The additional facet of immunoscore: immunoprofiling as a possible predictive tool for cancer treatment

Recent investigations of the tumor microenvironment have shown that many tumors are infiltrated by inflammatory and lymphocytic cells. Increasing evidence suggests that the number, type and location of these tumor-infiltrating lymphocytes in primary tumors has prognostic value, and this has led to the development of an 'immunoscore. As well as providing useful prognostic information, the immunoscore concept also has the potential to help predict response to treatment, thereby improving decision- making with regard to choice of therapy. This predictive aspect of the tumor microenvironment forms the basis for the concept of immunoprofiling, which can be described as 'using an individual's immune system signature (or profile) to predict that patient's response to therapy' The immunoprofile of an individual can be genetically determined or tumor-induced (and therefore dynamic). Ipilimumab is the first in a series of immunomodulating antibodies and has been shown to be associated with improved overall survival in patients with advanced melanoma. Other immunotherapies in development include anti-programmed death 1 protein (nivolumab), anti-PD-ligand 1, anti-CD137 (urelumab), and anti-OX40. Biomarkers that can be used as predictive factors for these treatments have not yet been clinically validated. However, there is already evidence that the tumor microenvironment can have a predictive role, with clinical activity of ipilimumab related to high baseline expression of the immune-related genes FoxP3 and indoleamine 2,3-dioxygenase and an increase in tumor-infiltrating lymphocytes. These biomarkers could represent the first potential proposal for an immunoprofiling panel in patients for whom anti-CTLA-4 therapy is being considered, although prospective data are required. In conclusion, the evaluation of systemic and local immunological biomarkers could offer useful prognostic information and facilitate clinical decision making. The challenge will be to identify the individual immunoprofile of each patient and the consequent choice of optimal therapy or combination of therapies to be used.

Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins.

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and beta-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins.

CELLULAR AND DEVELOPMENTAL FUNCTIONS OF THE XENOPUS ARVCF-CATENIN:KAZRIN COMPLEX

Xenopus ARVCF (xARVCF), a member of p120-catenin subfamily, binds cadherin cytoplasmic domains to enhance cadherin metabolic stability, or when dissociated, modulates Rho-family GTPases. We previously found that xARVCF binds directly to Xenopus KazrinA (xKazrinA), a widely expressed, conserved protein that bears little homology to established protein families. xKazrinA is also known to influence keratinocyte proliferation-differentiation and cytoskeletal activity. In my study, I first evaluated the expression pattern of endogenous Kazrin RNA and protein in Xenopus embryogenesis as well as in adult tissues. We then collaboratively predicted the helical structure of Kazrin’s coiled-coil domain, and I obtained evidence of Kazrin’s dimerization/oligomerization. In considering the intracellular localization of the xARVCF-catenin:xKazrin complex, I did not resolve xKazrinA in a larger ternary complex with cadherin, nor did I detect its co-precipitation with core desmosomal components. Instead, screening revealed that xKazrinA binds spectrin. This suggested a potential means by which xKazrinA localizes to cell-cell junctions, and indeed, biochemical assays confirmed a ternary xARVCF:xKazrinA:xβ2-spectrin complex. Functionally, I demonstrated that xKazrin stabilizes cadherins by negatively modulating the RhoA small-GTPase. I further revealed that xKazrinA binds to p190B RhoGAP (an inhibitor of RhoA), and enhances p190B’s association with xARVCF. Supporting their functional interaction in vivo, Xenopus embryos depleted of xKazrin exhibited ectodermal shedding, a phenotype that could be rescued with exogenous xARVCF. Cell shedding appeared to be caused by RhoA activation, which consequently altered actin organization and cadherin function. Indeed, I was capable of rescuing Kazrin depletion with ectopic expression of p190B RhoGAP. In addition, I obtained evidence that xARVCF and xKazrin participate in craniofacial development, with effects observed upon the neural crest. Finally, I found that xKazrinA associates further with delta-catenin and p0071-catenin, but not with p120-catenin, suggesting that Kazrin interacts selectively with additional members of the p120-catenin sub-family. Taken together, my study supports Kazrin’s essential role in development, and reveals KazrinA’s biochemical and functional association with ARVCF-catenin, spectrin and p190B RhoGAP.

