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Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms—(1) release of lytic granules (containing perforin and granzymes), and (2) Fas ligand (FasL)/Fas or TNF-initiated apoptosis. We have examined mechanisms of target cell lysis by effector T cells from gene-targeted and mutant mice using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH67 dye. Cell death was estimated by 7-AAD inclusion and annexin V-PE binding. A direct correlation has been found between the percentage of dead target cells in FC Assay and the results of 111In release cytotoxicity assay when effector T cells from either Pfp −/− (perforin knockout) or gld (non-functional Fas Ligand) mice were used. As shown by the 4 h FC assay, the granule-mediated mechanism was utilized by T cells from gld mice. In contrast, T cells from Pfp −/− mice used death receptor-mediated lysis. Therefore, cytotoxic cells from gene-targeted and mutant mice can serve as valuable tools for studying different mechanisms of cell-mediated cytotoxicity, and the FC assay could be applied irrespective of which cytotoxic effector pathway is involved. [Copyright &y& Elsevier]

Background: Interactions of cells with the extracellular matrix (ECM) are critical for the establishment and maintenance of stem cell self-renewal and differentiation. However, the ECM is a complex mixture of matrix molecules; little is known about the role of ECM components in human embryonic stem cell (hESC) differentiation into neural progenitors and neurons. Results: A reproducible protocol was used to generate highly homogenous neural progenitors or a mixed population of neural progenitors and neurons from hESCs. This defined adherent culture system allowed us to examine the effect of ECM molecules on neural differentiation of hESCs. hESC-derived differentiating embryoid bodies were plated on Poly-D-Lysine (PDL), PDL/ fibronectin, PDL/laminin, type I collagen and Matrigel, and cultured in neural differentiation medium. We found that the five substrates instructed neural progenitors followed by neuronal differentiation to differing degrees. Glia did not appear until 4 weeks later. Neural progenitor and neuronal generation and neurite outgrowth were significantly greater on laminin and laminin-rich Matrigel substrates than on other 3 substrates. Laminin stimulated hESC-derived neural progenitor expansion and neurite outgrowth in a dose-dependent manner. The laminin-induced neural progenitor expansion was partially blocked by the antibody against integrin α6 or β1 subunit. Conclusion: We defined laminin as a key ECM molecule to enhance neural progenitor generation, expansion and differentiation into neurons from hESCs. The cell-laminin interactions involve α6β1 integrin receptors implicating a possible role of laminin/α6β1 integrin signaling in directed neural differentiation of hESCs. Since laminin acts in concert with other ECM molecules in vivo, evaluating cellular responses to the composition of the ECM is essential to clarify further the role of cell-matrix interactions in neural derivation of hESCs. [ABSTRACT FROM AUTHOR]

Background: Genes associated with energy metabolism are decreased in schizophrenia brain and human and rodent diabetic skeletal muscle. These and other similarities between diabetes and schizophrenia suggest that an insulin signaling deficit may underlie schizophrenia. We determined with human SH-SY5Y neuroblastoma and astrocyte cell lines whether insulin or other molecules could modulate genes opposite to their change reported in schizophrenia brain. Methods: Both cell lines were treated with insulin, insulin-like growth factor (IGF)-1, IGF-2, or brain-derived neurotrophic factor (BDNF). Genes whose expression was found with microarrays to be changed by insulin in a reciprocal manner to their change in schizophrenia were used in a 16-gene miniarray to identify small molecules that might mimic insulin. Results: Insulin phosphorylated its receptor in the neuroblastoma cells but not in astrocytes and, like IGF-1, increased ERK1/2 and Akt phosphorylation. Insulin and IGF-1 increased the expression of genes decreased in schizophrenia, including those involved in mitochondrial functions, glucose and energy metabolism, hydrogen ion transport, and synaptic function. These gene effects were confirmed and shown to be dose related with the 16-gene miniarrays. Most of 1940 pharmacologically unique compounds failed to alter gene expression, with the exception of muscarinic agonists, which mimicked insulin and IGF-1, and which were blocked by the muscarinic antagonists atropine and telenzepine. Conclusions: Stimulation of muscarinic and insulin/IGF-1 receptors alter genes associated with metabolic and synaptic functions in a manner reciprocal to their changes in schizophrenia. Pharmacologic activation of these receptors may normalize genomic alterations in schizophrenia and better address root causes of this disease. [Copyright &y& Elsevier]

Background: The interferon-γ (IFN-γ) ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-γ secretion is not limited to only cytolytic cells. Granzyme B (GrB) is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-γ secretion. Methods: We assessed whether the GrB ELISPOT assay is a viable alternative to the 51Cr-release and IFN-γ ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (aEN-EBV and aJY) and an in vitro stimulated anti-Flu matrix peptide (FMP)-specific CTL. Results: When the GrB ELISPOT was directly compared to the IFN-γ ELISPOT and 51Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN- γ secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the 51Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. Conclusion: Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-γ ELISPOT assay, the GrB ELISPOT directly measures the release of a cytotolytic protein. Detection of low frequency tumor-specific CTL and their specific effector functions can provide valuable insight with regards to immunological responses. [ABSTRACT FROM AUTHOR]