Characterization and optimization of antigen-specific T cell responses during ex vivo expansion of melanoma tumor-infiltrating lymphocytes

Treatment of metastatic melanoma with tumor reactive T cells (adoptive T cell therapy, ACT) is a promising approach associated with a high clinical response rate. However, further optimization of this treatment modality is required to increase the clinical response after this therapy. ACT in melanoma involves an initial phase (pre-REP) of tumor-infiltrating lymphocyte (TIL) expansion ex vivo from tumor isolates followed by a second phase, “rapid expansion protocol” (REP) generating the billions of cells used as the TIL infusion product. The main question addressed in this thesis was how the currently used REP affected the responsiveness of the CD8+ T cells to defined melanoma antigens. We hypothesized that the REP drives the TIL to further differentiate and become hyporesponsive to antigen restimulation, therefore, proper cytokine treatment or other ways to expand TIL is required to improve upon this outcome. We evaluated the response of CD8+ TIL to melanoma antigen restimulation using MART-1 peptide-pulsed mature DC in vitro. Post-REP TILs were mostly hypo-responsive with poor proliferation and higher apoptosis. Phenotypic analysis revealed that the expression of CD28 was significantly reduced in post-REP TILs. By sorting experiment and microarray analysis, we confirmed that the few CD28+ post-REP TILs had superior survival capacity and proliferated after restimulation. We then went on to investigate methods to maintain CD28 expression during the REP and improve TIL responsiveness. Firstly, IL-15 and IL-21 were found to synergize in maintaining TIL CD28 expression and antigenic responsiveness during REP. Secondly, we found IL-15 was superior as compared to IL-2 in supporting the long-term expansion of antigen-specific CD8+ TIL after restimulation. These results suggest that current expansion protocols used for adoptive T-cell therapy in melanoma yield largely hyporesponsive products containing CD8+ T cells unable to respond in vivo to re-stimulation with antigen. A modification of our current approaches by using IL-15+IL-21 as supporting cytokines in the REP, or/and administration of IL-15 instead of IL-2 after TIL infusion, may enhance the anti-tumor efficacy and long-term persistence of infused T cells in vivo.

Mechanisms regulating the p120-catenin/Kaiso pathway

The Wnt pathways contribute to many processes in cancer and developmental biology, with β-catenin being a key canonical component. P120-catenin, which is structurally similar to β-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ/ zinc-finger transcription factor Kaiso. In my first project, employing Xenopus embryos and mammalian cell lines, I found that the degradation machinery of the canonical Wnt pathway modulates p120-catenin protein stability, especially p120 isoform-1, through mechanisms shared with b-catenin. Exogenous expression of destruction-complex components such as GSK3b or Axin promotes p120-catenin degradation, and consequently, is able to rescue developmental phenotypes resulting from p120 over-expression during early Xenopus embryonic development. Conversely, as predicted, the in vivo depletion of either Axin or GSK3b coordinately increased p120 and b-catenin levels, while p120 levels decreased upon LRP5/6 depletion, which are positive modulators in the canonical Wnt pathway. At the primary sequence level, I resolved conserved GSK3b phosphorylation sites in p120’s (isoform 1) amino-terminal region. Point-mutagenesis of these residues inhibited the association of destruction complex proteins including those involved in ubiquitination, resulting in p120-catenin stabilization. Importantly, we found that two additional p120-catenin family members, ARVCF-catenin and d-catenin, in common with b-catenin and p120, associate with Axin, and are degraded in Axin’s presence. Thus, by similar means, it appears that canonical Wnt signals coordinately modulate multiple catenin proteins having roles in development and conceivably disease states. In my second project, I found that the Dyrk1A kinase exhibits a positive effect upon p120-catenin levels. That is, unlike the negative regulator GSK3b kinase, a candidate screen revealed that Dyrk1A kinase enhances p120-catenin protein levels via increased half-life. Dyrk1A is encoded by a gene located within the trisomy of chromosome 21, which contributes to mental retardation in Down Syndrome patients. I found that Dyrk1A expression results in increased p120 protein levels, and that Dyrk1A specifically associates with p120 as opposed to other p120-catenin family members or b-catenin. Consistently, Dyrk1A depletion in mammalian cell lines and Xenopus embryos decreased p120-catenin levels. I further confirmed that Dyrk overexpression and knock-down modulates both Siamois and Wnt11 gene expression in the expected manner based upon the resulting latered levels of p120-catenin. I determined that Dyrk expression rescues Kaiso depletion effects (gastrulation failure; increased endogenous Wnt11 expression), and vice versa. I then identified a putative Dyrk phosphorylation region within the N-terminus of p120-catenin, which may also be responsible for Dyrk1A association. I went on to make a phosphomimic mutant, which when over-expressed, had the predicted enhanced capacity to positively modulate endogenous Wnt11 and Siamois expression, and thereby generate gastrulation defects. Given that Dyrk1A modulates Siamois expression through stabilization of p120-catenin, I further observed that ectopic expression of Dyrk can positively influence b-catenin’s capacity to generate ectopic dorsal axes when ventrally expressed in early Xenopus embryos. Future work will investigate how Dyrk1A modulates the Wnt signaling pathway through p120-catenin, and possibly begin to address how dysfunction of Dyrk1A with respect to p120-catenin might relate to aspects of Down syndrome. In summary, the second phase of my graduate work appears to have revealed a novel aspect of Dyrk1A/p120-catenin action in embryonic development, with a functional linkage to canonical Wnt signaling. What I have identified as a “Dyrk1A/p120-catenin/Kaiso pathway” may conceivably assist in our larger understanding of the impact of Dyrk1A dosage imbalance in Down syndrome.

