Monitoring tumor antigen specific T-cell responses in cancer patients and phase I clinical trials of peptide-based vaccination.

Numerous phase I and II clinical trials testing the safety and immunogenicity of various peptide vaccine formulations based on CTL-defined tumor antigens in cancer patients have been reported during the last 7 years. While specific T-cell responses can be detected in a variable fraction of immunized patients, an even smaller but significant fraction of these patients have objective tumor responses. Efficient therapeutic vaccination should aim at boosting naturally occurring antitumor T- and B-cell responses and at sustaining a large number of tumor antigen specific and fully functional effector T cells at tumor sites. Recent progress in our ability to quantitatively and qualitatively monitor tumor antigen specific CD8 T-cell responses will greatly help in making rapid progress in this field.

Clear Zone – A Synthesis of Practice and an Evaluation of the Benefi ts of Meeting the 10-ft Clear Zone Goal on Urban Streets, November 2008

In urban communities, there are often limited amounts of right-of-way available for establishing a large setback distance from the curb for fixed objects. Urban communities must constantly weigh the cost of purchasing additional right-of-way for clear zones against the risk of fixed object crashes. From 2004 to 2006, this type of crash on curbed roads represented 15% of all fatal crashes and 3% of all crashes in the state of Iowa. Many states have kept the current minimum AASHTO recommendations as their minimum clear zone standards; however, other states have decided that these recommendations are insufficient and have increased the required minimum clear zone distance to better suit the judgment of local designers. This report presents research on the effects of the clear zone on urban curbed streets. The research was conducted in two phases. The first phase involved a synthesis of practice that included a literature review and a survey of practices in jurisdictions that have developmental and historical patterns similar to those of Iowa. The second phase involved investigating the benefits of a 10 ft clear zone, which included examining urban corridors in Iowa that meet or do not meet the 10 ft clear zone goal. The results of this study indicate that a consistent fixed object offset results in a reduction in the number of fixed object crashes, a 5 ft clear zone is most effective when the goal is to minimize the number of fixed object c ashes, and a 3 ft clear zone is most effective when the goal is to minimize the cost of fixed object crashes.

The small RNA PhrS stimulates synthesis of the Pseudomonas aeruginosa quinolone signal.

Quorum sensing, a cell-to-cell communication system based on small signal molecules, is employed by the human pathogen Pseudomonas aeruginosa to regulate virulence and biofilm development. Moreover, regulation by small trans-encoded RNAs has become a focal issue in studies of virulence gene expression of bacterial pathogens. In this study, we have identified the small RNA PhrS as an activator of PqsR synthesis, one of the key quorum-sensing regulators in P. aeruginosa. Genetic studies revealed a novel mode of regulation by a sRNA, whereby PhrS uses a base-pairing mechanism to activate a short upstream open reading frame to which the pqsR gene is translationally coupled. Expression of phrS requires the oxygen-responsive regulator ANR. Thus, PhrS is the first bacterial sRNA that provides a regulatory link between oxygen availability and quorum sensing, which may impact on oxygen-limited growth in P. aeruginosa biofilms.

Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and colorectal carcinoma patients.

Carcinoembryonic antigen (CEACAM5) is commonly overexpressed in human colon cancer. Several antigenic peptides recognized by cytolytic CD8+ T-cells have been identified and used in colon cancer phase-I vaccination clinical trials. The HLA-A*0201-binding CEA(694-702) peptide was recently isolated from acid eluted MHC-I associated peptides from a human colon tumor cell line. However, the immunogenicity of this peptide in humans remains unknown. We found that the peptide CEA(694-702) binds weakly to HLA-A*0201 molecules and is ineffective at inducing specific CD8+ T-cell responses in healthy donors. Immunogenic-altered peptide ligands with increased affinity for HLA-A*0201 were identified. Importantly, the elicited cytolytic T lymphocyte (CTL) lines and clones cross-reacted with the wild-type CEA(694-702) peptide. Tumor cells expressing CEA were recognized in a peptide and HLA-A*0201 restricted fashion, but high-CEA expression levels appear to be required for CTL recognition. Finally, CEA-specific T-cell precursors could be readily expanded by in vitro stimulation of peripheral blood mononuclear cell (PBMC) from colon cancer patients with altered CEA peptide. However, the CEA-specific CD8+ T-cell clones derived from cancer patients revealed low-functional avidity and impaired tumor-cell recognition. Together, using T-cells to demonstrate the processing and presentation of the peptide CEA694-702, we were able to corroborate its presentation by tumor cells. However, the low avidity of the specific CTLs generated from cancer patients as well as the high-antigen expression levels required for CTL recognition pose serious concerns for the use of CEA694-702 in cancer immunotherapy.

