Development of technology for hot drape forming and nozzles for torus sections Final report, Jun. 1963 - Dec. 1965

Hot drape forming and heat treatment of aluminum base alloy toroidal shell segment

Peptide Ligands Incorporated into the Threefold Spike Capsid Domain to Re-Direct Gene Transduction of AAV8 and AAV9 In Vivo

Efficiency and specificity of viral vectors are vital issues in gene therapy. Insertion of peptide ligands into the adeno-associated viral (AAV) capsid at receptor binding sites can re-target AAV2-derived vectors to alternative cell types. Also, the use of serotypes AAV8 and -9 is more efficient than AAV2 for gene transfer to certain tissues in vivo. Consequently, re-targeting of these serotypes by ligand insertion could be a promising approach but has not been explored so far. Here, we generated AAV8 and -9 vectors displaying peptides in the threefold spike capsid domain. These peptides had been selected from peptide libraries displayed on capsids of AAV serotype 2 to optimize systemic gene delivery to murine lung tissue and to breast cancer tissue in PymT transgenic mice (PymT). Such peptide insertions at position 590 of the AAV8 capsid and position 589 of the AAV9 capsid changed the transduction properties of both serotypes. However, both peptides inserted in AAV8 did not result in the same changes of tissue tropism as they did in AAV2. While the AAV2 peptides selected on murine lung tissue did not alter tropism of serotypes 8 and -9, insertion of the AAV2-derived peptide selected on breast cancer tissue augmented tumor gene delivery in both serotypes. Further, this peptide mediated a strong but unspecific in vivo gene transfer for AAV8 and abrogated transduction of various control tissues for AAV9. Our findings indicate that peptide insertion into defined sites of AAV8 and -9 capsids can change and improve their efficiency and specificity compared to their wild type variants and to AAV2, making these insertion sites attractive for the generation of novel targeted vectors in these serotypes

The Dynamics of T-Cell Receptor Repertoire Diversity Following Thymus Transplantation for DiGeorge Anomaly

T cell populations are regulated both by signals specific to the T-cell receptor (TCR) and by signals and resources, such as cytokines and space, that act independently of TCR specificity. Although it has been demonstrated that disruption of either of these pathways has a profound effect on T-cell development, we do not yet have an understanding of the dynamical interactions of these pathways in their joint shaping of the T cell repertoire. Complete DiGeorge Anomaly is a developmental abnormality that results in the failure of the thymus to develop, absence of T cells, and profound immune deficiency. After receiving thymic tissue grafts, patients suffering from DiGeorge anomaly develop T cells derived from their own precursors but matured in the donor tissue. We followed three DiGeorge patients after thymus transplantation to utilize the remarkable opportunity these subjects provide to elucidate human T-cell developmental regulation. Our goal is the determination of the respective roles of TCR-specific vs. TCR-nonspecific regulatory signals in the growth of these emerging T-cell populations. During the course of the study, we measured peripheral blood T-cell concentrations, TCRβ V gene-segment usage and CDR3-length spectratypes over two years or more for each of the subjects. We find, through statistical analysis based on a novel stochastic population-dynamic T-cell model, that the carrying capacity corresponding to TCR-specific resources is approximately 1000-fold larger than that of TCR-nonspecific resources, implying that the size of the peripheral T-cell pool at steady state is determined almost entirely by TCR-nonspecific mechanisms. Nevertheless, the diversity of the TCR repertoire depends crucially on TCR-specific regulation. The estimated strength of this TCR-specific regulation is sufficient to ensure rapid establishment of TCR repertoire diversity in the early phase of T cell population growth, and to maintain TCR repertoire diversity in the face of substantial clonal expansion-induced perturbation from the steady state

Phase I Evaluation of STA-1474, a Prodrug of the Novel HSP90 Inhibitor Ganetespib, in Dogs with Spontaneous Cancer