Structural plasticity can produce metaplasticity.

BACKGROUND: Synaptic plasticity underlies many aspect of learning memory and development. The properties of synaptic plasticity can change as a function of previous plasticity and previous activation of synapses, a phenomenon called metaplasticity. Synaptic plasticity not only changes the functional connectivity between neurons but in some cases produces a structural change in synaptic spines; a change thought to form a basis for this observed plasticity. Here we examine to what extent structural plasticity of spines can be a cause for metaplasticity. This study is motivated by the observation that structural changes in spines are likely to affect the calcium dynamics in spines. Since calcium dynamics determine the sign and magnitude of synaptic plasticity, it is likely that structural plasticity will alter the properties of synaptic plasticity. METHODOLOGY/PRINCIPAL FINDINGS: In this study we address the question how spine geometry and alterations of N-methyl-D-aspartic acid (NMDA) receptors conductance may affect plasticity. Based on a simplified model of the spine in combination with a calcium-dependent plasticity rule, we demonstrated that after the induction phase of plasticity a shift of the long term potentiation (LTP) or long term depression (LTD) threshold takes place. This induces a refractory period for further LTP induction and promotes depotentiation as observed experimentally. That resembles the BCM metaplasticity rule but specific for the individual synapse. In the second phase, alteration of the NMDA response may bring the synapse to a state such that further synaptic weight alterations are feasible. We show that if the enhancement of the NMDA response is proportional to the area of the post synaptic density (PSD) the plasticity curves most likely return to the initial state. CONCLUSIONS/SIGNIFICANCE: Using simulations of calcium dynamics in synaptic spines, coupled with a biophysically motivated calcium-dependent plasticity rule, we find under what conditions structural plasticity can form the basis of synapse specific metaplasticity.

Analysis of endogenous retinoic acid receptor expression and function in murine melanoma cells

There have been numerous reports over the past several years on the ability of vitamin A analogs (retinoids) to modulate cell proliferation, malignant transformation, morphogenesis, and differentiation in a wide variety of cell types and organisms. Two families of nuclear retinoid-inducible, trans-acting, transcription-enhancing receptors that bear strong DNA sequence homology to thyroid and steroid hormone receptors have recently been discovered. The retinoic acid receptors (RARs) and retinoid X receptors (RXRs) each have at least three types designated $\alpha,$ $\beta,$ and $\gamma,$ which are encoded by separate genes and expressed in a tissue and cell type-specific manner. We have been interested in the mechanism by which retinoids inhibit tumor cell proliferation and induce differentiation. As a model system we have employed several murine melanoma cell lines (S91-C2, K1735P, and B16-F1), which are sensitive to the growth-inhibitory and differentiation-inducing effects of RA, as well as a RA-resistant subclone of one of the cell lines (S91-C154), in order to study the role of the nuclear RARs in these effects. The initial phase of this project consisted of the characterization of the expression pattern of the three known RAR and RXR types in the murine melanoma cell lines in order to determine whether any differences exist which may elucidate a role for any of the receptors in RA-induced growth inhibition and differentiation. The novel finding was made that the RAR-$\beta$ gene is rapidly induced from undetectable levels by RA treatment at the mRNA and protein level, and that the induction of RAR-$\beta$ by other biologically active retinoids correlated with their ability to inhibit the growth of the highly RA-sensitive S91-C2 cell line. This suggests a role for RAR-$\beta$ in the growth inhibiting effect of retinoids. The second phase of this project involves the stable expression of RAR-$\beta$ in the S91-C2 cells and the RAR-$\beta$ receptor-null cell line, K1735P. These studies have indicated an inverse correlation between RAR-$\beta$ expression and proliferation rate. ^