Durable, Cost-Effective Pavement Markings Phase I: Synthesis of Current Research, June 2001

Pavement marking technology is a continually evolving subject. There are numerous types of materials used in the field today, including (but not limited to) paint, epoxy, tape, and thermoplastic. Each material has its own set of unique characteristics related to durability, retro reflectivity, installation cost, and life-cycle cost. The Iowa Highway Research Board was interested in investigating the possibility of developing an ongoing program to evaluate the various products used in pavement marking. This potential program would maintain a database of performance and cost information to assist state and local agencies in determining which materials and placement methods are most appropriate for their use. The Center for Transportation Research and Education at Iowa State University has completed Phase I of this research: to identify the current practice and experiences from around the United States to recommend a further course of action for the State of Iowa. There has been a significant amount of research completed in the last several years. Research from Michigan, Pennsylvania, South Dakota, Ohio, and Alaska all had some common findings: white markings are more retro reflective than yellow markings; paint is by-and-large the least expensive material; paint tends to degrade faster than other materials; thermoplastic and tapes had higher retro reflective characteristics. Perhaps the most significant program going on in the area of pavement markings is the National Transportation Product Evaluation Program (NTPEP). This is an ongoing research program jointly conducted by the American Association of State Highway and Transportation Officials and its member states. Field and lab tests on numerous types of pavement marking materials are being conducted at sites representing four climatological areas. These results are published periodically for use by any jurisdiction interested in pavement marking materials performance.At this time, it is recommended that the State of Iowa not embark on a test deck evaluation program. Instead, close attention should be paid to the ongoing evaluations of the NTPEP program. Materials that fare well on the NTPEP test de cks should be considered for further field studies in Iowa.

Inhibition of cell migration and invasion mediated by the TAT-RasGAP317-326 peptide requires the DLC1 tumor suppressor.

TAT-RasGAP317-326, a peptide corresponding to the 317-326 sequence of p120 RasGAP coupled with a cell-permeable TAT-derived peptide, sensitizes the death response of various tumor cells to several anticancer treatments. We now report that this peptide is also able to increase cell adherence, prevent cell migration and inhibit matrix invasion. This is accompanied by a marked modification of the actin cytoskeleton and focal adhesion redistribution. Interestingly, integrins and the small Rho GTP-binding protein, which are well-characterized proteins modulating actin fibers, adhesion and migration, do not appear to be required for the pro-adhesive properties of TAT-RasGAP317-326. In contrast, deleted in liver cancer-1, a tumor suppressor protein, the expression of which is often deregulated in cancer cells, was found to be required for TAT-RasGAP317-326 to promote cell adherence and inhibit migration. These results show that TAT-RasGAP317-326, besides its ability to favor tumor cell death, hampers cell migration and invasion.

The Design of Forest Taxation: A Synthesis with New Directions

Summary

Lentivector immunization induces tumor antigen-specific B and T cell responses in vivo.

