Staphylococcus aureus employs an array of virulence factors to aid in its pathogenesis. A subset of cytotoxins termed bi-component leucocidins have been characterized as important determinants in the host-pathogen interaction in S. aureus infections. While they have been studied over a century, bi-component leucocidins’ complex mechanisms in pathogenesis have not been fully elucidated. Secreted as monomers, with the exception of LukAB/GH, many combinations of the S- (HlgA, HlgC, LukE, LukS, LukA/H) and F- (HlgB, LukD, LukF, LukB/G) components have demonstrated cell lysis via pore formation and a magnified proinflammatory response at sublytic concentrations. While studies have described various host cellular receptors and therapeutic strategies to evade leucocidin binding, a common receptor for all the leucocidins has yet to be classified. Challenges in data extrapolation have occurred due to non-native protein expression methods and species specificity of the leucocidin components. In turn, developing successful therapeutic strategies has proven problematic with a need for multimodal therapy. Thus, our studies aimed to clarify the bi-component leucocidins’ cytotoxic effects on multiple subsets of leukocytes using a native protein expression system and to identify a novel human leukocyte receptor common to all leucocidins. Overall, combinations with HlgA and LukE demonstrated the highest degrees of cytotoxicity against PMNs and PBMCs. Coronin 1A was discovered as a common receptor for all cognate pairs of bi-component leucocidins, except LukAB/GH. In conclusion, our results have expanded the knowledge of the cellular targets for leucocidin cytotoxicity and have described a common leucocidin receptor as a potential therapeutic target against the bi-component leucocidins.

No description available.

No description available.

No description available.

The immunotoxic effects of multiple pesticide exposure were evaluated. C57BL/6 mouse thymocytes were exposed to lindane, malathion, and permethrin, either separately or in mixtures of two pesticides, in concentrations ranging from 37.5 uM to 1mM. These exposures caused both apoptotic and necrotic cell death in thymocytes as evaluated by 7-aminoactinomycin-D, Annexin-V/PI, and lactate dehydrogenase release assays. When cells were exposed to lindane+malathion, or lindane+permethrin, a significantly greater-than-additive cytotoxicity was observed. The pesticide exposure caused DNA ladder formation with increased laddering in mixtures. Further, the effect of these pesticides on thymocyte oxidative stress was investigated. Thymocytes treated with any of these pesticides generated superoxide and H2O2. The lindane + malathion caused more-than-additive increase in superoxide production compared to single treatments of these pesticides. However, the effect of the lindane + permethrin was not significantly different from individual components of this mixture. The effects of pesticides on antioxidant enzymes were also investigated and only mixtures were found to have significant effects. Alteration in transcription factor NFkB level was measured as an indicator of oxidative stress in thymocytes following 12 h pesticide exposure, in vitro. Only lindane + malathion was found to increase the protein level. Furthermore, the effects of pesticides and their mixtures on immune functions of mice were studied in vivo. Animals (8-12 week old, male mice) were randomly divided into groups of six and injected intraperitoneally with three different doses (one-half, one-third, one-fourth, or one-eight of LD50) of individual pesticides. Exposure to individual pesticides did not alter the thymus/body or spleen/body weight ratios, thymic or splenic cell counts, or CD4/CD8 or CD45/CD90 ratios. However, anti-sRBC plaque forming cell (PFC) counts were significantly lowered with all treatments. Two other groups of animals were injected with lindane + malathion or lindane + permethrin at one-third of the LD50 of each pesticide. Exposure to pesticide mixtures did not alter the CD4/CD8 or CD45/CD90 ratios. However, the thymus/ and spleen/body weight ratios, thymic and splenic cell counts, and PFC counts were significantly lowered. These data indicate that lindane, malathion, and permethrin are immunotoxic and their mixtures can cause higher toxicity compared to individual exposures. In addition, these data support the hypothesis that oxidative stress were induced in thymocytes by exposure to these pesticides in vitro. / Ph. D.

Natural killer (NK) cell deficiency (NKD) occurs when an individual’s major clinical immunodeficiency derives from abnormal NK cells and is associated with several genetic etiologies. Three categories of β actin-related diseases with over 60 ACTB variants have previously been identified, none with a distinct NK cell phenotype. An individual with mild developmental delay, macrothrombocytopenia, susceptibility to infections, molluscum, and EBV-associated lymphoma had functional NK cell deficiency for over a decade. A de novo ACTB variant encoding G342D β actin was identified and was consistent with the individual’s developmental and platelet phenotype. This novel variant also was found to have a direct impact in NK cells, as its expression in YTS (YTS-NKD) cells caused increased cell spreading in lytic immune synapses created on activating surfaces. YTS-NKD cells were able to degranulate and perform cytotoxicity, but demonstrated defective serial killing owing to prolonged conjugation to the killed target cell and thus were effectively unable to terminate lytic synapses. G342D β actin results in a novel mechanism of functional NKD via increased synaptic spreading and defective lytic synapse termination with resulting impaired serial killing leading to overall reductions in NK cell cytotoxicity.