Comparison of cost and outcome of care in patients with low back pain who are managed by physicians or physical therapists in private practice

The purpose of this study was to determine if there were differences in the cost and outcome of care in patients with low back pain who were managed by physicians or physical therapists in private practice in the state of Arizona. A secondary purpose was to describe the current status of private practice physical therapy clinicians who treat patients with low back pain.^ A Survey on Practice was mailed to 194 physical therapists who were listed by the American Physical Therapy Association as being in private practice in Arizona. Eighty-three percent of the surveys were returned after three attempts. Of those which were returned, 72 were complete and included in the analysis.^ The 72 practices were screened to determine those eligible for the second phase of the study. Those eligible for the second phase numbered 52 clinics. Twenty-six practices agreed to participate; however, only 21 did participate. Clinics which participated were sent packets of information which were to be kept on each patient seen with a complaint of low back pain during a three month period. Packets contained a patient-oriented survey on functional activity to be completed before and after the physical therapy course of treatment, as well as a log which was completed by the physical therapist on the type of care given to the patient and an assessment of the outcome of treatment. The patient was asked to fill out a satisfaction survey relative to the care received from the physical therapist and physician, if applicable.^ Although 259 patients were entered into the study, 210 patient logs were available for analysis. Results indicated that generally, there was no difference in cost or outcome as measured by the final functional score, change between the initial and final functional scores, or the therapist-rated outcome between the patients who were managed by physicians or physical therapists when controlling for age and length of time the patient was experiencing pain. Patients were more satisfied with care received from physical therapists as compared to physicians. Age and length of pain were good predictors of the type of referral patients received according to a logistic regression procedure. The initial disability score (IRS) and the time spent in the facility predicted therapist-rated outcome, a good or poor final disability score (FRS), and a good or poor change score. In addition, age predicted FRS and change scores. The time that the therapist spent in direct contact with the patient also predicted the change score.^ These findings of no difference in the cost and outcome of care were discussed as they relate to the practice of medicine and physical therapy. ^

Analysis of galectin expression and identification of endogenous ligands in a human colon carcinoma cell line

The 14.5 kDa (galectin-1) and 31 kDa (galectin-3) lectins are the most well characterized members of a family of vertebrate carbohydrate-binding proteins known as the galectins. Evidence has been obtained implicating these galectins in events as diverse as cell-cell and cell-extracellular matrix interactions, growth regulation, transformation, differentiation, and programmed cell death. In the present study, sodium butyrate was found to be a potent inducer of galectin-1 in the KM12 human colon carcinoma cell line. Prior to treatment with butyrate this cell line expresses only galectin-3. These cells were utilized as an in vitro model system to study galectin expression as well as that of their endogenous ligands. The initial phase of this project involved the examination of the induction of galectin-1 by butyrate at the protein level. These studies indicated that galectin-1 induction by butyrate was relatively rapid reaching nearly maximal levels after only 24 hours. Additionally, the induction was found to be reversible upon the removal of butyrate and to precede the increase in expression of the well characterized differentiation marker, carcinoembryonic antigen (CEA). The second phase of this project involved the characterization of potential glycoprotein ligands for galectin-1 and galectin-3. This work demonstrated that the polylactosaminoglycan-containing glycoproteins laminin, CEA, and the lysosome-associated glycoproteins-1 and -2 (LAMPs-1 and -2) are capable of serving as ligands for both galectin-1 and -3. The third phase of this project involved the analysis of the induction of the galectin-1 promoter by butyrate. Through the analysis of deletion constructs transiently transfected into KM12 cells, the region of the galectin-1 promoter mediating a high level of induction by butyrate was localized primarily within a proximal portion of the promoter containing a CCAAT element and an Sp1 binding site. The CCAAT-binding activity in the KM12 nuclear extracts was subsequently dentified as NF-Y by gel shift analysis. These studies suggest that: (1) the galectins may be involved in modulating adhesive interactions in human colon carcinoma cells through the binding of several polylactosaminoglycans shown to play a role in adhesion and (2) high level induction of the galectin-1 promoter by butyrate can proceed through a discreet, proximal element containing an NF-Y-binding CCAAT box and an Sp1 site. ^

Functional studies of the homeobox gene {\it Goosecoid\/} during mouse embryogenesis

