for almost 2000 serum samples revealed a marked inter-laboratory variation for HI titres (up to 5.8-fold) and even more for neutralisation titres (up to 7.0-fold). When the absolute titres were adjusted relative to the calibrated International Antibody Standard 09/194 variation was drastically reduced and acceptable agreement of results from different laboratories could be achieved. Hence, inclusion of an appropriate calibrated antibody standard for adjustment of original titres is a powerful tool to substantially increase reproducibility of serological results from different laboratories and to significantly improve regulatory evaluation of influenza vaccine efficacy.
    Document Type:
    Reference
    Product Catalog Number:
    MAB8258
    Product Catalog Name:
    Anti-Influenza A Antibody, nucleoprotein, clone A3

  • Efficiency of laryngeal motor nerve repair is greater with bulbar than with mucosal olfactory ensheathing cells. 21168497

    The real ability of OECs provided by olfactory mucosa cultures (OM-OECs) and those from olfactory bulb cultures (OB-OECs) must be better characterized in order to propose their future clinical application. Therefore, we used a lesion of the vagus nerve (VN), which constitutes a severe motor denervation due to long distance of the muscular targets (4.5 cm). We performed a section/anastomosis surgery of the VN, at the third tracheal ring. Then, OM-OECs and OB-OECs were injected in matrigel around the lesion site. Three months after surgery, laryngeal muscle activity, synkinesis phenomena and latency were evaluated by videolaryngoscopy and electromyography recordings. To complete these procedures, axonal morphometric study of the right recurrent nerve was performed to assess axonal regrowth and tracking of green fluorescent protein positive cells was performed. Recurrent nerve is the motor branch innervating the laryngeal muscles, and is located distally to the lesion, near the muscular targets (0.7 cm). These analyses permitted to compare the ability of these two populations to improve functional recovery and axonal regrowth. Our results show that, OM-OECs improved electrical muscular activity and nervous conduction with significant tissue healing but induced aberrant movement and poor functional recovery. In contrast, OB-OECs induced a partial functional recovery associated with an increase in the number of myelinated fibers and nervous conduction. Our study suggests that, as recently reported in a microarray study, OM-OECs and OB-OECs express different properties. In particular, OM-OECs could regulate inflammation processes and extracellular matrix formation but have a poor regeneration potential, whereas, OB-OECs could improve functional recovery by inducing targeted axonal regrowth.
    Document Type:
    Reference
    Product Catalog Number:
    MAB365
    Product Catalog Name:
    Anti-Nerve Growth Factor Receptor Antibody, extracellular, clone 192-IgG

  • TGF-β-stimulated CTGF production enhanced by collagen and associated with biogenesis of a novel 31-kDa CTGF form in human corneal fibroblasts. 20393108

    Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-β) after corneal wounding. This study addressed the role of the extracellular matrix in the induction of CTGF by TGF-β.Human corneal fibroblasts (HCFs) were grown on fibronectin (FN), vitronectin (VN), or collagen (CL) in supplemented serum-free media alone or with TGF-β1 or fibroblast growth factor plus heparin. CTGF mRNA was analyzed by qPCR and protein expression by Western blot analysis of Triton X-100 (TX-100)-soluble and TX-100-insoluble cell lysates using antibodies to N-terminal, mid, and C-terminal CTGF regions. Immunocytochemistry was performed on nonconfluent or scrape-wounded confluent HCFs.TGF-β-treated HCFs grown on CL produced five times more 38-kDa CTGF than untreated controls (72 hours). TGF-β-treated HCFs on CL secreted twofold more CTGF than those on FN or VN. Furthermore, a 31-kDa CTGF form, lacking the N-terminal domain, was detected in Triton X-100 insoluble fractions in Western blot analysis. Immunodetectable extracellular CTGF formed linear arrays parallel to, but not colocalized with, CL or FN. It also did not colocalize with FAK, vinculin, or integrins α(v)β(3) and α(5)β(1). Intracellular CTGF was detected in the Golgi apparatus and vesicles, including endosomes.Enhanced CTGF secretion induced by TGF-β in CL-grown cells may contribute to positive feedback in which CL is overexpressed in CTGF-induced fibrosis. N-terminal CTGF fragments in the plasma of patients with severe fibrotic disease may be a product of CTGF proteolysis that also produces the newly identified 31-kDa CTGF that remains cell associated and may have its impact by non-integrin signaling pathways.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Role of MT1-MMP in the osteogenic differentiation. 19027888