Expression of the cancer/germ-line antigen NY-ESO-1 by tumors elicits spontaneous humoral and cellular immune responses in some cancer patients. Development of vaccines capable of stimulating such comprehensive immune responses is desirable. We have produced recombinant lentivectors directing the intracellular synthesis of NY-ESO-1 (rLV/ESO) and have analyzed the in vivo immune response elicited by this vector. Single injection of rLV/ESO into HLA-A2-transgenic mice elicited long-lasting B and T cell responses against NY-ESO-1. CD8+ T cells against the HLA-A2-restricted peptide NY-ESO-1(157-165) were readily detectable ex vivo and showed restricted TCR Vbeta usage. Moreover, rLV/ESO elicited a far greater anti-NY-ESO-1(157-165) CD8+ T cell response than peptide- or protein-based vaccines. Anti-NY-ESO-1 antibodies were rapidly induced after immunization and their detection preceded that of the antigen-specific CD8+ T cells. The rLV/ESO also induced CD4+ T cells. These cells played an essential role as their depletion completely abrogated B cell and CD8+ T cell responses against NY-ESO-1. The induced CD4+ T cells were primarily directed against a single NY-ESO-1 epitope spanning amino acids 81-100. Altogether, our study shows that rLV/ESO induces potent and comprehensive immune responses in vivo.

Metabolic engineering of plants for the synthesis of polyhydroxyalkanoates

Synthesis of polyhydroxyalkanoates (PHAs) in crop is viewed as an attractive approach for the production of this family of biodegradable plastics in large quantities and at low costs. Synthesisof PHAs containing various monomers has so far been demonstrated in the cytosol, plastids, and peroxisomes of plants. Several biochemical pathways have been modifies to achieve this, including the isoprenois pathway, the fatty acid biosynthetic pathway, and the fatty acid

Differential peptide binding to CD40 evokes counteractive responses.

The antigen-presenting cell-expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)-12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.

Synthesis, structure, and magnetic studies on self-assembled BiFeO3-CoFe2O4 nanocomposite thin films

Self-assembled (0.65)BiFeO3-(0.35)CoFe2O4 (BFO-CFO) nanostructures were deposited on SrTiO3 (001) and (111) substrates by pulsed laser deposition at various temperatures from 500 to 800°C. The crystal phases and the lattice strain for the two different substrate orientations have been determined and compared. The films grow epitaxial on both substrates but separation of the spinel and perovskite crystallites, without parasitic phases, is only obtained for growth temperatures of around 600-650°C. The BFO crystallites are out-of-plane expanded on STO(001), whereas they are almost relaxed on (111). In contrast, CFO crystallites grow out-of-plane compressed on both substrates. The asymmetric behavior of the cell parameters of CFO and BFO is discussed on the basis of the role of the epitaxial stress caused by the substrate and the spinel-perovskite interfacial stress. It is concluded that interfacial stress dominates the elastic properties of CFO crystallites and thus it may play a fundamental on the interface magnetoelectric coupling in these nanocomposites.

Synthesis, structural order and magnetic behavior of self-assembled epsilon-Co nanocrystal arrays

The synthesis of magnetic nanoparticles with monodispere size distributions, their self assembly into ordered arrays and their magnetic behavior as a function of structural order (ferrofluids and 2D assemblies) are presented. Magnetic colloids of monodispersed, passivated, cobalt nanocrystals were produced by the rapid pyrolysis of cobalt carbonyl in solution. The size, size distribution (std. dev.< 5%) and the shape of the nanocrystals were controlled by varying the surfactant, its concentration, the reaction rate and the reaction temperature. The Co particles are defect-free single crystals with a complex cubic structure related to the beta phase of manganese (epsilon-Co). In the 2D assembly, a collective behavior was observed in the low-field susceptibility measurements where the magnetization of the zero field cooled process increases steadily and the magnetization of the field cooling process is independent the temperature. This was different from the observed behavior in a sample comprised of disordered interacting particles. A strong paramagnetic contribution appears at very low temperatures where the magnetization increases drastically after field cooling the sample. This has been attributed to the Co surfactant-particle interface since no magnetic atomic impurities are present in these samples.

Synthesis of sodium-zinc spinel ferrites

The synthesis of spinel ferrites with composition Zn1-2xNaxFe2+xO4has been performed and the composition range in which single phase samples are obtained has been defined. The characterization of the samples has been carried out from atomic absorption and X-ray fluorescence analyses, X-ray diffraction patterns, Mössbauer spectroscopy and thermomagnetic measurements. It is show that significant loss of Na does exist when the synthesis is performed at high temperatures. When the Na volatilization is avoided spinel oxides with Na content up to 0.25 atoms per unit formula can be obtained. In this case the increase of the interatomic distances leads to differing fundamental magnetic properties as compared to the equivalent lithium-zinc ferrites.