Cancer, the second leading cause of death worldwide, is characterized by uncontrolled and abnormal cell growth. Even though researchers have made significant progress in its treatment over the past several decades, innovative therapeutic approaches that both improve patient survival and lessen the many debilitating side effects of conventional cancer treatments are vital. Accordingly, we first investigated the mechanism of interaction of a bimetallic complex, Ru(II)-Rh(III), with DNA. Non-covalent binding of Ru(II)-Rh(III) is strong and involves electrostatic and, potentially, groove binding interactions. Ru(II)-Rh(III) photobinds and photocleaves DNA through an O2-independent, metal-center mediated mechanism that could be beneficial in hypoxic tumors. Furthermore, the extent of covalent binding and cleavage of DNA, which inhibit PCR amplification, is dependent upon the strength of the non-covalent interactions. These results suggest that the toxicity of Ru(II)-Rh(III) could be selectively generated in tissues irradiated with light (e.g., a tumor). Secondly, we identified protein combinations selectively present in melanoma, which could be utilized in heteromultivalency. Heteromultivalent scaffolds display higher affinity towards cells that express a protein combination in comparison to those with only one of the proteins, which facilitates cell discrimination. Using an empirically-optimized threshold-based screening method and expression profiles of melanoma patients and normal tissues, we identified surface proteins and protein combinations that are selectively found in melanoma patients and not in normal tissues. After a preliminary validation process using the scientific literature, we used immunofluorescence to confirm differential expression of some of these combinations in established melanoma cell lines in comparison to immortalized keratinocytes controls. Finally, we investigated the resazurin assay, a method used for the evaluation of proliferation and cytotoxicity in more than 2,000 publications. We found that only ~14% of these utilized validated assay conditions, while ~40% failed to report essential analytical parameters needed for their replication. We evaluated assay parameters needed for accurate estimation of cell number in eight cell lines, and found that these are highly variable and independent of tissue type, growth kinetics, and energetic parameters. Furthermore, we obtained some insights into the biochemical reduction of resazurin and proposed minimum reporting standards, along with a sample protocol for assay validation. / PHD / Cancer, a group of diseases characterized by uncontrolled and abnormal cell growth, is the second-leading cause of death worldwide. Even though researchers have made significant progress in its treatment over the past several decades, innovative therapeutic approaches that both improve survival outcome and lessen the many debilitating side-effects of conventional cancer treatments are vital. First, we investigated the mechanism of interaction of a particular molecule, Ru(II)- Rh(III), with DNA. We found that Ru(II)-Rh(III) is strongly attracted to DNA due to its charge and an interaction with the indentations along its helix. Upon light activation only, Ru(II)-Rh(III) binds to and cleaves DNA without the need for molecular oxygen, which is scarce in tumors and can limit the activity of other drugs, and to an extent that is affected by the concentration of ions in the solution. Thus, the cytotoxic effect of Ru(II)-Rh(III) might be selectively activated in those tissues that are irradiated with light (e.g., a tumor). Secondly, we identified protein combinations selectively present in melanoma, which could be utilized in heteromultivalency. Heteromultivalent scaffolds bind strongly to cells that express a combination of proteins rather than one protein at a time, making them excellent candidates for delivering a payload in a selective manner. Using expression profiles of melanoma and normal tissues, we identified surface proteins and protein combinations that are selectively found in melanoma patients and not in normal tissues. After a preliminary validation process using the scientific literature, we used confirmed differences in the expression intensities of some of these combinations in melanoma cell lines in comparison to normal skin controls. Finally, we investigated the resazurin assay, a method used for the evaluation of cell growth and drug candidates in more than 2,000 publications. We found that only ~14% of these utilized validated assay conditions, while ~40% failed to report essential analytical parameters needed for their replication. We evaluated assay conditions for eight cell lines, and found that these are highly variable and independent of tissue type and some metabolic parameters. Furthermore, we obtained insights into the mechanism through which cells react with resazurin and proposed minimum reporting standards for publications, along with a protocol for assay validation.