    Metalloproteinase MT1-MMP is induced and Pro-MMP-2 up modulated early in rat preosteoblasts (ROB) set to differentiate. We here show that the induction of MMPs, accompanied by activation of Pro-MMP-2, occurs by 6 h of adhesion on endogenous extracellular matrix (ECM), Fibronectin (FN) and Collagen type I (CI). These events do not occur after adhesion on Collagen III (CIII), Vitronectin (VN) or BSA. Within the first hour on inducing substrata or plastic, FAK is unchanged and ERK(1,2), is activated, but this activation is not sufficient for MT1-MMP induction. The function of p38 MAPK and PTKs is not required for the induction by substrata of MMPs. Six hours after plating preosteoblasts on MMP-inducing substrata, complexes of beta1 integrin with MT1-MMP are formed, that contain integrin dimers specifically engaged by the substratum, alpha4 and alpha5 chains for cells plated on FN, and alpha2 chain for cells plated on CI and ECM. Induction of MT1-MMP and its expression during osteogenesis pleiotropically regulate alkaline phosphatase (AP) expression. During differentiation, variant clones derived from preosteoblasts and MMPs-over-expressing osteoblasts show high MT1-MMP level associated with high AP level both persisting in time, while inhibition of MMPs is accompanied by inhibition of AP. Up or down modulation of AP, transcriptionally or by inhibition of the enzyme activity, has no effect on level or timing of expression of MT1-MMP and Pro-MMP-2. The persistence in expression of MT1-MMP during differentiation, and the associated persistence in expression of AP, as well as their inhibition, both impair the formation of nodules and mineral deposition. A transient pattern of expression of MT1-MMP is required for the establishment of nodules, and MT1-MMP decrease is permissive for nodule mineralization. The expression of AP is required for nodule formation and its level modulates the mineralization. MT1-MMP has multiple functions and is implicated in multiple steps of the differentiation process, acting to regulate homeostasis of the osteogenic differentiation.
    Document Type:
    Reference
    Product Catalog Number:
    AB1928
    Product Catalog Name:
    Anti-Integrin α5 Antibody, CT, Intracellular

  • Cationic surface charge combined with either vitronectin or laminin dictates the evolution of human embryonic stem cells/microcarrier aggregates and cell growth in agitat ... 24641164

    The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 μm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 μm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Inhibition of vascular smooth muscle cell adhesion and migration by c7E3 Fab (abciximab): a possible mechanism for influencing restenosis. 11090842

    OBJECTIVES: Brief intravenous administration of chimeric antibody c7E3 Fab during coronary angioplasty has been shown in some studies to provide long term protection against coronary events. Smooth muscle cell (SMC) adhesion and migration are key initial steps in the development of restenosis. The purpose of this study was to investigate the effect of c7E3 Fab on adhesion and migration of SMC to the extracellular matrix (ECM) proteins osteopontin (Opn) and vitronectin (Vn). METHODS: Adhesion of human vascular SMCs to ECM proteins was quantified using a CyQUANT assay kit. Migration of SMCs to Vn, Opn and PDGF was studied using a modified Boyden's chamber migration assay. Integrin expression was determined by immunoprecipitation. RESULTS: c7E3 Fab reduced SMC adhesion on Vn and Opn to 69.2+/-3.3% (P0.001) and 52.5+/-4.8% (P0.001) respectively, compared to adhesion without antibody present. This reduction was the same as that for anti-alpha(v)beta(3) integrin antibody LM609 (P=0.5). The combination of anti-alpha(v)beta(5) integrin antibody and c7E3 Fab had a greater effect than either antibody alone (P0.001). c7E3 Fab reduced SMC migration to Vn and Opn to 51.6+/-8.9% (P0.001) and 20.3+/-6.1% (P0.001) respectively, compared to migration in the absence of antibodies. Again, similar results were seen with LM609. PDGF-induced SMC migration was also inhibited by c7E3 Fab (P=0.004) and LM609 (P=0.001), but to much less an extent. The migration SMCs from a culture found not to express the alpha(v)beta(3) integrin was unaffected by these antibodies, strengthening the argument that c7E3 Fab inhibits SMC function via this integrin. CONCLUSIONS: c7E3 Fab inhibits the adhesion and migration of SMCs via the alpha(v)beta(3) integrin. The inhibition, however, is partial, and varied depending on type of ECM protein and alpha(v)beta(3) integrin expression. Some of the clinical benefits of c7E3 Fab may be due to its effect on SMCs.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • ECM regulates MT1-MMP localization with beta1 or alphavbeta3 integrins at distinct cell compartments modulating its internalization and activity on human endothelial cell ... 12427871

    Regulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) by different extracellular matrices (ECMs) on human endothelial cells (ECs) has been investigated. First, MT1-MMP is found at the intercellular contacts of confluent ECs grown on beta1 integrin-dependent matrix such as type 1 collagen (COL I), fibronectin (FN), or fibrinogen (FG), but not on gelatin (GEL) or vitronectin (VN). The novel localization of MT1-MMP at cell-cell contacts is assessed by confocal videomicroscopy of MT1-MMP-GFP-transfected ECs. Moreover, MT1-MMP colocalizes with beta1 integrins at the intercellular contacts, whereas it is preferentially found with alphavbeta3 integrin at motility-associated structures on migrating ECs. In addition, clustered integrins recruit MT1-MMP and neutralizing anti-beta1 or anti-alphav integrin mAb displace MT1-MMP from its specific sites, pointing to a biochemical association that is finally demonstrated by coimmunoprecipitation assays. On the other hand, COL I, FN, or FG up-regulate cell surface MT1-MMP on confluent ECs by an impairment of its internalization, whereas expression and internalization are not modified on GEL or VN. In addition, MT1-MMP activity is diminished in confluent ECs on COL I, FN, or FG. Finally, MT1-MMP participates and cooperates with beta1 and alphavbeta3 integrins in the migration of ECs on different ECM. These data show a novel mechanism by which ECM regulates MT1-MMP association with beta1 or alphavbeta3 integrins at distinct cellular compartments, thus modulating its internalization, activity, and function on human ECs.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Translating human embryonic stem cells from 2D to 3D cultures in a defined medium on laminin and vitronectin coated surfaces. 22034857

    While defining the environment for human embryonic stem cell (hESC) culture on 2D surfaces has made rapid progress, the industrial-scale implementation of this technology will benefit from translating this knowledge into a 3D system, thus enabling better control, automation, and volumetric scale up in bioreactors. The present study describes a system with defined conditions that are capable of supporting the long-term 2D culture of hESC, and the transposing of these conditions to 3D microcarrier (MC) cultures. Vitronectin (VN) and Laminin (LN) were chosen as matrices for the long-term propagation of hESC in a defined culture medium (STEMPRO®) for conventional 2D culture. Adsorption of these proteins onto 2D tissue culture polystyrene (TCPS) indicated that surface density saturation of 510 and 850 ng/cm2 for VN and LN, respectively, was attained above 20µg/ml deposition solution concentration. Adsorption of these proteins onto spherical (97 ± 10 µm), polystyrene MC followed a similar trend and coating surface densities of 450 and 650 ng/cm2 for VN and LN, respectively, were used to support hESC propagation. The long-term expansion of hESC was equally successful on TCPS and MC, with consistently high expression (>90%) of pluripotent markers (OCT-4, Mab84 & TRA-1-60) over 20 passages and maintenance of karyotypic normality. The average fold-increase in cell numbers on VN-coated MC per serial passage was 8.5 ± 1.0, which was similar to LN-coated MC (8.5 ±0.9). Embryoid body differentiation assays and teratoma formation confirmed that hESC retained the ability to differentiate into lineages of all three germ layers, thus demonstrating the first translation to a fully defined environment for the MC-based expansion of hESC.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1637
    Product Catalog Name:
    Anti-Tubulin Antibody, beta III isoform, CT, clone TU-20 (Similar to TUJ1)

