Anti-idiotype responses abrogate anti-CD4–induced tolerance to a tumor-specific antigen and promote systemic tumor immunity

Purpose: Immunologic-based cancer treatment modalities represent an active area of investigation. Included in these strategies are passive administration of monoclonal antibodies which recognize tumor-associated antigens and active vaccination with identified tumor antigens. However, several problems associated with these types of treatment strategies have been identified. Methods: In this report, we address certain issues by employing a murine model for experimental pulmonary metastasis and a tumor antigen vaccination strategy that induces complete tumor immunity in this system. Utilizing this model, we attempt to address issues related to unresponsiveness to tumor antigen immunization induced by passive administration of a rat monoclonal anti-CD4 and the induction of anti-idiotype responses to a passively administered monoclonal antibody and the effects on the induction of tumor immunity. Results: The results presented indicate that passive administration of rat monoclonal anti-CD4 exhibits immunosuppressive effects that inhibit the production of antibodies to the tumor antigen immunization and abolishes tumor immunity. Repeated administration of the rat monoclonal anti-CD4 results in an anti-idiotype response that can abrogate unresponsiveness to tumor antigen immunization and promote systemic tumor immunity. Conclusions: The data examine a number of potential problems associated with immunologic-based treatments for cancer. These problems include the potential for tolerance to the tumor antigen and establishing an immunocompromised state where immunization with a tumor antigen failed to generate tumor immunity. Approaches to eliminate tolerant T cells by targeting anti-CD4 via anti-idiotype responses that could be generated in vivo without CD4⁺ T cells allowed for recovery of nontolerant T cells, and an antibody response to the tumor antigen that results in tumor immunity.Abbreviations CTL Cytotoxic T lymphocyte - FITC Fluorescein isothiocyanate - OD Optical density - PBS Phosphate-buffered saline - SV40 Simian virus 40     

Rational siRNA design for RNA interference

Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3'-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.     

Essential role for integrin linked kinase in Akt-mediated integrin survival signaling in hippocampal neurons

Activation of integrin receptors in neurons can promote cell survival and synaptic plasticity, but the underlying signal transduction pathway(s) is unknown. We report that integrin signaling prevents apoptosis of embryonic hippocampal neurons by a mechanism involving integrin-linked kinase (ILK) that activates Akt kinase. Activation of integrins using a peptide containing the amino acid sequence EIKLLIS derived from the alpha chain of laminin protected hippocampal neurons from apoptosis induced by glutamate or staurosporine, and increased Akt activity in a beta1 integrin-dependent manner. Transfection of neurons with a plasmid encoding dominant negative Akt blocked the protective effect of the integrin-activating peptide, as did a chemical inhibitor of Akt. Although inhibitors of phosphoinositide-3 (PI3) kinase blocked the protective effect of the peptide, we found no increase in PI3 kinase activity following integrin stimulation suggesting that PI3 kinase was necessary for Akt activity but was not sufficient for the increase in Akt activity following integrin activation. Instead, we show a requirement for ILK in integrin receptor-induced Akt activation. ILK was activated following integrin stimulation and dominant negative ILK blocked integrin-mediated Akt activation and cell survival. Activation of ILK and Akt were also required for neuroprotection by substrate-associated laminin. These results establish a novel pathway that signals cell survival in neurons in response to integrin receptor activation.     

The SRM12/ADA1 gene acts as a cis-active factor when inserted into recombinant plasmids and makes them more stable in Saccharomyces

A DNA fragment containing the SRM12/ADA1 gene sequence inserted into a recombinant circular plasmid improves its maintenance in budding yeast (Saccharomyces cerevisiae) cells. Plasmid stabilization caused by the integrated SRM12 sequence does not require the SRM12 function complementing the srm12 mutation and depends on the orientation of the inserted fragment in the vector. This stabilization is mainly due to a decrease in spontaneous plasmid underreplication/copy loss rather than an increase in the fidelity of mitotic plasmid segregation.     