DEVELOPMENTAL AND CELLULAR FUNCTIONS OF DELTA-CATENIN

Catenins have diverse and powerful roles in embryogenesis, homeostasis or disease progression, as best exemplified by the well-known beta-catenin. The less studied delta-catenin likewise contains a central Armadillo-domain. In common with other p120 sub-class members, it acts in a variety of intracellular compartments and modulates cadherin stability, small GTPase activities and gene transcription. In mammals, delta-catenin exhibits neural specific expression, with its knock-out in mice correspondingly producing cognitive defects and synaptic dysfunctions. My work instead employed the amphibian, Xenopus laevis, to explore delta-catenin’s physiological functions in a distinct vertebrate system. Initial isolation and characterization indicated delta-catenin’s expression in Xenopus. Unlike the pattern observed for mammals, delta-catenin was detected in most adult Xenopus tissues, although enriched in embryonic structures of neural fate as visualized using RNA in-situ hybridization. To determine delta-catenin’s requirement in amphibian development, I employed anti-sense morpholinos to knock-down gene products, finding that delta-catenin depletion results in developmental defects in gastrulation, neural crest migration and kidney tubulogenesis, phenotypes that were specific based upon rescue experiments. In biochemical and cellular assays, delta-catenin knock-down reduced cadherin levels and cell adhesion, and impaired activation of RhoA and Rac1, small GTPases that regulate actin dynamics and morphogenetic movements. Indeed, exogenous C-cadherin, or dominant-negative RhoA or dominant-active Rac1, significantly rescued delta-catenin depletion. Thus, my results indicate delta-catenin’s essential roles in Xenopus development, with contributing functional links to cadherins and Rho family small G proteins. In examining delta-catenin’s nuclear roles, I identified delta-catenin as an interacting partner and substrate of the caspase-3 protease, which plays critical roles in apoptotic as well as non-apoptotic processes. Delta-catenin’s interaction with and sensitivity to caspase-3 was confirmed using assays involving its cleavage in vitro, as well as within Xenopus apoptotic extracts or mammalian cell lines. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo-repeat 6 of delta-catenin, was identified through peptide sequencing. Cleavage thus generates an amino- (1-816) and carboxyl-terminal (817-1314) fragment each containing about half of the central Armadillo-domain. I found that cleavage of delta-catenin both abolishes its association with cadherins, and impairs its ability to modulate small GTPases. Interestingly, the carboxyl-terminal fragment (817-1314) possesses a conserved putative nuclear localization signal that I found is needed to facilitate delta-catenin’s nuclear targeting. To probe for novel nuclear roles of delta-catenin, I performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating its interaction with an uncharacterized KRAB family zinc finger protein I named ZIFCAT. My results indicate that ZIFCAT is nuclear, and suggest that it may associate with DNA as a transcriptional repressor. I further determined that other p120 sub-class catenins are similarly cleaved by caspase-3, and likewise bind ZIFCAT. These findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120 sub-family members, facilitating the coordinate modulation of cadherins, small GTPases and nuclear functions. Together, my work suggested delta-catenin’s essential roles in Xenopus development, and has revealed its novel contributions to cell junctions (via cadherins), cytoskeleton (via small G proteins), and nucleus (via ZIFCAT). Future questions include the larger role and gene targets of delta-catenin in nucleus, and identification of upstream signaling events controlling delta-catenin’s activities in development or disease progression.

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.

Cardiomyocyte PDGFR-beta signaling is an essential component of the mouse cardiac response to load-induced stress.

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. Recently, however, several anticancer drugs that inhibit PDGFR signaling have been associated with clinical heart failure. Understanding this effect of PDGFR inhibitors has been difficult because the role of PDGFR signaling in the heart remains largely unexplored. As described herein, we have found that PDGFR-beta expression and activation increase dramatically in the hearts of mice exposed to load-induced cardiac stress. In mice in which Pdgfrb was knocked out in the heart in development or in adulthood, exposure to load-induced stress resulted in cardiac dysfunction and heart failure. Mechanistically, we showed that cardiomyocyte PDGFR-beta signaling plays a vital role in stress-induced cardiac angiogenesis. Specifically, we demonstrated that cardiomyocyte PDGFR-beta was an essential upstream regulator of the stress-induced paracrine angiogenic capacity (the angiogenic potential) of cardiomyocytes. These results demonstrate that cardiomyocyte PDGFR-beta is a regulator of the compensatory cardiac response to pressure overload-induced stress. Furthermore, our findings may provide insights into the mechanism of cardiotoxicity due to anticancer PDGFR inhibitors.