A Cell Permeable Rasgap-derived Peptide Sensitizes Cancer Cells To Radiotherapy

Purpose/Objective(s): Radiotherapy is an effective treatment modality against cancer. Despite recent technical progresses in radiation delivery precision, toxicity to healthy tissues remains the main limiting factor. RasGAP is a regulator of the Ras and Rho pathway; it has either a pro- or anti-apoptotic activity depending on the level of caspase expressed in the cell. The RasGAP derived peptide: TAT-RasGAP317 - 326 is the minimal sequence known to sensitize cancer cells, but not healthy cells, to genotoxin-induced apoptosis. In this study the TAT-RasGAP317 - 326 radio-sensitizing effect was tested in vitro and in vivo.Materials/Methods: Two weeks clonogenic forming assays with 5 human cancer cells (PANC-1, HCT116, U87, U251 and HeLa) and a non tumorigenic cell line (HaCaT) were performed. Cells were exposed to 0, 1, 2 and 4 Gy with or without 20 mMTAT-RasGAP317 - 326. Twenty mMTAT peptide was also used as control. TAT-RasGAP317 - 326 effect was also tested in tumor xenograft mouse models. Mice bearing HCT116 tumors (WT or p53 mutant) received 1.65 mg/kg TAT-RasGAP317 - 326 i.p. injected and were locally irradiated for 10 days with 3 Gy. Tumor volume was then followed during a minimum of 20 days. Control mice were treated with a single modality, either with TAT-RasGAP317 - 326 or with radiotherapy.Results: At all the tested radiation doses TAT-RasGAP317 - 326 showed a significant supra additive radio-sensitizing effect on all the tested tumor cell lines. Furthermore, it showed no sensitizing effect on the non tumorigenic cell line. In vivo, TAT-RasGAP317 - 326 also showed a significantly radio-sensitizing effect as shown by a significant higher reduction in tumor volume as much as by a significant tumor growth delay.Conclusions: Taken together our data suggest that TAT-RasGAP317 - 326 has a radio-sensitizing effect on in vivo and in vitro tumors without any effect on healthy tissues. Therefore TAT-RasGAP317 - 326 should be considered as a novel and attractive sensitizer compound allowing an improvement of the therapeutic interval.

Toward synthetic combinatorial peptide libraries in positional scanning format (PS-SCL)-based identification of CD8+ Tumor-reactive T-Cell Ligands: a comparative analysis of PS-SCL recognition by a single tumor-reactive CD8+ cytolytic T-lymphocyte clone.

The use of synthetic combinatorial peptide libraries in positional scanning format (PS-SCL) has emerged recently as an alternative approach for the identification of peptides recognized by T lymphocytes. The choice of both the PS-SCL used for screening experiments and the method used for data analysis are crucial for implementing this approach. With this aim, we tested the recognition of different PS-SCL by a tyrosinase 368-376-specific CTL clone and analyzed the data obtained with a recently developed biometric data analysis based on a model of independent and additive contribution of individual amino acids to peptide antigen recognition. Mixtures defined with amino acids present at the corresponding positions in the native sequence were among the most active for all of the libraries. Somewhat surprisingly, a higher number of native amino acids were identifiable by using amidated COOH-terminal rather than free COOH-terminal PS-SCL. Also, our data clearly indicate that when using PS-SCL longer than optimal, frame shifts occur frequently and should be taken into account. Biometric analysis of the data obtained with the amidated COOH-terminal nonapeptide library allowed the identification of the native ligand as the sequence with the highest score in a public human protein database. However, the adequacy of the PS-SCL data for the identification for the peptide ligand varied depending on the PS-SCL used. Altogether these results provide insight into the potential of PS-SCL for the identification of CTL-defined tumor-derived antigenic sequences and may significantly implement our ability to interpret the results of these analyses.