The novel water soluble compound STA-1474 is metabolized to ganetespib (formerly STA-9090), a potent HSP90 inhibitor previously shown to kill canine tumor cell lines in vitro and inhibit tumor growth in the setting of murine xenografts. The purpose of the following study was to extend these observations and investigate the safety and efficacy of STA-1474 in dogs with spontaneous tumors.This was a Phase 1 trial in which dogs with spontaneous tumors received STA-1474 under one of three different dosing schemes. Pharmacokinetics, toxicities, biomarker changes, and tumor responses were assessed. Twenty-five dogs with a variety of cancers were enrolled. Toxicities were primarily gastrointestinal in nature consisting of diarrhea, vomiting, inappetence and lethargy. Upregulation of HSP70 protein expression was noted in both tumor specimens and PBMCs within 7 hours following drug administration. Measurable objective responses were observed in dogs with malignant mast cell disease (n = 3), osteosarcoma (n = 1), melanoma (n = 1) and thyroid carcinoma (n = 1), for a response rate of 24% (6/25). Stable disease (>10 weeks) was seen in 3 dogs, for a resultant overall biological activity of 36% (9/25).This study provides evidence that STA-1474 exhibits biologic activity in a relevant large animal model of cancer. Given the similarities of canine and human cancers with respect to tumor biology and HSP90 activation, it is likely that STA-1474 and ganetespib will demonstrate comparable anti-cancer activity in human patients

Identification of Mammalian Protein Quality Control Factors by High-Throughput Cellular Imaging

Protein Quality Control (PQC) pathways are essential to maintain the equilibrium between protein folding and the clearance of misfolded proteins. In order to discover novel human PQC factors, we developed a high-content, high-throughput cell-based assay to assess PQC activity. The assay is based on a fluorescently tagged, temperature sensitive PQC substrate and measures its degradation relative to a temperature insensitive internal control. In a targeted screen of 1591 siRNA genes involved in the Ubiquitin-Proteasome System (UPS) we identified 25 of the 33 genes encoding for 26S proteasome subunits and discovered several novel PQC factors. An unbiased genome-wide siRNA screen revealed the protein translation machinery, and in particular the EIF3 translation initiation complex, as a novel key modulator of misfolded protein stability. These results represent a comprehensive unbiased survey of human PQC components and establish an experimental tool for the discovery of genes that are required for the degradation of misfolded proteins under conditions of proteotoxic stress

At What Stage of Neural Processing Does Cocaine Act to Boost Pursuit of Rewards?

Dopamine-containing neurons have been implicated in reward and decision making. One element of the supporting evidence is that cocaine, like other drugs that increase dopaminergic neurotransmission, powerfully potentiates reward seeking. We analyze this phenomenon from a novel perspective, introducing a new conceptual framework and new methodology for determining the stage(s) of neural processing at which drugs, lesions and physiological manipulations act to influence reward-seeking behavior. Cocaine strongly boosts the proclivity of rats to work for rewarding electrical brain stimulation. We show that the conventional conceptual framework and methods do not distinguish between three conflicting accounts of how the drug produces this effect: increased sensitivity of brain reward circuitry, increased gain, or decreased subjective reward costs. Sensitivity determines the stimulation strength required to produce a reward of a given intensity (a measure analogous to the KM of an enzyme) whereas gain determines the maximum intensity attainable (a measure analogous to the vmax of an enzyme-catalyzed reaction). To distinguish sensitivity changes from the other determinants, we measured and modeled reward seeking as a function of both stimulation strength and opportunity cost. The principal effect of cocaine was a two-fourfold increase in willingness to pay for the electrical reward, an effect consistent with increased gain or decreased subjective cost. This finding challenges the long-standing view that cocaine increases the sensitivity of brain reward circuitry. We discuss the implications of the results and the analytic approach for theories of how dopaminergic neurons and other diffuse modulatory brain systems contribute to reward pursuit, and we explore the implications of the conceptual framework for the study of natural rewards, drug reward, and mood