Eluding antibiotic resistance: capitalizing on antimicrobial peptides interaction with the lipid bilayer

It is widely accepted that the emergence of drug-resistant pathogens is the result of the overuse and misuse of antibiotics. Infectious Disease Society of America, Center for Disease Control and World Health Organization continue to view, with concern, the lack of antibiotics in development, especially those against Gram-negative bacteria. Antimicrobial peptides (AMPs) have been proposed as an alternative to antibiotics due to their selective activity against microbes and minor ability to induce resistance. For example, the Food and Drug Administration approved Daptomycin (DAP) in 2003 for treatment of severe skin infections caused by susceptible Gram-positive organisms. Currently, there are 12 to 15 examples of modified natural and synthetic AMPs in clinical development. But most of these agents are against Gram-positive bacteria. Therefore, there is unmet medical need for antimicrobials used to treat infections caused by Gram-negative bacteria. In this study, we show that a pro-apoptotic peptide predominantly used in cancer therapy, (KLAKLAK)2, is an effective antimicrobial against Gram-negative laboratory strains and clinical isolates. Despite the therapeutic promise, AMPs development is hindered by their susceptibility to proteolysis. Here, we demonstrate that an all-D enantiomer of (KLAKLAK)2, resistant to proteolysis, retains its activity against Gram-negative pathogens. In addition, we have elucidated the specific site and mechanism of action of D(KLAKLAK)2 through a repertoire of whole-cell and membrane-model assays. Although it is considered that development of resistance does not represent an obstacle for AMPs clinical development, strains with decreased susceptibility to these compounds have been reported. Staphylococci resistance to DAP was observed soon after its approval for use and has been linked to alterations of the cell wall (CW) and cellular membrane (CM) properties. Immediately following staphylococcal resistance, Enterococci resistance to DAP was seen, yet the mechanism of resistance in enterococci remains unknown. Our findings demonstrate that, similar to S. aureus, development of DAP-resistance in a vancomycin-resistant E. faecalis isolate is associated with alterations of the CW and properties of the CM. However, the genes linked to these changes in enterococci appear to be different from those described in S. aureus.

Molecular architecture of basement membranes

Basement membranes are specialized extracellular matrices with support, sieving, and cell regulatory functions. The molecular architectures of these matrices are created through specific binding interactions between unique glycoprotein and proteoglycan protomers. Type IV collagen chains, using NH2-terminal, COOH-terminal, and lateral association, form a covalently stabilized polygonal framework. Laminin, a four-armed glycoprotein, self-assembles through terminal-domain interactions to form a second polymer network, Entactin/nidogen, a dumbbell-shaped sulfated glycoprotein, binds laminin near its center and interacts with type IV collagen, bridging the two. A large heparan sulfate proteoglycan, important for charge-dependent molecular sieving, is firmly anchored in the basement membrane and can bind itself through a core-protein interaction to form dimers and oligomers and bind laminin and type IV collagen through its glycosaminoglycan chains. Heterogeneity of structure and function occur in different tissues, in development, and in response to different physiological needs. The molecular architecture of these matrices may be regulated during or after primary assembly through variations in compositions, isoform substitutions, and the modifying influence of exogenous macromolecules such as heparin and heparan sulfate.

Characterization of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp. mycoides small colony type.

With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli. The expressed protein was of the same apparent molecular mass as that produced by the parent strain. The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE. Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II-membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells. An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M. capricolum subsp. capricolum but not on that of Mycoplasma genitalium. The P72 gene was detected in 11/11 M. mycoides subsp. mycoides SC strains. Antiserum against recombinant P72 reacted strongly with 12/12 strains of M. mycoides subsp. mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the 'mycoides cluster' or closely related mycoplasmas. Cows experimentally contact-infected with M. mycoides subsp. mycoides SC developed a humoral response against P72 within 35 d. P72 is a specific antigenic membrane lipoprotein of M. mycoides subsp. mycoides SC with potential for use in development of diagnostic reagents. It seems to belong to a family of lipoproteins of the "mycoides cluster'.

