Enhancement of IL-2-induced T cell proliferation by a novel factor(s) present in murine spleen dendritic cell-T cell culture supernatants

The mechanism(s) underlying the potent accessory cell function of dendritic cells (DC) remains unclear. The possibility was considered that a soluble factor(s) released during the interaction of DC and T cells might contribute to the potent T cell activating function of DC. Culture supernatants were generated from mixtures of murine spleen DC and periodate-treated spleen T cells and were examined for the presence of known cytokine activities and factors capable of enhancing T cell responsiveness to IL-2. Serum-free supernatants from 24 h DC-T cell co-cultures exhibited high levels of IL-2, detectable levels of IL-3, and negligible levels of IL-1, -4, -5, -6, and TNF. A factor(s) was also identified with an apparent Mr of 12.5 to 17.0 kDa, henceforth designated IL-2 enhancing factor (IL-2EF), which enhanced the IL-2-induced proliferation of murine thymocytes, CTLL, and HT-2 cells by approximately three- to fourfold. This enhancement was also observed in the presence of neutralizing antibodies to murine IL-1 alpha, -1 beta, -3, -4, -5, -6, granulocyte-macrophage (GM)-CSF, TNF, and IFN-gamma. However, IL-2EF failed to enhance: 1) the activity of IL-1, -3, -4, -5, or -6 on cells responsive to these cytokines; 2) IL-2-augmented, IL-5-induced BCL1 proliferation; and 3) either PHA- or Con A-stimulated thymocyte proliferation. Moreover, neither IFN-gamma nor GM-CSF exhibited IL-2EF activity. When DC and T cells were cultured separately (after an initial 12 h co-culture period), IL-2EF activity resided predominantly in the T cell-derived supernatants. These and other data indicate that IL-2EF, a heat-labile T cell-derived 12.5 to 17.0 kDa protein, is distinct from IL-1 alpha, -1 beta, -2, -3, -4, -5, -6, TNF, IFN-gamma, GM-CSF, and previously described factors that co-stimulate thymocyte proliferation in the presence of Con A or PHA. It is suggested that IL-2EF functions to specifically enhance IL-2-driven T cell proliferation and contributes to the potent activation of T cells induced by DC.     

Phylogenetic comparative analysis of RNA structure on Macintosh computers

A Macintosh Hypertalk program (Hypercard ‘stack’)for use in phylogenetic comparative analysis of RNA structureis described. The program identifies covariations and compensatorychanges in RNA sequence alignments, for use in the constructionof secondary structure models or the identification of tertiaryinteractions. The results of an analysis are presented eitheras a list of positions in the alignment which covary, or asa 2-dimensional matrix in which potential helices in the secondarystructure appear as diagonal patterns. Received on January 7, 1991; accepted on March 19, 1991     

Glioma IL13Rα2 Is Associated with Mesenchymal Signature Gene Expression and Poor Patient Prognosis

A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the biological context of IL-13 receptor α2 (IL13Rα2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13Rα2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13Rα2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13Rα2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13Rα2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13Rα2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13Rα2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13Rα2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials.     

Multiple mechanisms contribute to myenteric plexus ablation induced by benzalkonium chloride in the guinea-pig ileum

Ablation of rat myenteric plexus with benzalkonium chloride has provided a model of intestinal aganglionosis, but the degenerative responses are not well understood. We examined the effects of this detergent on neurons and glia, including expression of c-Myc, c-Jun, JunB, and c-Fos, and on immunocytes in the guinea-pig ileum. Benzalkonium chloride (0.1%) or saline was applied to the serosal surface of distal ileum. Tissues were analyzed 2, 3, or 7 days later and compared with cyclosporine-treated and untreated animals. More than 90% of myenteric neurons were destroyed in ileal segments 3–7 days after benzalkonium-chloride treatment. Glia withdrew processes from around neurons after 2 days and were mostly gone after 3 days. Neuronal c-Myc began to disappear while c-Fos, c-Jun, and JunB were evident in some neuronal nuclei after 2 or 3 days. After 3 days, widespread apoptosis was evident in the myenteric plexus. Populations of T cells, B cells, and macrophage-like cells in untreated and saline-treated myenteric plexuses were substantially increased 3 and 7 days after benzalkonium-chloride treatment. Cyclosporine delayed significant neuronal loss. We conclude that a variety of degenerative mechanisms may be active in this model, including an immune response which may actively contribute to tissue destruction. Received: 13 September 1996 / Accepted: 20 January 1997     

Regulatory and essential light-chain-binding sites in myosin heavy chain subfragment-1 mapped by site-directed mutagenesis

Site-directed mutagenesis of the cloned subfragment-1 (S-1) region of the unc-54 gene, encoding the myosin heavy chain B (MHC B) from Caenorhabditis elegans, has been used to locate binding sites for the regulatory and essential light chains. MHC B S-1 synthesized in Escherichia coli co-migrated with rabbit skeletal muscle myosin S-1 (Mr 90,000), was recognized by anti-nematode myosin antiserum on immunoblots, and specifically bound to 125I-labelled regulatory and essential light chains in a gel overlay assay. Deletion of 102 residues from the C terminus (mutant 655) reduced regulatory and essential light-chain binding to about 30% and 20% of wild-type levels, respectively. Similar reductions in relative binding of the two light chains were seen with mutant 534, in which 38 residues were deleted from the C terminus. Potential binding sites within 75 residues of the C terminus of S-1 were mapped by construction of five other mutant S-1 clones (398, 399, 400, 409 and 411) containing internal deletions of ten to 12 amino acid residues. These showed up to 30% reductions in their ability to bind essential light chains, but did not differ significantly from wild-type in their ability to bind regulatory light chains. Another mutant, 415, containing a deletion of a conserved acidic hexapeptide, E-D-I-R-D-E, showed enhancement of binding of regulatory and essential light chains to 150% and 165% of wild-type levels. Hence, the major binding sites for both light chains are within 38 amino acid residues of the C terminus.