Stereotypical Chronic Lymphocytic Leukemia B-Cell Receptors Recognize Survival Promoting Antigens on Stromal Cells

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Survival of CLL cells depends on their close contact with stromal cells in lymphatic tissues, bone marrow and blood. This microenvironmental regulation of CLL cell survival involves the stromal secretion of chemo- and cytokines as well as the expression of adhesion molecules. Since CLL survival may also be driven by antigenic stimulation through the B-cell antigen receptor (BCR), we explored the hypothesis that these processes may be linked to each other. We tested if stromal cells could serve as an antigen reservoir for CLL cells, thus promoting CLL cell survival by stimulation through the BCR. As a proof of principle, we found that two CLL BCRs with a common stereotyped heavy chain complementarity-determining region 3 (previously characterized as “subset 1”) recognize antigens highly expressed in stromal cells – vimentin and calreticulin. Both antigens are well-documented targets of autoantibodies in autoimmune disorders. We demonstrated that vimentin is displayed on the surface of viable stromal cells and that it is present and bound by the stereotyped CLL BCR in CLL-stroma co-culture supernatant. Blocking the vimentin antigen by recombinant soluble CLL BCR under CLL-stromal cell co-culture conditions reduces stroma-mediated anti-apoptotic effects by 20–45%. We therefore conclude that CLL BCR stimulation by stroma-derived antigens can contribute to the protective effect that the stroma exerts on CLL cells. This finding sheds a new light on the understanding of the pathobiology of this so far mostly incurable disease

Resistance to HSP90 inhibition involving loss of MCL1 addiction

YesInhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality

Recurrent, Robust and Scalable Patterns Underlie Human Approach and Avoidance

BACKGROUND. Approach and avoidance behavior provide a means for assessing the rewarding or aversive value of stimuli, and can be quantified by a keypress procedure whereby subjects work to increase (approach), decrease (avoid), or do nothing about time of exposure to a rewarding/aversive stimulus. To investigate whether approach/avoidance behavior might be governed by quantitative principles that meet engineering criteria for lawfulness and that encode known features of reward/aversion function, we evaluated whether keypress responses toward pictures with potential motivational value produced any regular patterns, such as a trade-off between approach and avoidance, or recurrent lawful patterns as observed with prospect theory. METHODOLOGY/PRINCIPAL FINDINGS. Three sets of experiments employed this task with beautiful face images, a standardized set of affective photographs, and pictures of food during controlled states of hunger and satiety. An iterative modeling approach to data identified multiple law-like patterns, based on variables grounded in the individual. These patterns were consistent across stimulus types, robust to noise, describable by a simple power law, and scalable between individuals and groups. Patterns included: (i) a preference trade-off counterbalancing approach and avoidance, (ii) a value function linking preference intensity to uncertainty about preference, and (iii) a saturation function linking preference intensity to its standard deviation, thereby setting limits to both. CONCLUSIONS/SIGNIFICANCE. These law-like patterns were compatible with critical features of prospect theory, the matching law, and alliesthesia. Furthermore, they appeared consistent with both mean-variance and expected utility approaches to the assessment of risk. Ordering of responses across categories of stimuli demonstrated three properties thought to be relevant for preference-based choice, suggesting these patterns might be grouped together as a relative preference theory. Since variables in these patterns have been associated with reward circuitry structure and function, they may provide a method for quantitative phenotyping of normative and pathological function (e.g., psychiatric illness).National Institute on Drug Abuse (14118, 026002, 026104, DABK39-03-0098, DABK39-03-C-0098); The MGH Phenotype Genotype Project in Addiction and Mood Disorder from the Office of National Drug Control Policy - Counterdrug Technology Assessment Center; MGH Department of Radiology; the National Center for Research Resources (P41RR14075); National Institute of Neurological Disorders and Stroke (34189, 05236