Treatment of echinococcosis: albendazole and mebendazole - what else?

The search for novel therapeutic options to cure alveolar echinococcosis (AE), due to the metacestode of Echinococcus multilocularis, is ongoing, and these developments could also have a profound impact on the treatment of cystic echinococcosis (CE), caused by the closely related Echinococcus granulosus s.l. Several options are being explored. A viable strategy for the identification of novel chemotherapeutically valuable compounds includes whole-organism drug screening, employing large-scale in vitro metacestode cultures and, upon identification of promising compounds, verification of drug efficacy in small laboratory animals. Clearly, the current focus is targeted towards broad-spectrum anti-parasitic or anti-cancer drugs and compound classes that are already marketed, or that are in development for other applications. The availability of comprehensive Echinococcus genome information and gene expression data, as well as significant progress on the molecular level, has now opened the door for a more targeted drug discovery approach, which allows exploitation of defined pathways and enzymes that are essential for the parasite. In addition, current in vitro and in vivo models that are used to assess drug efficacy should be optimized and complemented by methods that give more detailed information on the host-parasite interactions that occur during drug treatments. The key to success is to identify, target and exploit those parasite molecules that orchestrate activities essential to parasite survival.

Transcriptional regulation of tenascin genes

Extracellular matrix proteins of the tenascin family resemble each other in their domain structure, and also share functions in modulating cell adhesion and cellular responses to growth factors. Despite these common features, the 4 vertebrate tenascins exhibit vastly different expression patterns. Tenascin-R is specific to the central nervous system. Tenascin-C is an "oncofetal" protein controlled by many stimuli (growth factors, cytokines, mechanical stress), but with restricted occurrence in space and time. In contrast, tenascin-X is a constituitive component of connective tissues, and its level is barely affected by external factors. Finally, the expression of tenascin-W is similar to that of tenascin-C but even more limited. In accordance with their highly regulated expression, the promoters of the tenascin-C and -W genes contain TATA boxes, whereas those of the other 2 tenascins do not. This article summarizes what is currently known about the complex transcriptional regulation of the 4 tenascin genes in development and disease.

Hypersensitive granulomatous response to mycobacterial glycolipid trehalose 6,6'-dimycolate

Protection against Mycobacterium tuberculosis infection requires an effective cell mediated immune response leading to granuloma formation and organism containment. Trehalose 6,6'-dimycolate (TDM), a glycolipid present on the mycobacterial cell wall, has been implicated as a key component in establishment of the granulomatous response. TDM has potent immunoregulatory and inflammatory properties; the acute response to TDM produces pathology resembling early Mycobacterium tuberculosis infection. We have further developed this model to study TDM-specific cell mediated immune responses that may play a role in the later stages of infection and pathology. Lungs from mice immunized with TDM in the form of a water-oil-water (w/o/w) emulsion demonstrate heightened histological damage, inflammation, lymphocytic infiltration, and vascular endothelial cell damage upon subsequent challenge with TDM. This exacerbated response can be adoptively transferred to naïve mice via transfer of non-adherent lymphocytes from TDM immunized mice. To identify the cell phenotype(s) regulating this response, purified non-adherent cell populations (CD4+ and CD8+ T cells; CD19 + B cells) were isolated from TDM immunized mice, adoptively transferred into naive mice, and subsequently challenged with TDM. Lung histopathology and cytokine production identified CD4+ cells as the critical cell phenotype regulating the TDM-specific hypersensitive response. The role of CD1d in presentation of TDM was examined. CD1d, a molecule known to present lipids to T cells, was identified as critical in development of the hypersensitive response. CD4+ cells were isolated from TDM-immunized CD1d -/- mice and adoptively transferred into naive wild type mice, followed by TDM challenge. These mice were deficient in development of the hypersensitive granulomatous response, signifying the importance of CD1d in the generation of TDM-specific CD4+ cells. The experiments presented in this dissertation provide further evidence for involvement of TDM-specific cell mediated immune response in elicitation of pathological damage during Mycobacterium tuberculosis infection. ^