Solution structures of antimalarial drug-heme complexes

Paramagnetic metal centers [such as Fe(III) found within ferriprotoporphyrin IX heme (FPIX)] exert through space effects on the relaxation rate of nearby proton spins that depend critically on the metal-proton distance. We have measured these effects for all protons of several antimalarial drugs that bind to FPIX by systematically varying the drug:heme molar ratio in high field NMR experiments. These measurements allow us to determine precise FPIX Fe-drug H distances for the solution structures of noncovalent complexes formed between FPIX mu-oxo dimers and the antimalarial drugs chloroquine (CQ), quinine (QN), and quinidine (QD). Using these distances, we then performed distance restraint calculations to determine the lowest-energy solution structures of these complexes. Structures were solved for neutral, monoprotic (+1), and diprotic (+2) forms of the drugs. Analysis of these structures allows us to visualize for the first time the stereospecific differences between QN and QD binding to FPIX and the differences in populations of QN and QD solution structures upon changes in digestive vacuolar pH for drug resistant malarial parasites [Dzekunov, S. M., et al. (2000) Mol. Biochem. Parasitol. 110, 107-124]. The data indicate a previously unrecognized key role for the CQ aliphatic chain in stabilizing FPIX-CQ complexes, and suggest how lengthening or shortening the chain might perturb stability. We also define FPIX:drug stoichiometries of 2:1 for the complexes formed at physiological FPIX concentrations, in contrast to the 4:1 and 5:1 stoichiometries previously determined at higher FPIX concentrations [Dorn, A., et al. (1998) Biochem. Pharmacol. 55, 727-736]. These atomic resolution antimalarial drug-heme structures should help elucidate how these drugs inhibit formation of hemozoin during metabolism of heme within the malarial parasite Plasmodium falciparum and assist ongoing development of strategies for circumventing antimalarial drug resistance.     

Disruption of hsp90 function results in degradation of the death domain kinase, receptor-interacting protein (RIP), and blockage of tumor necrosis factor-induced nuclear factor-kappaB activation

The death domain kinase, receptor interacting protein (RIP), is one of the major components of the tumor necrosis factor receptor 1 (TNFR1) complex and plays an essential role in tumor necrosis factor (TNF)-mediated nuclear factor kappaB (NF-kappaB) activation. The activation of NF-kappaB protects cells against TNF-induced apoptosis. Heat-shock proteins (Hsps) are chaperone molecules that confer protein stability and help to restore protein native folding following heat shock and other stresses. The most abundant Hsp, Hsp90, is also involved in regulating the stability and function of a number of cell-signaling molecules. Here we report that RIP is a novel Hsp90-associated kinase and that disruption of Hsp90 function by its specific inhibitor, geldanamycin (GA), selectively causes RIP degradation and the subsequent inhibition of TNF-mediated IkappaB kinase and NF-kappaB activation. MG-132, a specific proteasome inhibitor, abrogated GA-induced degradation of RIP but failed to restore the activation of IkappaB kinase by TNF, perhaps because, in the presence of GA and MG-132, RIP accumulated in a detergent-insoluble subcellular fraction. Most importantly, the degradation of RIP sensitizes cells to TNF-induced apoptosis. These data indicate that Hsp90 plays an important role in TNF-mediated NF-kappaB activation by modulating the stability and solubility of RIP. Thus, inhibition of NF-kappaB activation by GA may be a critical component of the anti-tumor activity of this drug.     

Role of hydroxycinnamic acids in food flavor: a brief overview

The influence of hydroxycinnamic acids (HCAs) on food flavor is reviewed. In coffee, whole-grain foods and related model systems, the HCAs have been reported to contribute to the flavor profile by multiple mechanisms, such as to impart taste attributes, to generate aroma and taste-active compounds by phenolic degradation, as well as to alter the mechanisms of the Maillard reaction and related flavor development. Consequently the role of HCAs on the flavor properties of food products is complex, multifaceted and can be related to the chemistry and fate of HCAs during thermal processing.