Protective anti-tumor immunity induced by vaccination with recombinant adenoviruses encoding multiple tumor-associated cytotoxic T lymphocyte epitopes in a string-of-beads fashion

Vaccines harboring genes that encode functional oncoproteins are intrinsically hazardous, as their application may lead to introduction of these genes into normal cells and thereby to tumorigenesis. On the other hand, oncoproteins are especially attractive targets for immunotherapy of cancer, as their expression is generally required for tumor growth, making the arisal of tumor variants lacking these antigens unlikely. Using murine tumor models, we investigated the efficacy of polyepitope recombinant adenovirus (rAd) vaccines, which encode only the immunogenic T cell epitopes derived from several oncogenes, for the induction of protective anti-tumor immunity. We chose to employ rAd, as these are safe vectors that do not induce the side effects associated with, for example, vaccinia virus vaccines. A single polyepitope rAd was shown to give rise to presentation of both H-2 and human leukocyte antigen-restricted cytotoxic T lymphocyte (CTL) epitopes. Moreover, vaccination with a rAd encoding H-2-restricted CTL epitopes, derived from human adenovirus type 5 early region 1 and human papilloma virus type 16-induced tumors, elicited strong tumor-reactive CTL and protected the vaccinated animals against an otherwise lethal challenge with either of these tumors. The protection induced was superior compared with that obtained by vaccination with irradiated tumor cells. Thus, vaccination with polyepitope rAd is a powerful approach for the induction of protective anti-tumor immunity that allows simultaneous immunization against multiple tumor-associated T cell epitopes, restricted by various major histocompatibility complex haplotypes.

The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two virulence determinants appear to be closely associated and coordinately regulated.

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

The algT (algU) gene of Pseudomonas aeruginosa, a key regulator involved in alginate biosynthesis, encodes an alternative sigma factor (sigma E).

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is the leading cause of mortality among cystic fibrosis (CF) patients. During the course of sustained infection, the production of an alginate capsule protects the bacteria and allows them to persist in the CF lung. One of the key regulators of alginate synthesis is the algT (algU) gene encoding a putative alternative sigma factor (sigma E). AlgT was hyperproduced and purified from Escherichia coli. The N-terminal sequence of the purified protein matched perfectly with that predicted from the DNA sequence. The purified protein, in the presence of E. coli RNA polymerase core enzyme, was able to initiate transcription of an algT promoter. Deletion of the -35 region of this promoter abolished this activity in vitro as well as in vivo. These data indicate that the algT gene encodes a sigma factor that is autoregulatory.

Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway

The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin⋅FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.

The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4+ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4+ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the β cell-reactive CD4+ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4+ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and/or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.

Polyvalent binding to carbohydrates immobilized on an insoluble resin

Numerous studies have established that polyvalency is a critical feature of cell surface carbohydrate recognition. Nevertheless, carbohydrate–protein interactions are typically evaluated by using assays that focus on the behavior of monovalent carbohydrate ligands in solution. It has generally been assumed that the relative affinities of monovalent carbohydrate ligands in solution correlate with their polyvalent avidities. In this paper we show that carbohydrate ligands synthesized directly on TentaGel beads interact with carbohydrate-binding proteins in a polyvalent manner. The carbohydrate-derivatized beads can, therefore, be used as model systems for cell surfaces to evaluate polyvalent carbohydrate–protein interactions. By using a combinatorial approach to synthesize solid-phase libraries of polyvalent carbohydrates, one can rapidly address key issues in the area of cell surface carbohydrate recognition. For example, studies reported herein demonstrate that there is an unanticipated degree of specificity in recognition processes involving polyvalent carbohydrates. However, the correlation between polyvalent avidities and solution affinities is poor. Apparently, the presentation of carbohydrates on the polymer surface has a profound influence on the interaction of the ligand with the protein receptor. These findings have implications for how carbohydrates function as recognition signals in nature, as well as for how polyvalent carbohydrate–protein interactions should be studied.

