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Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

A HEp-2 cell culture model was used to investigate the invasive properties of Campylobacter species. Two of twenty-five Campylobacter isolates did not invade HEp-2 cells, and one of these isolates did not adhere to the epithelial cells. Penetration of HEp-2 epithelial cells by C. jejuni was significantly (P < 0.05) inhibited with C. jejuni lysates and a MAb (1B4) in competitive inhibition studies. Immunogold electron microscopic studies revealed that the 1B4 MAb bound to the flagella and cell surface of low passage (invasive) C. jejuni M 96, whereas only the flagella of high passage (non-invasive) C. jejuni were labelled. Western blot analysis revealed that the 1B4 MAb identified an epitope on antigens ranging in size from 66 to 44 kDa in invasive and non-invasive organisms. Antigens were also recognized in lysates prepared only from invasive strains from 42 to 38 kDa. Sodium meta-periodate chemical treatment of C. jejuni lysates significantly (P < 0.05) affected its inhibitory capacity. Additionally, proteinase K and sodium meta-periodate treatment of lysates changed the mobility of antigens recognized by the 1B4 MAb. This suggests that the antigens required for epithelial cell penetration by C. jejuni may be glycoprotein in nature and that the functional binding site is dependent upon an intact carbohydrate moiety. Co-infection of HEp-2 epithelial cells with coxsackievirus B3, echovirus 7, polio virus (LSc type 1), porcine enterovirus and Campylobacter isolates was performed to determine if a synergistic effect could be obtained. The invasiveness of C. jejuni was significantly increased for HEp-2 cells pre-infected with echovirus 7, coxsackievirus B3, and UV-inactivated (non-infectious) coxsackievirus B3 particles. Polio and porcine enterovirus had no effect on C. jejuni adherence and invasiveness. C. hyointestinalis and C. mucosalis, two non-invasive isolates, did not invade virus-infected HEp-2 cells. The increase of invasiveness of C. jejuni appears to be the result of specific interactions between the virus and the HEp-2 cell membrane. The data suggest that the invasiveness of Campylobacter is dependent upon the inherent properties of the organism. Virus-induced cell alterations can potentiate the invasiveness of virulent Campylobacter but are not sufficient to allow internalization by non-invasive bacteria.

Enhanced transport of microbes in subsurface is a focus in bioaugmentation applications for remediation of groundwater. In this study, the effect of low-concentration monorhamnolipid biosurfactant on transport of Pseudomonas aeruginosa ATCC 9027 in natural porous media (silica sand and a sandy soil) with or without hexadecane as the nonaqueous phase liquids (NAPLs) was studied with miscible-displacement experiments using artificial groundwater as the background solution. Transport of two types of cells was investigated, glucose-grown and hexadecane-grown cells with lower and higher cell surface hydrophobicity (CSH), respectively. A clean-bed colloid deposition model was used to calculate deposition rate coefficients (k) for quantitative assessment on the effect of the rhamnolipid on the transport. In the absence of NAPLs, significant cell retention was observed in the sand (81% and 82% for glucose-grown and hexadecane-grown cells, respectively). Addition of low-concentration rhamnolipid enhanced cell transport, with 40 mg/L of rhamnolipid reducing retention to 50% and 60% for glucose-grown and hexadecane-grown cells, respectively. The k values for both glucose-grown and hexadecane-grown cells correlated linearly with rhamnolipid-dependent CSH quantitatively measured using a bacterial-adhesion-to-hydrocarbon method. Retention of cells by the soil was nearly complete (>99%). Forty milligrams per liter of rhamnolipid reduced the retention to 95%. The presence of NAPLs in the sand enhanced the retention of hexadecane-grown cells with higher CSH. Transport of cells in the presence of NAPLs was enhanced by rhamnolipid at all concentrations tested, and the relative enhancement was greater than in the absence of NAPLs. This study shows the importance of hydrophobic interaction on bacterial transport in natural porous media and the potential of using low-concentration rhamnolipid for facilitating cell transport in subsurface for bioaugmentation efforts.