A fundamental question in developmental biology is to understand the mechanisms that govern the development of an adult individual from a single cell. Goosecoid (Gsc) is an evolutionarily conserved homeobox gene that has been cloned in vertebrates and in Drosophila. In mice, Gsc is first expressed during gastrulation stages where it marks anterior structures of the embryo, this pattern of expression is conserved among vertebrates. Later, expression is observed during organogenesis of the head, limbs and the trunk. The conserved pattern of expression of Gsc during gastrulation and gain of function experiments in Xenopus suggested a function for Gsc in the development of anterior structures in vertebrates. Also, its expression pattern in mouse suggested a role in morphogenesis of the head, limbs and trunk. To determine the functional requirement of Gsc in mice a loss of function mutation was generated by homologous recombination in embryonic stem cells and mice mutant for Gsc were generated.^ Gsc-null mice survived to birth but died hours after delivery. Phenotypic analysis revealed craniofacial and rib cage abnormalities that correlated with the second phase of Gsc expression in the head and trunk but no anomalies were found that correlated with its pattern of expression during gastrulation or limb development.^ To determine the mode of action of Gsc during craniofacial development aggregation chimeras were generated between Gsc-null and wild-type embryos. Chimeras were generated by the aggregation of cleavage stage embryos, taking advantage of two different Gsc-null alleles generated during gene targeting. Chimeras demonstrated a cell-autonomous function for Gsc during craniofacial development and a requirement for Gsc function in cartilage and mesenchymal tissues.^ Thus, during embryogenesis in mice, Gsc is not an essential component of gastrulation as had been suggested in previous experiments. Gsc is required for craniofacial development where it acts cell autonomously in cartilage and mesenchymal tissues. Gsc is also required for proper development of the rib cage but it is dispensable for limb development in mice. ^

Proactive aggression: A two part study

Aggressive behavior can be divided into the subtypes: reactive and proactive. Reactive aggressive acts occur in response to a stimulus or provocation. Proactive aggressive acts occur without provocation and are goal-directed. A number of findings have suggested that individuals displaying proactive aggression may be discerned from individuals not displaying proactive aggression on measures of personality, psychopathology and psychopathy, as well as on aggressive histories and type and severity of aggressive behaviors committed. The current study was conducted in two phases; phase 1 and 2. This was because phase 1 compared proactive aggressive, reactive aggressive and non-aggressive subjects on questionnaire measures, while phase 2 observed the acute effects of the benzodiazepine alprazolam on only proactive aggressive subjects. The phase 1 hypotheses were that proactive aggressive subjects would show greater numbers of personality disorders and have greater psychopathy relative to reactive and non-aggressive subjects. To verify these hypotheses subjects were recruited from the community and classified as proactive (n = 20), reactive (n = 20) or non-aggressive (n = 10) via laboratory behavioral testing. Classified subjects were administered a battery of questionnaires pertaining to personality disorders (SCID-II, OMNI-IV), psychopathy (PCL-R) and aggression history. The results of these questionnaire measures were subjected to statistical analyses, which confirmed the hypotheses. In the second phase, the acute effects of three doses of the benzodiazepine alprazolam were evaluated in proactive aggressive subjects on proactive aggressive responding in the computer-based Point Subtraction Aggression Paradigm (PSAP). In phase 2 it was hypothesized that alprazolam would produce dose dependent decreases in aggressive responding. Subjects were never provoked in this phase, and aggressive responding was classified as proactive. Studies of drugs acting on the GABA system have frequently found decreases in aggression in animals and humans, although there have also been findings of increased (paradoxical) aggression. The hypothesis was tested by statistical analysis of proactive aggressive responding under placebo vs. under alprazolam. The hypothesis was supported by six of seven subjects. Aggressive responding was significantly, decreased under alprazolam relative to placebo in six subjects. One subject showed increases in aggressive responding.^

A comprehensive framework for evaluating funder's performance

Evaluation methods for assessing the performance of non-profit funders are lacking. The purpose of the research was to create a comprehensive framework that systematically assesses the goals and objectives of a funder, how these relate to the funder's allocation of resources, and the potential impact of programs and services selected by the funder for resource allocation to address organizational goals and objectives. The Houston Affiliate of Susan G. Komen for the Cure, a local chapter of a national breast cancer awareness advocacy organization, was selected as the funding agency whose performance assessment was to assist in the creation of this framework. Evaluation approaches from the government sector were adapted and incorporated into the research to guide the methods used to answer the three research questions corresponding to the three phases of research within the study: (1) what are the funding goals and objectives of the Affiliate?; (2) what allocation scheme does the organization use to address these goals and objectives and select programs for funding?; and, (3) to what extent do the programs funded by the Affiliate have potential long-term impact? ^ Within the first stage of the research, document reviews of the Affiliate's mission-based documents and bylaws and interviews with organizational and community informants revealed a highly latent constellation of broad objectives that were not formalized into one guiding document, thus creating gaps in management and governance. Within the second phase of the research, reviews of grant applications from the 2008-2009 funding cycle and interviews with employees and volunteers familiar with the funding process revealed competing ideas regarding resource allocation in light of vague organizational documents describing funding goals and objectives. Within the final stage of the research, these findings translated to the Affiliate selecting programs with highly varying potential long-term impact with regards to addressing goals and objectives relating to breast cancer education, screening, diagnostics, treatment, and support. The resulting performance assessment framework, consisting of three phases of research utilizing organizational documents and key informant interviews, demonstrated the importance of clearly defined funding goals and objectives, reference documents and committee participation within the funding process, and regular reviews of potential long-term impact for selected programs, all supported by the active participation and governance of a funder's Board of Directors.^

