Human pluripotent stem cells are a promising source of differentiated cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for ...intensely studied cell types like spinal motor neurons is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that within 3 weeks induce motor neurons at up to 50% abundance and with defined subtype identities of relevance to neurodegenerative disease. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1, and column-specific markers that mirror those observed in vivo in human embryonic spinal cord. They also exhibited spontaneous and induced activity, and projected axons toward muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1(+)/LHX3(-)). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays.
We tested the hypothesis that bispecific Abs (Bsab) with increased binding affinity for tumor Ags augment retargeted antitumor cytotoxicity. We report that an increase in the affinity of Bsab for the ...HER2/neu Ag correlates with an increase in the ability of the Bsab to promote retargeted cytotoxicity against HER2/neu-positive cell lines. A series of anti-HER2/neu extracellular domain-directed single-chain Fv fragments (scFv), ranging in affinity for HER2/neu from 10(-7) to 10(-11) M, were fused to the phage display-derived NM3E2 human scFV: NM3E2 associates with the extracellular domain of human FcgammaRIII (CD16). The resulting series of Bsab promoted cytotoxicity of SKOV3 human ovarian carcinoma cells overexpressing HER2/neu by human PBMC preparations containing CD16-positive NK cells. The affinity for HER2/neu clearly influenced the ability of the Bsab to promote cytotoxicity of (51)Cr-labeled SKOV3 cells. Lysis was 6.5% with an anti-HER2/neu K(D) = 1.7 x 10(-7) M, 14.5% with K(D) = 5.7 x 10(-9) M, and 21.3% with K(D) = 1.7 x 10(-10) M at 50:1 E:T ratios. These scFv-based Bsab did not cross-link receptors and induce leukocyte calcium mobilization in the absence of tumor cell engagement. Thus, these novel Bsab structures should not induce the dose-limiting cytokine release syndromes that have been observed in clinical trials with intact IgG BSAB: Additional manipulations in Bsab structure that improve selective tumor retention or facilitate the ability of Bsab to selectively cross-link tumor and effector cells at tumor sites should further improve the utility of this therapeutic strategy.
The bispecific murine monoclonal antibody (MAb) 1A10 has specificity for the human transferrin receptor (TfR) and the human tumor-associated antigen gp40. This antibody, therefore, functions as an ..."antigen fork" by binding to two distinct antigens on the same malignant cell. Highly purified 1A10 inhibits the growth of cells coexpressing high levels of human TfR and the tumor-associated antigen gp40 by binding to both target antigens. In SW948 cells, the majority of 1A10 binding is via its gp40 specificity, and half-maximal inhibition of cell growth by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay requires 20-30-micrograms/ml concentrations of 1A10. The binding of 1A10 correlates with growth inhibition in the cell lines HT-29, SK-OV-3, OVCAR-2, and OVCAR-3. The growth of OVCAR-10 cells, which express little gp40 and TfR, is not inhibited by 1A10. However, SK-BR-3 cells, which express abundant gp40 and extremely high levels of TfR, are insensitive to the effects of 1A10. In some cell lines, combined exposure to 1A10 and the iron chelator deferoxamine mesylate has synergistic antiproliferative effects. A single i.p. dose of 600 micrograms 1A10 is sufficient to achieve an estimated tumor concentration of at least 30 micrograms/ml for 7 days in C.B17/Icr-scid mice bearing SW948 human tumor xenografts. Treatment of scid mice bearing day 2 or day 4 SW948 xenografts with single or multiple 1A10 doses inhibits tumor growth in a dose-related fashion. Antitumor effects are not seen with therapy using either parental antibody of 1A10. The antiproliferative properties of 1A10 in tumor cells overexpressing gp40 and TfR suggest avenues for the development of new bispecific antibody-promoted treatment strategies.
Human L-ficolin is a soluble protein of the innate immune system able to sense pathogens through its fibrinogen (FBG) recognition domains and to trigger activation of the lectin complement pathway ...through associated serine proteases. L-Ficolin has been previously shown to recognize pneumococcal clinical isolates, but its ligands and especially its molecular specificity remain to be identified. Using solid-phase binding assays, serum and recombinant L-ficolins were shown to interact with serotype 2 pneumococcal strain D39 and its unencapsulated R6 derivative. Incubation of both strains with serum triggered complement activation, as measured by C4b and C3b deposition, which was decreased by using ficolin-depleted serum. Recombinant L-ficolin and its FBG-like recognition domain bound to isolated pneumococcal cell wall extracts, whereas binding to cell walls depleted of teichoic acid (TA) was decreased. Both proteins were also shown to interact with two synthetic TA compounds, each comprising part structures of the complete lipoteichoic acid molecule with two PCho residues. Competition studies and direct interaction measurements by surface plasmon resonance identified PCho as a novel L-ficolin ligand. Structural analysis of complexes of the FBG domain of L-ficolin and PCho revealed that the phosphate moiety interacts with amino acids previously shown to define an acetyl binding site. Consequently, binding of L-ficolin to immobilized acetylated BSA was inhibited by PCho and synthetic TA. Binding of serum L-ficolin to immobilized synthetic TA and PCho-conjugated BSA triggered activation of the lectin complement pathway, thus further supporting the hypothesis of L-ficolin involvement in host antipneumococcal defense.