The growth and morphology of fibroblasts cultured on a physically and chemically modified surface was investigated. The need to understand cellular relationships with surface topography and chemistry is essential in the fields of biomedical engineering and biotechnology. It is well documented that mammalian cell behavior senses and responds to the surrounding micro- and nano- scale environment, but the research defining the chemistry, surface architecture, and material properties for control of this behavior is still in its infancy. The cell response to a substrate is complex, involving membrane proteins, extracellular matrix (ECM), cytoskeletal rearrangement, and changes in gene expression. Conventional cell culture is carried out on two-dimensional (2-D) cell culture platforms, such as polystyrene (PS) or glass, and forces cell behavior to adapt and adhere to an unnatural, planar environment. The biological behavior of these cells is used as a starting point for drug screening, implant design, and metabolic processes, but this is a misrepresentation of cells in their native environment. This discrepancy may be hampering biological research or initiating experimental efforts that are invalid. This body of work seeks to address these issues and contains established protocols for inexpensive, pseudo three-dimensional (3-D) culture scaffolds. The research described offers a multi-disciplinary approach for fabrication of biomaterials to achieve user defined or in vivo cell behavior using human fibroblasts. To provide insight into the design of alternative cell culture templates we have analyzed cell-surface interactions and characterized the surface properties. The substrates fabricated utilized micro-roughened surface topography with 2 – 6 µm wide features and surface chemistry as a method for controlling cell behavior. Surface roughness was templated onto polydimethylsiloxane (PDMS) and PS. The fabricated polymer surfaces have been characterized by atomic force microscopy (AFM), contact angle goniometry, fluorescence microscopy, and infrared (IR) spectroscopy. Initial studies of the textured surface yielded a super-hydrophobic surface with a 154° contact angle and high surface adhesion that was investigated using surface free energy calculations. This was followed by modification of the micro-roughness with self-assembled monolayers (SAMs), proteins, or thin films of polymer for use as a culture platform for cells. Cell behavior on the modified polymers was compared and analyzed against unmodified surfaces and tissue culture PS dishes. Cell morphology on rough PDMS surface was altered by the surface topography decreasing the average cell area to 1760 µm2 compared to an average cell area of 3410 µm2 on smooth PDMS. Gene expression changes were also noted with a 2.3 fold increase in the matrix metalloproteinase, MMP14, in cells on the rough surface compared to cells cultured on Petri dishes. Surface roughness was also combined with other surface modification methods for cell culture, including cell alignment and cell sheet engineering. 50 µm wide lines of fibronectin (FN) patterned on the rough PDMS induced cell directionality while still maintaining a pseudo 3-D culture system creating the first cell culture surface of its kind. The micro-roughness was also templated onto PS and chemically modified with a thermo-responsive polymer. This novel surface produced confluent cell sheets that detached from the surface when cooled below 32°C. Cell sheets cultured on the modified PS surfaces had an increase in FN fibril formation stimulated by the surface roughness when compared to cell sheets detached from a smooth, control surface. The minor alterations to surface topology were proven to be effective in modifying cell biochemical response compared to cells cultured on flat substrates. Differences in surface topography and chemistry stimulated changes in cell adhesion, cytoskeletal arrangement, ECM composition, and gene expression. These cell properties were used as markers for comparison to native cell systems and other reports of 3 D culture scaffolds. The mechanism of altering cell response is discussed in each chapter with respect to the specific type of surface used and compared to cell response and behavior on planar culture systems. New fabrication procedures are described that include the incorporation of other surface modification techniques such as SAMs, surface patterning, and thermo-responsive polymer grafting with surface roughness for original cell culture platforms to mimic an in vivo environment. The research presented here demonstrates that micro- and nano- changes to surface topography have large impacts on the cell-surface relationship which have important implications for research and medical applications involving adherent cells.

Tumor angiogenesis, the ability of tumor cells to stimulate blood vessel growth, is one the most critical steps of tumor progression. To support the growth of the expanding tumor, the “angiogenic switch” is turned on, which is often triggered by hypoxia (i.e., low oxygen)-mediated events such as expression of vascular endothelial growth factor (VEGF), causing normally quiescent endothelial cells to proliferate and sprout. An emerging picture of angiogenesis suggests that while governed by complex mechanisms, cell adhesion molecules (CAMs) plays a pivotal role in the regulation of angiogenesis. Our laboratory recently identified multiple previously unknown proteins including, transmembrane and immunoglobulin domain containing 1 (TMIGD1) and immunoglobulin-containing and proline-rich receptor 1 (IGPR1). Immunoglobulin-containing and cysteine-rich receptor 1 (IGCR1) represents the third remember of IGPR-1 family proteins. To investigate the expression and function of IGCR1, we have developed a rabbit polyclonal anti-IGCR1 antibody and demonstrated that IGCR1 is expressed in the endothelial cells of human blood vessels. To examine possible function of IGCR1, we have generated porcine aortic endothelial (PAE) cells over-expressing IGCR1. We demonstrate that IGCR1 expression in PAE cells inhibited cell proliferation and capillary tube formation as measured by colorimetric MTT and matrigel tube formation assays, respectively. In contrast, over-expression of IGCR1 in PAE cells inhibited cell migration as measured by wounding assay. Taken together, this study identifies IGCR1 as a novel regulator of angiogenesis. Given, angiogenesis is a highly coordinated cellular processes controlled spatially and temporally by a myriad of cell surface receptors and ligands, IGCR1 by modulating the rate of endothelial cell proliferation and migration, plays a significant role in the formation of blood vessels. / 2018-07-11T00:00:00Z

