Visualization of p53(264-272)/HLA-A*0201 complexes naturally presented on tumor cell surface by a multimeric soluble single-chain T cell receptor

Intracellular Ags are processed into small peptides that are presented on cell surfaces in the context of HLA class I molecules. These peptides are recognized by TCRs displayed by CD8+ T lymphocytes (T cells). To date, direct identification and quantitation of these peptides has relied primarily on mass spectrometry analysis, which is expensive and requires large quantities of diseased tissues to obtain useful results. Here we demonstrate that multimerization of a soluble single-chain TCR (scTCR), recognizing a peptide from p53 presented in the context of HLA-A2.1, could be used to directly visualize and quantitate peptide/MHC complexes on unmanipulated human tumor cells. Tumor cells displaying as few as 500 peptide/MHC complexes were readily detectable by flow cytometry. The scTCR/multimers exhibited exquisite recognition capability and could distinguish peptides differing in as little as a single amino acid. We also demonstrate that scTCR/multimers could specifically stain human tumors generated in mice, as well as tumors obtained from patient biopsies. Thus, scTCR/multimers represent a novel class of immunostaining reagents that could be used to validate, quantitate, or monitor epitope presentation by cancer cells.     

TLR3 ligation activates an antiviral response in human fetal astrocytes: a role for viperin/cig5

TLR3 functions as a viral nucleic acid sentinel activated by dsRNA viruses and virus replication intermediates within intracellular vesicles. To explore the spectrum of genes induced in human astrocytes by TLR3, we used a microarray approach and the analog polyriboinosinic polyribocytidylic acid (pIC) as ligand. As expected for TLR activation, pIC induced a wide array of cytokines and chemokines known for their role in inflammatory responses, as well as up-regulation of the receptor itself. The data also showed activation of a broad spectrum of antiviral response genes. To determine whether pIC induced an antiviral state in astrocytes, a pseudotyped HIV viral particle, vesicular stomatitis virus g-env-HIV-1, was used. pIC significantly abrogated HIV-1 replication, whereas IL-1, which also potently activates astrocytes, did not. One of the most highly up-regulated genes on microarray was the protein viperin/cig5. We found that viperin/cig5 expression was dependent on IFN regulatory factor 3 and NF-kappaB signaling, and that repetitive stimulation with pIC, but not IL-1, further increased expression. Viperin induction could also be substantially inhibited by neutralizing Abs to IFN-beta, as could HIV-1 replication. To explore a role for viperin in IFN-beta-mediated inhibition of HIV-1, we used an RNA interference (RNAi) approach. RNAi directed against viperin, but not a scrambled RNAi, significantly inhibited viperin expression, and also significantly reversed pIC-induced inhibition of HIV-1 replication. We conclude that viperin contributes to the antiviral state induced by TLR3 ligation in astrocytes, supporting a role for astrocytes as part of the innate immune response against infection in the CNS.     

Identification of Jmjd1a as a STAT3 downstream gene in mES cells

Mouse embryonic stem (mES) cells can be maintained in undifferentiated state in the presence of a cytokine, leukemia inhibitory factor (LIF). Many investigators found that STAT3 activation is important for the maintenance of pluripotency by LIF. However, the downstream pathways of STAT3 activation are still unknown. To look for STAT3-downstream target genes, we performed DD-RT PCR in the presence or absence of LIF. Through further confirmation, we finally selected 8 genes whose expressions were significantly dependent upon the presence of LIF. Among them, Jmjd1a was down-regulated after LIF withdrawal, and it was selected for further investigation. Its expression started to decrease 1 day after the removal of LIF, and disappeared on day 3. It was also shown that STAT3 could bind to the promoter region of Jmjd1a gene. These data demonstrate that Jmjd1a might be a critical signaling molecule underlying the maintenance of pluripotency in mES cells.     