Fcγ receptor (FcγR) engagement is pivotal for many effector functions of macrophages, polymorphonuclear neutrophils (PMN), and natural killer (NK) cells. Mice transgenic for the A and B isoforms of ...human (h) FcγRIII on macrophages, PMN, and NK cells were constructed to permit the study of mechanisms and potential in vivo strategies to utilize the cytotoxic effector and antigen-presenting functions of cells expressing the hFcγR. The present report characterizes the phenotypic and functional expression of hFcγRIII in transgenic mice derived by crossing hFcγRIIIA and hFcγRIIIB transgenic mice. Interleukin-2 (IL-2) induces hFcγRIII expression by myeloid cells and their precursors, and these transgenic receptors promote in vitro cytotoxicity and anti-hFcγRIII antibody internalization. Splenocytes from untreated and IL-2-treated hFcγRIIIA, hFcγRIIIB, and hFcγRIIIA/B mice exhibited enhanced in vitro cytotoxicity toward HER-2/
neu
-overexpressing SK-OV-3 human ovarian carcinoma cells when incubated with the murine bispecific mAb 2B1, which has specificity for HER-2/
neu
and hFcγRIII. These results indicate that hFcγRIII transgenes are expressed on relevant murine cellular subsets, exhibit inducible up-regulation patterns similar to those seen in humans, and code for functional proteins. hFcγRIII transgenic mice exhibiting specific cellular subset expression will permit the examination of strategies designed to enhance hFcγRIII-dependent immunological effector functions and will provide a model system in which to evaluate preclinically potential candidate molecules that recognize hFcγRIII for the immunotherapy of cancer.
2B1 is a bispecific murine monoclonal antibody (bsmAb) targeting the c-erbB-2 and CD16 (Fc gamma RIII) antigens. c-erbB-2 is over-expressed by a variety of adenocarcinomas, and CD16, the low-affinity ...Fc gamma receptor for aggregated immunoglobulins, is expressed by polymorphonuclear leukocytes (PMN), natural killer (NK) cells and differentiated mononuclear phagocytes. 2B1 potentiates the in vitro lysis of c-erb-2 over-expressing tumors by NK cells and macrophages. In this report, the interactions between 2B1 and PMN were investigated to assess the impact of these associations on in vitro 2B1-promoted tumor cytotoxicity by human NK cells. The peak binding of 2B1 to PMN was observed at a concentration of 10 microgram/ml 2B1. However, 2B1 rapidly dissociated from PMN in vitro at 37 degrees C in non-equilibrium conditions. This dissociation was not caused by CD16 shedding. When PMN were labeled witn 125I-2B1 and incubated at 37 degrees C and the supernatants examined by HPLC analysis, the Fab regions of dissociated 2B1 were not complexed with shed CD16 extracellular domain. While most of the binding of 2B1 PMN was solely attributable to Fab-directed binding to Fc gamma RIII, PMN-associated 2B1 also bound through Fc gamma-domain/Fc gamma RII interactions. 2B1 did not promote in vitro PMN cytotoxicity against c-erbB-2-expressing SK-OV-3 tumor cells. When PMN were coincubated with peripheral blood lymphocytes, SK-OV-3 tumor and 2B1, the concentration of 2B1 required for maximal tumor lysis was lowered. Although PMN may serve as a significant competitive binding pool of systemically administered 2B1 in vivo, the therapeutic potential of the targeted cytotoxicity properties of this bsmAb should not be compromised.
As the world population grows, fisheries practitioners will be under increased pressure to address global challenges in data-limited fisheries management. With a focus on addressing localized and ...case-specific management needs, we provide a practical guide to the design and development of multi-indicator frameworks for fishery management. In a data-limited context, indicators are observations or estimates of the state of the fishery resource that are typically proxies for variables of interest, rather than quantities such as stock biomass estimated from data-rich stock assessments. Indicator frameworks structure the integration and interpretation of indicators to guide tactical fishery decision-making, often when the application of more formal analytical assessments is not feasible, yet where indicators in combination provide insight into stock status. With a focus on multi-indicator frameworks, we describe a pragmatic approach for their development
via
a set of organizational steps, considering a wide spectrum of types and severity of information limitations. We highlight where multi-indicator frameworks can be insightful and informative in relation to single indicator approaches but also point to potential pitfalls, with emphasis on critical evaluation and detection of performance flaws during the design phase using methods such as management strategy evaluation.
Recent decades have seen rapid development of new analytical methods to investigate patterns of interspecific variation. Yet these cutting‐edge statistical analyses often rely on data of questionable ...origin, varying accuracy, and weak comparability, which seem to have reduced the reproducibility of studies. It is time to improve the transparency of comparative data while also making these improved data more widely available. We, the authors, met to discuss how transparency, usability, and reproducibility of comparative data can best be achieved. We propose four guiding principles: 1) data identification with explicit operational definitions and complete descriptions of methods; 2) inclusion of metadata that capture key characteristics of the data, such as sample size, geographic coordinates, and nutrient availability (for example, captive versus wild animals); 3) documentation of the original reference for each datum; and 4) facilitation of effective interactions with the data via user friendly and transparent interfaces. We urge reviewers, editors, publishers, database developers and users, funding agencies, researchers publishing their primary data, and those performing comparative analyses to embrace these standards to increase the transparency, usability, and reproducibility of comparative studies.
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