<p>The chaperone protein GRP78 is primarily expressed in the endoplasmic reticulum, but it is also aberrantly expressed on the surface of cells under pathological conditions. One the cell membrane, GRP78 acts as a signaling molecule with unique properties. The amino-terminal domain acts as a growth factor receptor-like protein, while the carboxyl-terminal domain functions as a death-signaling receptor-like protein to extrinsically induce apoptosis. Autoantibodies that react with cell-surface GRP78 on many tumor cell lines occur in the sera of patients with prostate cancer, melanoma, and ovarian cancer. These autoantibodies are a negative prognostic factor in prostate cancer and melanoma, and when purified, stimulate tumor cell proliferation in vitro. It is unclear, however, whether these IgGs are merely a biomarker, or if they actually promote tumor growth in vivo. We immunized C57Bl/6 mice with recombinant GRP78 and then implanted the B16F1 murine melanoma cell line as flank tumors. We employed the antisera from these mice for in vitro cell signaling and proliferation assays. The immunodominant epitope in human cancer patients was well represented in the antibody repertoire of these immunized mice. We observed significantly accelerated tumor growth, as well as shortened survival in GRP78-immunized mice as compared to controls. Furthermore, antisera from these mice, as well as purified anti-GRP78 IgG from similarly immunized mice, stimulate Akt phosphorylation and proliferation in B16F1 and human DM6 melanoma cells in culture. These studies demonstrate a causal link between a humoral response to GRP78 and the progression of cancer in a murine melanoma model. They support the hypothesis that such autoantibodies are involved in the progression of human cancers and are not simply a biomarker. Because GRP78 is present on the surface of many types of cancer cells, this hypothesis has broad clinical and therapeutic implications.</p><p>We generated and characterized a panel of monoclonal murine antibodies (mAbs) against GRP78 with the goal of identifying therapeutic candidate IgGs. We developed three stable hybridomas that produce interesting antibodies. The N88 IgG reacts with the NH2-terminal domain and is an agonist. The C38 IgG reacts with the COOH-terminal domain and is an antagonist of NH2-terminal signaling. The C107 IgG binds the COOH-terminal domain and induces apoptosis. </p><p>We examined the effect of these three mAbs on the growth of B16 flank tumors. N88 accelerated and C107 slowed tumor growth, while C38 had no net effect. We are currently developing these antibodies and derivatives thereof as therapeutics for melanoma as well as for cancers of the brain, breast, ovary, and prostate. In fact, any tumor cell that over-expresses GRP78 on its surface is a potential therapeutic target for our future studies.</p> / Dissertation

ABSTRACT Carbohydrates in various forms play vital roles in numerous critical biological processes including cell-cell adhesion and communication, embryo development, immune response, etc. Fluorescent sensors for such carbohydrates have a wide range of potential applications including glucose concentration determination, cell labeling and targeting based on carbohydrate biomarkers, as in vitro diagnostic tools, and biomarker-directed cellular imaging. Our group has been interested in the design and synthesis of multi-boronic acid compounds with well-defined three-dimensional scaffolding for the specific recognition of selected carbohydrate biomarkers. Aberrant expression of carbohydrate antigens such as sialyl Lewis X (sLex), sialyl Lewis A (sLea), Lewis X (Lex), and Lewis Y (Ley) have been associated with tumor formation and metastasis in various cancer types.1-4 As such, for our initial design, we have selected sialyl Lewis X (sLex) as our potential target due to implication in the development of liver and colon cancer.5, 6 Herein, we describe the design, synthesis and evaluation of four such compounds, each having about ten linear steps in its synthesis. In addition to the design of fluorescent probes for cell surface carbohydrates, we also have designed lipophilic boronic acid derivatives as potential fusogenic agents. Due to boronic acid¡¯s ability to bind to 1,2 and 1,3 cis diols, we hypothesize that the aliphatic chain should be able to insert into lipid cellular membrane and the boronic acid units should allow for the ¡°attachment to neighboring cells¡± through complexation with cell surface glycans. Such interactions should allow the boronic acid compounds to bring two or more cells together for fusion. Herein, we have described the methodologies of the design of such compounds. INDEX WORDS: Boronic acid, sialyl Lewis X probe, boronolectin, fluorescence, sensor, cell-cell fusion, fusogen, immunotherapy.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 162-172).