  • Complement activation and inflammatory processes in Drusen formation and age related macular degeneration. 11846519

    Recent studies implicate inflammation and complement mediated attack as early events in drusen biogenesis. The investigations described here sought to determine whether primary sites of complement activation could be identified within drusen substructure, and whether known inhibitors of the terminal pathway of complement are present in drusen and/or retinal pigmented epithelial (RPE) cells that lie in close proximity to drusen. Immunohistochemical examination shows two fluid phase regulators of the terminal pathway, vitronectin (Vn, S-protein) and clusterin (apolipoprotein J), to be present in drusen; Vn also accumulates in the cytoplasm of RPE cells that are closely associated with drusen. The membrane associated complement inhibitor, complement receptor 1, is also localized in drusen, but it is not detected in RPE cells immunohistochemically. In contrast, a second membrane associated complement inhibitor, membrane cofactor protein, is present in drusen associated RPE cells, as well as in small, spherical substructural elements within drusen. These previously unidentified elements also show strong immunoreactivity for proteolytic fragments of complement component C3 that are characteristically deposited at sites of complement activation. It is proposed that these structures represent residual debris from degenerating RPE cells that are the targets of complement attack. It is likely that RPE cell debris entrapped between the RPE monolayer and Bruch's membrane serves as a chronic inflammatory stimulus and a potential nucleation site for drusen formation. Thus, the process of drusen biogenesis may be envisaged as a secondary manifestation of primary RPE pathology that is exacerbated by consequences of local inflammatory processes.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1945
    Product Catalog Name:
    Anti-Vitronectin Antibody, clone BV2

  • Diverse effects of Purkinje cell loss on deep cerebellar and vestibular nuclei neurons in Purkinje cell degeneration mutant mice: a possible compensatory mechanism. 9259491

    The genetic defect in the Purkinje cell degeneration (PCD) mutant mouse completely disrupts the cerebellar corticonuclear connection through intrinsic action on the final integrating unit of the cerebellar cortex, the Purkinje cell (PC). The postsynaptic target neurons of the PC in the deep cerebellar nuclei (DCN) and the vestibular nuclei (VN) are denervated by this PC loss by more than two-thirds of their total y-aminobutyric acid (GABA)-ergic innervation. This massive disinhibition should be reflected in an increased and thus electrophysiologically detectable activity of the respective neurons. To address this question, we performed extracellular recordings of PCD mutant and corresponding wild-type VN neurons under sinusoidal vestibular stimulation. The response amplitudes (neuronal response to sinusoidal rotation) of VN neurons in PCD mutant mice showed a decrease rather than the expected increase. The same was true for the mean resting rate, whereas the phase relationships were unaffected for the most part. This finding is a clear indication of compensatory reactions in the VN that substitute quantitatively for the lost PC inhibition. The expression of the calcium-binding protein parvalbumin (Parv) is assumed to correlate with the physiological activity of neurons, and Parv is localized predominantly in inhibitory neurons. Because inhibitory inter- or projecting neurons are also largely denervated by the PC loss, Parv immunocytochemistry also was performed. In wild-type mice, only very few Parv-immunopositive (Parv+) somata were present in the VN, and none were present in the DCN. In PCD mutant mice, a substantial number of Parv+ neuronal somata were visible in the VN, and even more were visible in the DCN. This increase in Parv+ somata in PCD mutant mice is closely related temporally and spatially to the extent of denervation caused by the PCD. Parv+ neuronal somata are first visible in the dentate nucleus at postnatal day (P) 24 and appear in the other cerebellar and VN up to P29. Direct double labeling of Parv and GABA and of Parv and glycine reveals that the large majority of Parv + neurons colocalize GABA, glycine, or both inhibitory transmitters. These results show that neurons that are postsynaptic to cerebellar PC develop diverse physiological and biochemical reactions in the course of genetically determined PCD. These mechanisms are likely to contribute to the phenotypically mild motor disturbances observed in PCD mutant mice.
    Document Type:
    Reference
    Product Catalog Number:
    AB139