Loss and recovery of protein productivity in genetically modified plant cell lines

This paper addresses the problem of decreasing protein expression levels in genetically modified plant cells. A modeling approach was used to explain the loss of productivity over successive generations. Using this model, productivity losses were simulated for two cell lines. Although the lines were relatively stable, the protein production level could decrease by more than 80% after a large number of generations. Motivated by this problem, a dispersion method was developed to isolate productive cells from existing cell suspensions. Dilution of transgenic cells in a feeder layer of nonproducing cells at a ratio of 1:1000 facilitated the recovery of distinct, separate daughter colonies. Applying this method, it was possible to recover high producing cell lines.     

Regulation of microglial activities by glial cell line derived neurotrophic factor

Much attention has been paid to the ability of glial cell line-derived neurotrophic factor (GDNF) to protect neurons from neurotoxic insults in the central nervous system (CNS). However, little is known about GDNF action on CNS glia that also can express GDNF receptor systems. In this study, we examined the effects of GDNF on primary rat microglia that function as resident macrophages in the CNS and as the source of proinflammatory mediators upon activation. We found that treatment of primary rat microglia with GDNF had no effect on the secretion of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), but it increased the nitric oxide (NO) production to some extent. In addition, GDNF increased the enzymatic activity of superoxide dismutase (SOD), the gene expression of surface antigen intercellular adhesion molecule-1 (ICAM-1), the production of the integrin alpha5 subunit, and the phagocytotic capability in primary rat microglia. Furthermore, inhibition of mitogen-activated protein kinase (Erk-MAPK) in the mouse microglial cell line BV2 by U0126 indicated that the MAP kinase signaling pathway may be involved in the regulation of NO and integrin alpha5 production by GDNF. In vivo evidence also showed that amoeboid cells with integrin alpha5 or with ED1 immunoreactivity appeared in GDNF-treated spinal cord tissues at the lesion site 1 week post spinal cord injury (SCI). Furthermore, inhibition of Erk-MAPK in the mouse microglial cell line BV2 by U0126 indicated that the MAP kinase signaling pathway may be involved in the regulation of NO and integrin alpha5 production by GDNF. Taken together, our results indicate that GDNF has a positive regulatory effect on microglial activities, such as phagocytosis and the upregulation of adhesion molecules.     

CREB-dependent cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression is mediated by protein kinase C and calcium

Cellular production of prostaglandins (PGs) is controlled by the concerted actions of cyclooxygenases (COX) and terminal PG synthases on arachidonic acid in response to agonist stimulation. Recently, we showed in an ileal epithelial cell line (IEC-18), angiotensin II-induced COX-2-dependent PGI2 production through p38MAPK, and calcium mobilization (J. Biol. Chem. 280: 1582-1593, 2005). Agonist binding to the AT1 receptor results in activation of PKC activity and Ca2+ signaling but it is unclear how each pathway contributes to PG production. IEC-18 cells were stimulated with either phorbol-12,13-dibutyrate (PDB), thapsigargin (TG), or in combination. The PG production and COX-2 and PG synthase expression were measured. Surprisingly, PDB and TG produced PGE2 but not PGI2. This corresponded to induction of COX-2 and mPGES-1 mRNA and protein. PGIS mRNA and protein levels did not change. Activation of PKC by PDB resulted in the activation of ERK1/2, JNK, and CREB whereas activation of Ca2+ signaling by TG resulted in the delayed activation of ERK1/2. The combined effect of PKC and Ca2+ signaling were prolonged COX-2 and mPGES-1 mRNA and protein expression. Inhibition of PKC activity, MEK activity, or Ca2+ signaling blocked agonist induction of COX-2 and mPGES-1. Expression of a dominant negative CREB (S133A) blocked PDB/TG-dependent induction of both COX-2 and mPGES-1 promoters. Decreased CREB expression by siRNA blocked PDB/TG-dependent expression of COX-2 and mPGES-1 mRNA. These findings demonstrate a coordinated induction of COX-2 and mPGES-1 by PDB/TG that proceeds through PKC/ERK and Ca2+ signaling cascades, resulting in increased PGE2 production.