Synthesis of Various Classes of Cyanine Fluorophores and Their Application In In Vivo Tissue Imaging

A novel series of near-infrared fluorescent contrast agents was developed and characterized. Their physicochemical and optical properties were measured. By altering functional groups of cyanine fluorophores, the selective targeting of endocrine glands, exocrine glands, cartilage and bone using NIR fluorescence to visualize the targeted tissue has been reported. These agents have high specificity for tissue targeting inherent to the chemical structure of the fluorophore. After a single low-dose intravenous injection these agents have high specificity for tissue targeting inherent to the chemical structure of the fluorophore. The results lay the foundation for future improvements in optical imaging in endocrine surgery, tissue engineering, joint surgery, and cartilage-specific drug development

Unsymmetrical Trimethine Cyanine Dyes: Synthesis, Optical Properties, and Evaluations as Inhibitors of Protein Arginine Methyl Transferases

Carbocyanine dyes are a class of organic compounds that possess two nitrogen containing heterocycles that act as electron donors and acceptors connected by a conjugated methine bridge. This thesis will present the synthetic methodology of symmetrical and unsymmetrical trimethine cyanine dyes in three chapters. The first chapter is a review on the synthesis and application of unsymmetric cyanine dyes. The second will describe the synthesis of unsymmetrical trimethine cyanine dyes and how their optical properties differ from symmetrical dyes. The third chapter will not only discuss the synthetic procedure for synthesis of symmetrical trimethine cyanine dyes, but also will show how varying the N-alkyl substituents and hydrophobicity of the heterocycles affects the dyes interaction with and ability to be used as inhibitors for protein arginine methyltransferases (PRMTs). Several synthesized compounds have displayed lower IC50 values for the inhibition of PRMT1 and PRMT5 comparable to that of current inhibitors

Benz[c,d]indolium-containing Monomethine Cyanine Dyes: Synthesis and Photophysical Properties

Asymmetric monomethine cyanines have been extensively used as probes for nucleic acids among other biological systems. Herein we report the synthesis of seven monomethine cyanine dyes that have been successfully prepared with various heterocyclic moieties such as quinoline, benzoxazole, benzothiazole, dimethyl indole, and benz[e]indole adjoining benz[c,d]indol-1-ium, which was found to directly influence their optical and energy profiles. In this study the optical properties vs. structural changes were investigated using nuclear magnetic resonance and computational approaches. The twisted conformation unique to monomethine cyanines was exploited in DNA binding studies where the newly designed sensor displayed an increase in fluorescence when bound in the DNA grooves compared to the unbound form

Multiplicative disadvantage of being an unmarried and inadequately insured woman living in poverty with colon cancer: historical cohort exploration in California

Background: Many Americans diagnosed with colon cancer do not receive indicated chemotherapy. Certain unmarried women may be particularly disadvantaged. A 3-way interaction of the multiplicative disadvantages of being an unmarried and inadequately insured woman living in poverty was explored. Methods: California registry data were analyzed for 2,319 women diagnosed with stage II to IV colon cancer between 1996 and 2000 and followed until 2014. Socioeconomic data from the 2000 census classified neighborhoods as high poverty (≥30% of households poor), middle (5–29%) or low poverty (\u3c5% poor). Primary health insurance was private, Medicare, Medicaid or none. Comparisons of chemotherapy rates used standardized rate ratios (RR). We respectively used logistic and Cox regression models to assess chemotherapy and survival. Results: A statistically significant 3-way marital status by health insurance by poverty interaction effect on chemotherapy receipt was observed. Chemotherapy rates did not differ between unmarried (39.0%) and married (39.7%) women who lived in lower poverty neighborhoods and were privately insured. But unmarried women (27.3%) were 26% less likely to receive chemotherapy than were married women (37.1%, RR = 0.74, 95% CI 0.58, 0.95) who lived in high poverty neighborhoods and were publicly insured or uninsured. When this interaction and the main effects of health insurance, poverty and chemotherapy were accounted for, survival did not differ by marital status. Conclusions: The multiplicative barrier to colon cancer care that results from being inadequately insured and living in poverty is worse for unmarried than married women. Poverty is more prevalent among unmarried women and they have fewer assets so they are probably less able to absorb the indirect and direct, but uncovered, costs of colon cancer care. There seem to be structural inequities related to the institutions of marriage, work and health care that particularly disadvantage unmarried women that policy makers ought to be cognizant of as future reforms of the American health care system are considered

Paradoxical Immune Responses in Non-HIV Cryptococcal Meningitis

The fungus Cryptococcus is a major cause of meningoencephalitis in HIV-infected as well as HIV-uninfected individuals with mortalities in developed countries of 20% and 30%, respectively. In HIV-related disease, defects in T-cell immunity are paramount, whereas there is little understanding of mechanisms of susceptibility in non-HIV related disease, especially that occurring in previously healthy adults. The present description is the first detailed immunological study of non-HIV-infected patients including those with severe central nervous system (s-CNS) disease to 1) identify mechanisms of susceptibility as well as 2) understand mechanisms underlying severe disease. Despite the expectation that, as in HIV, T-cell immunity would be deficient in such patients, cerebrospinal fluid (CSF) immunophenotyping, T-cell activation studies, soluble cytokine mapping and tissue cellular phenotyping demonstrated that patients with s-CNS disease had effective microbiological control, but displayed strong intrathecal expansion and activation of cells of both the innate and adaptive immunity including HLA-DR+ CD4+ and CD8+ cells and NK cells. These expanded CSF T cells were enriched for cryptococcal-antigen specific CD4+ cells and expressed high levels of IFN-gamma as well as a lack of elevated CSF levels of typical T-cell specific Th2 cytokines -- IL-4 and IL-13. This inflammatory response was accompanied by elevated levels of CSF NFL, a marker of axonal damage, consistent with ongoing neurological damage. However, while tissue macrophage recruitment to the site of infection was intact, polarization studies of brain biopsy and autopsy specimens demonstrated an M2 macrophage polarization and poor phagocytosis of fungal cells. These studies thus expand the paradigm for cryptococcal disease susceptibility to include a prominent role for macrophage activation defects and suggest a spectrum of disease whereby severe neurological disease is characterized by immune-mediated host cell damage