A categorical data analysis approach to estimation of demographic characteristics for Texas county populations

The need for timely population data for health planning and Indicators of need has Increased the demand for population estimates. The data required to produce estimates is difficult to obtain and the process is time consuming. Estimation methods that require less effort and fewer data are needed. The structure preserving estimator (SPREE) is a promising technique not previously used to estimate county population characteristics. This study first uses traditional regression estimation techniques to produce estimates of county population totals. Then the structure preserving estimator, using the results produced in the first phase as constraints, is evaluated.^ Regression methods are among the most frequently used demographic methods for estimating populations. These methods use symptomatic indicators to predict population change. This research evaluates three regression methods to determine which will produce the best estimates based on the 1970 to 1980 indicators of population change. Strategies for stratifying data to improve the ability of the methods to predict change were tested. Difference-correlation using PMSA strata produced the equation which fit the data the best. Regression diagnostics were used to evaluate the residuals.^ The second phase of this study is to evaluate use of the structure preserving estimator in making estimates of population characteristics. The SPREE estimation approach uses existing data (the association structure) to establish the relationship between the variable of interest and the associated variable(s) at the county level. Marginals at the state level (the allocation structure) supply the current relationship between the variables. The full allocation structure model uses current estimates of county population totals to limit the magnitude of county estimates. The limited full allocation structure model has no constraints on county size. The 1970 county census age - gender population provides the association structure, the allocation structure is the 1980 state age - gender distribution.^ The full allocation model produces good estimates of the 1980 county age - gender populations. An unanticipated finding of this research is that the limited full allocation model produces estimates of county population totals that are superior to those produced by the regression methods. The full allocation model is used to produce estimates of 1986 county population characteristics. ^

THE IMPLEMENTATION OF FEDERAL FAMILY PLANNING POLICY: AN ANALYSIS OF FACTORS AFFECTING STATE EXPENDITURES, FISCAL YEARS 1976-1981 (WOMEN, BUDGET, POPULATION)

The purpose of this study was to analyze the implementation of national family planning policy in the United States, which was embedded in four separate statutes during the period of study, Fiscal Years 1976-81. The design of the study utilized a modification of the Sabatier and Mazmanian framework for policy analysis, which defined implementation as the carrying out of statutory policy. The study was divided into two phases. The first part of the study compared the implementation of family planning policy by each of the pertinent statutes. The second part of the study identified factors that were associated with implementation of federal family planning policy within the context of block grants.^ Implemention was measured here by federal dollars spent for family planning, adjusted for the size of the respective state target populations. Expenditure data were collected from the Alan Guttmacher Institute and from each of the federal agencies having administrative authority for the four pertinent statutes, respectively. Data from the former were used for most of the analysis because they were more complete and more reliable.^ The first phase of the study tested the hypothesis that the coherence of a statute is directly related to effective implementation. Equity in the distribution of funds to the states was used to operationalize effective implementation. To a large extent, the results of the analysis supported the hypothesis. In addition to their theoretical significance, these findings were also significant for policymakers insofar they demonstrated the effectiveness of categorical legislation in implementing desired health policy.^ Given the current and historically intermittent emphasis on more state and less federal decision-making in health and human serives, the second phase of the study focused on state level factors that were associated with expenditures of social service block grant funds for family planning. Using the Sabatier-Mazmanian implementation model as a framework, many factors were tested. Those factors showing the strongest conceptual and statistical relationship to the dependent variable were used to construct a statistical model. Using multivariable regression analysis, this model was applied cross-sectionally to each of the years of the study. The most striking finding here was that the dominant determinants of the state spending varied for each year of the study (Fiscal Years 1976-1981). The significance of these results was that they provided empirical support of current implementation theory, showing that the dominant determinants of implementation vary greatly over time. ^