Polycaps: Reversibly formed polymeric capsules

Described are assemblies consisting of polymeric capsules, “polycaps,” formed from two calix[4]arene tetraureas covalently connected at their lower rims. In these structures self-assembly leads to reversibly formed capsule sites along a chain, reminiscent of beads on a string. Their dynamic behavior is characterized by 1H NMR spectroscopy through encapsulation of guest species, reversible polymerization, and the formation of sharply defined hybrid capsules.

HLA class I and II antigens are partially co-clustered in the plasma membrane of human lymphoblastoid cells

Major histocompatibility complex (MHC) class II molecules displayed clustered patterns at the surfaces of T (HUT-102B2) and B (JY) lymphoma cells characterized by interreceptor distances in the micrometer range as detected by scanning force microscopy of immunogold-labeled antigens. Electron microscopy revealed that a fraction of the MHC class II molecules was also heteroclustered with MHC class I antigens at the same hierarchical level as described by the scanning force microscopy data, after specifically and sequentially labeling the antigens with 30- and 15-nm immunogold beads. On JY cells the estimated fraction of co-clustered HLA II was 0.61, whereas that of the HLA I was 0.24. Clusterization of the antigens was detected by the deviation of their spatial distribution from the Poissonian distribution representing the random case. Fluorescence resonance energy transfer measurements also confirmed partial co-clustering of the HLA class I and II molecules at another hierarchical level characterized by the 2- to 10-nm Förster distance range and providing fine details of the molecular organization of receptors. The larger-scale topological organization of the MHC class I and II antigens may reflect underlying membrane lipid domains and may fulfill significant functions in cell-to-cell contacts and signal transduction.

Phagocytosis of rod outer segments by retinal pigment epithelial cells requires αvβ5 integrin for binding but not for internalization

Phagocytosis of shed photoreceptor rod outer segments (ROS) by the retinal pigment epithelium (RPE) is essential for retinal function. Here, we demonstrate that this process requires αvβ5 integrin, rather than αvβ3 integrin utilized by systemic macrophages. Although adult rat RPE expressed both αvβ3 and αvβ5 integrins, only αvβ3 was expressed at birth, when the retina is immature and phagocytosis is absent. Expression of αvβ5 was first detected in RPE at PN7 and reached adult levels at PN11, just before onset of phagocytic activity. Interestingly, αvβ5 localized in vivo to the apical plasma membrane, facing the photoreceptors, and to intracellular vesicles, whereas αvβ3 was expressed basolaterally. Using quantitative fluorimaging to assess in vitro uptake of fluorescent particles by human (ARPE-19) and rat (RPE-J) cell lines, αvβ5 function-blocking antibodies were shown to reduce phagocytosis by drastically decreasing (85%) binding of ROS but not of latex beads. In agreement with a role for αvβ5 in phagocytosis, immunofluorescence experiments demonstrated codistribution of αvβ5 integrin with internalized ROS. Control experiments showed that blocking αvβ3 function with antibodies did not inhibit ROS phagocytosis and that αvβ3 did not colocalize with phagocytosed ROS. Taken together, our results indicate that the RPE requires the integrin receptor αvβ5 specifically for the binding of ROS and that phagocytosis involves internalization of a ROS-αvβ5 complex. αvβ5 integrin does not participate in phagocytosis by other phagocytic cells and is the first of the RPE receptors involved in ROS phagocytosis that may be specific for this process.

A hierarchical neuronal network for planning behavior

Planning a goal-directed sequence of behavior is a higher function of the human brain that relies on the integrity of prefrontal cortical areas. In the Tower of London test, a puzzle in which beads sliding on pegs must be moved to match a designated goal configuration, patients with lesioned prefrontal cortex show deficits in planning a goal-directed sequence of moves. We propose a neuronal network model of sequence planning that passes this test and, when lesioned, fails in a way that mimics prefrontal patients’ behavior. Our model comprises a descending planning system with hierarchically organized plan, operation, and gesture levels, and an ascending evaluative system that analyzes the problem and computes internal reward signals that index the correct/erroneous status of the plan. Multiple parallel pathways connecting the evaluative and planning systems amend the plan and adapt it to the current problem. The model illustrates how specialized hierarchically organized neuronal assemblies may collectively emulate central executive or supervisory functions of the human brain.