A novel vaccine platform using glucan particles for induction of protective responses against Francisella tularensis and other pathogens

Vaccines are considered the bedrock of preventive medicine. However, for many pathogens, it has been challenging to develop vaccines that stimulate protective, long-lasting immunity. We have developed a novel approach using beta-1,3-D-glucans (BGs), natural polysaccharides abundantly present in fungal cell walls, as a biomaterial platform for vaccine delivery. BGs simultaneously provide for receptor-targeted antigen delivery to specialized antigen-presenting cells together with adjuvant properties to stimulate antigen-specific and trained non-specific immune responses. This review focuses on various approaches of using BG particles (GPs) to develop bacterial and fungal vaccine candidates. A special case history for the development of an effective GP tularaemia vaccine candidate is highlighted

A population of Langerin-positive dendritic cells in murine Peyer\u27s patches involved in sampling beta-glucan microparticles

Glucan particles (GPs) are 2-4 mum hollow, porous shells composed of 1,3-beta-D-glucan that have been effectively used for oral targeted-delivery of a wide range of payloads, including small molecules, siRNA, DNA, and protein antigens. While it has been demonstrated that the transepithelial transport of GPs is mediated by Peyer\u27s patch M cells, the fate of the GPs once within gut-associated lymphoid tissue (GALT) is not known. Here we report that fluorescently labeled GPs administered to mice by gavage accumulate in CD11c+ DCs situated in Peyer\u27s patch sub-epithelial dome (SED) regions. GPs appeared in DCs within minutes after gavage and remained within the SED for days afterwards. The co-administration or sequential administration of GPs with differentially labeled GPs or poly(lactic-co-glycolic acid) nanoparticles demonstrated that the SED DC subpopulation in question was capable of internalizing particles of different sizes and material compositions. Phenotypic analysis identified the GP-containing DCs as being CD8alpha- and CD11blo/-, suggesting they are the so-called myeloid and/or double negative (DN) subset(s) of PP DCs. A survey of C-type lectin receptors (CLRs) known to be expressed by leukocytes within the intestinal mucosa revealed that GP-containing SED DCs were positive for Langerin (CD207), a CLR with specificity for beta-D-glucan and that has been shown to mediate the internalization of a wide range of microbial pathogens, including bacteria, viruses and fungi. The presence of Langerin+ DCs in the SED as determined by immunofluorescence was confirmed using Langerin E-GFP transgenic mice. In summary, our results demonstrate that following M cell-mediated transepithelial transport, GPs (and other micro/nanoparticles) are sampled by a population of SED DCs distinguished from other Peyer\u27s patch DC subsets by their expression of Langerin. Future studies will be aimed at defining the role of Langerin in antigen sampling and antigen presentation within the context of the GALT

Robust Stimulation of Humoral and Cellular Immune Responses following Vaccination with Antigen-Loaded beta-Glucan Particles

beta-Glucan particles (GPs) are purified Saccharomyces cerevisiae cell walls treated so that they are primarily beta1,3-d-glucans and free of mannans and proteins. GPs are phagocytosed by dendritic cells (DCs) via the Dectin-1 receptor, and this interaction stimulates proinflammatory cytokine secretion by DCs. As the hollow, porous GP structure allows for high antigen loading, we hypothesized that antigen-loaded GPs could be exploited as a receptor-targeted vaccine delivery system. Ovalbumin (OVA) was electrostatically complexed inside the hollow GP shells (GP-OVA). Incubation of C57BL/6J mouse bone marrow-derived DCs with GP-OVA resulted in phagocytosis, upregulation of maturation markers, and rapid proteolysis of OVA. Compared with free OVA, GP-OVA was \u3e100-fold more potent at stimulating the proliferation of OVA-reactive transgenic CD8(+) OT-I and CD4(+) OT-II T cells, as measured by in vitro [(3)H]thymidine incorporation using DCs as antigen-presenting cells. Next, immune responses in C57BL/6J mice following subcutaneous immunizations with GP-OVA were compared with those in C57BL/6J mice following subcutaneous immunizations with OVA absorbed onto the adjuvant alum (Alum/OVA). Vaccination with GP-OVA stimulated substantially higher antigen-specific CD4(+) T-cell lymphoproliferative and enzyme-linked immunospot (ELISPOT) responses than that with Alum/OVA. Moreover, the T-cell responses induced by GP-OVA were Th1 biased (determined by gamma interferon [IFN-gamma] ELISPOT assay) and Th17 biased (determined by interleukin-17a [IL-17a] ELISPOT assay). Finally, both the GP-OVA and Alum/OVA formulations induced strong secretions of IgG1 subclass anti-OVA antibodies, although only GP-OVA induced secretion of Th1-associated IgG2c antibodies. Thus, the GP-based vaccine platform combines adjuvanticity and antigen delivery to induce strong humoral and Th1- and Th17-biased CD4(+) T-cell responses