The p120-catenin/Kaiso signaling pathway

The canonical and non-canonical Wnt signaling pathways appear to interact with one another as a network in development, or when hyper-activated, in the progression of disease. A much studied key mediator of the canonical Wnt pathway, β-catenin, is characterized by a central armadillo-repeat domain that engages in multiple protein-protein interactions, such as those with cadherins functioning at cell-cell contact regions. In the nucleus, β-catenin forms a complex with the repressor TCF/LEF, promoting the activation of genes participating in processes such as proliferation, differentiation and stem cell survival. Somewhat similarly, the p120-catenin binds the distinct transcriptional repressor Kaiso, relieving Kaiso-mediated repression to promote gene activation. Here, employing Xenopus laevis, I report upon both downstream and upstream aspects of the p120-catenin/Kaiso pathway which was previously poorly understood. I first show that Kaiso, a BTB/POZ zinc-finger family member, directly represses canonical Wnt gene targets (Siamois, c-Fos, Cyclin-D1 and c-Myc) in conjunction with TCF. Depletion or dominant-negative inhibition of xKaiso results in Siamois de-repression, while xKaiso over-expression induces additional Siamois repression through recruitment of N-CoR co-repressor and chromatin modifications. Functional interdependencies are further corroborated by the capacity of Kaiso to suppress β-catenin-induced axis duplication. Thus, my work inter-relates the p120-catenin/Kaiso and β-catenin/TCF pathways at the level of specific gene promoters important in development and cancer progression. Regarding upstream aspects of the p120-catenin/Kaiso pathway, I collaboratively identified p120 in association with Frodo, a protein previously identified as a component of the canonical (β-catenin dependent) Wnt pathway. I determined that canonical Wnt signals result in Frodo-mediated stabilization of p120-catenin, resulting in the sequestration of Kaiso to the cytoplasm and thereby the activation (relief of repression) of gene targets. Developmental evidence supporting this view included findings that Frodo has the capacity to partially rescue Kaiso over-expression phenotypes in early Xenopus embryos. Taken together, my studies point to the convergence of p120-catenin/Kaiso and β-catenin/TCF signaling pathways at the level of gene transcription as well as at more upstream points during vertebrate development. ^

Policy analysis of the Americans with Disabilities Act of 1990 and federal funding concerning barrier-free playgrounds

The Americans with Disabilities Act (ADA) of 1990 was created to prohibit discrimination against disabled persons in our society. The goal of the ADA as a comprehensive civil rights law is to "ensure equal opportunity and complete participation, independent living and economic self-sufficiency" for disabled persons (U.S. Department of Justice, 2008). As part of Title II and III of the ADA, states and local governments are required to provide people with disabilities the same chance to engage in and benefit from all programs and services including recreational facilities and activities as every other citizen. Recreational facilities and related structures must comply with accessibility standards when creating new structures or renovating existing ones. Through a systematic literature review of articles accessed through online databases, articles relating to children with disabilities, their quality of life and their experience gained through play were reviewed, analyzed and synthesized. Additionally, the ADA's Final Rule regarding accessible playgrounds was evaluated through a descriptive analysis which yielded the following five components relating the importance of barrier-free playgrounds to children with disabilities: appropriate dimensions for children, integration of the play area, variety of activity and stimulation, availability of accessible play structures to communities, and financial feasibility. These components were used as evaluation criteria to investigate the degree to which the ADA's Final Rule document met these criteria. An evaluation of two federal funding sources, the Urban Parks and Recreation Renewal Program (UPARR) and the Land and Water Conservation Fund (LWCF), was also conducted which revealed three components relating the two programs' ability to support the realization of the ADA's Final Rule which included: current budget for the program, ability of local communities to attain funds, and level of ADA compliance required to receive funding. Majority of the evaluation of the Final Rule concluded it be adequate in development of barrier-free playgrounds although there are some portions of the guidelines that would benefit from further elucidation. Both funding programs were concluded to not adequately support the development of barrier-free playgrounds and therefore it was recommended that their funding be re-instated or increased as necessary. ^