Pressure-induced protein-folding/unfolding kinetics

We use an off-lattice minimalist model to describe the effects of pressure in slowing down the folding/unfolding kinetics of proteins when subjected to increasingly larger pressures. The potential energy function used to describe the interactions between beads in the model includes the effects of pressure on the pairwise interaction of hydrophobic groups in water. We show that pressure affects the participation of contacts in the transition state. More significantly, pressure exponentially decreases the chain reconfigurational diffusion coefficient. These results are consistent with experimental results on the kinetics of pressure-denaturation of staphylococcal nuclease.

The Small Mr Ras-like GTPase Rap1 and the Phospholipase C Pathway Act to Regulate Phagocytosis in Dictyostelium discoideum

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C–specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.

High Resolution Detection of Mechanical Forces Exerted by Locomoting Fibroblasts on the Substrate

We have developed a new approach to detect mechanical forces exerted by locomoting fibroblasts on the substrate. Cells were cultured on elastic, collagen-coated polyacrylamide sheets embedded with 0.2-μm fluorescent beads. Forces exerted by the cell cause deformation of the substrate and displacement of the beads. By recording the position of beads during cell locomotion and after cell removal, we discovered that most forces were radially distributed, switching direction in the anterior region. Deformations near the leading edge were strong, transient, and variable in magnitude, consistent with active local contractions, whereas those in the posterior region were weaker, more stable, and more uniform, consistent with passive resistance. Treatment of cells with cytochalasin D or myosin II inhibitors caused relaxation of the forces, suggesting that they are generated primarily via actin–myosin II interactions; treatment with nocodazole caused no immediate effect on forces. Immunofluorescence indicated that the frontal region of strong deformation contained many vinculin plaques but no apparent concentration of actin or myosin II filaments. Strong mechanical forces in the anterior region, generated by locally activated myosin II and transmitted through vinculin-rich structures, likely play a major role in cell locomotion and in mechanical signaling with the surrounding environment.

Isolation and Contraction of the Stress Fiber

Stress fibers were isolated from cultured human foreskin fibroblasts and bovine endothelial cells, and their contraction was demonstrated in vitro. Cells in culture dishes were first treated with a low-ionic-strength extraction solution and then further extracted using detergents. With gentle washes by pipetting, the nucleus and the apical part of cells were removed. The material on the culture dish was scraped, and the freed material was forced through a hypodermic needle and fractionated by sucrose gradient centrifugation. Isolated, free-floating stress fibers stained brightly with fluorescently labeled phalloidin. When stained with anti-α-actinin or anti-myosin, isolated stress fibers showed banded staining patterns. By electron microscopy, they consisted of bundles of microfilaments, and electron-dense areas were associated with them in a semiperiodic manner. By negative staining, isolated stress fibers often exhibited gentle twisting of microfilament bundles. Focal adhesion–associated proteins were also detected in the isolated stress fiber by both immunocytochemical and biochemical means. In the presence of Mg-ATP, isolated stress fibers shortened, on the average, to 23% of the initial length. The maximum velocity of shortening was several micrometers per second. Polystyrene beads on shortening isolated stress fibers rotated, indicating spiral contraction of stress fibers. Myosin regulatory light chain phosphorylation was detected in contracting stress fibers, and a myosin light chain kinase inhibitor, KT5926, inhibited isolated stress fiber contraction. Our study demonstrates that stress fibers can be isolated with no apparent loss of morphological features and that they are truly contractile organelle.