Stent is one of the coronary angioplasty techniques that expands the narrowed coronary arteries due to the accumulation of fat, cholesterol and other substances in the lumen of the arteries. The major complication of stent is restenosis. Current development of drug-eluting stents shows successfully reduce the occurrence of restenosis. Other than using drugs, electrostatic self assembled (ESAd) thin films may be the potential candidates to prevent restenosis. ESA is a process to fabricate thin films bases on the electrostatic attraction between two oppositely charges. We used this technique to fabricate four PVP films and four PEI films. All films were examined by XPS and AFM. XPS data showed our coatings were successfully fabricated on substrates. AFM images revealed PVP coating was uniform, but PEI coatings had different morphologies due to diffusion and pH during the process. Three preliminary hemocompatibility testes were performed to evaluate the hemocompatibility of the coatings. Platelet adhesion study showed the thin films inhibited platelet adhesion. All thin films were able to inhibit coagulation and were less cytotoxic. The studies suggested the ESA films were potentially hemocompatible. / Master of Science

Abelson murine leukemia virus (A-MuLV)-induced transformation was investigated to determine whether cells not sensitive to TNF-α could be made sensitive to the cytolytic action of TNF-α when infected with this retrovirus. Mouse embryonic fibroblast cell line CL.7 was found to be relatively insensitive to TNF-α. Upon transformation with A-MuLV, these cells gave rise to a clone (3R.1) which was found to be insensitive to TNF-α and another clone (6R.1) which had an increased sensitivity to TNF-α. The differential cytotoxicity was observed when cells were treated with TNF-α, for 18 hr, at 0 to 100 units/ml, at 37°C. The mechanism of this differential cytotoxicity was further investigated. Thus, TNF-R levels on the cell surface were found to be not correlated with the differential TNF-α response. The A-MuLV transformation suppressed the epidermal growth factor-receptor (EGF-R) in 3R.1 clone and induced its levels significantly in the 6R.1 clone (p<0.05). Cell surface EGF-receptor (EGF-R) levels in CL.7 and 3R.1 clones were lower than the 6R.1 clone (p<0.05). Although the EGF-R levels in all the clones were induced with TNF-α, the expression of EGF-R correlated with the susceptibility to TNF-α. The role of antioxidants, such as α-tocopherol and β-carotene, (known anti-cancer agents) in modulating TNF-α-induced EGF-R expression was investigated. In both the untransformed and the transformed clones, f-carotene suppressed the constitutive and the TNF-α induced EGF-R levels whereas α-tocopherol was found to have an enhancing effects. Studies with metabolic inhibitors on TNF-R and EGF-R expression indicate that inhibitors of the arachidonic acid cascade and modulators of protein kinase-C (PK-C), could influence the binding and internalization of TNF-α and thereby controlling the physiologic future of the cells. The A-MuLV specific V-abl protein, p120, tyrosine phosphorylation was determined by a radio-labelled anti-phosphotyrosine antibody in an antigen capture assay. TNF-α had little effect on p120 phosphotyrosine levels of TNF-α insensitive CL.7 and 3R.1 clones. The, TNF-α sensitive, 6R.1 clone, however, was found to induce its p120 specific phosphotyrosine upon exposure to TNF-α for 8 hr. Thus, TNF-α modulated the tyrosine phosphorylation of p120 only in the TNF-α-sensitive cell line. The mitochondrial toxicity of TNF-α was determined by monitoring the rate of quenching of a cationic spin probe CAT 16. Mitochondrial preparation from CL.7 and 3R.1 clones had higher ability to quench CAT 16 signal with TNF-α incubation time than mitochondria from the 6R.1 cells. This indicates that the differential TNF-α cytotoxicity manifested in A-MuLV transformed clones may, in part, be due to the differential mitochondrial toxicity of this cytokine. The hypothesis that TNF-α cytotoxicity was mediated via an oxidative process was tested on the TNF-α sensitive L929 cells. Using a flow cytometric detection system it was determined that TNF-α produced intracellular hydrogen peroxide in these cells which was sensitive to concentration and incubation time of TNF-α. Superoxide radicals were also generated during TNF-α action on L929 cells, as determined by the use of the spin trap PBN in conjunction with EPR spectroscopic techniques. The PBN-OOH spin adduct spectrum peaked at 9 hr of TNF-α incubation and was inhibitable upto 30 % with 10 µM of desferral-Mn complex (a known SOD mimic). These data indicate that superoxide and hydrogen peroxide are common events in TNF-α dependent cell killing process. The differential TNF-α cytotoxicity was found to depend on differences in the antioxidant status of the target clones. Thus, it was found that Cu/Zn-SOD, Mn-SOD, GSH-Peroxidase and GSH-Reductase enzymes were all induced significantly in the CL.7 clone (p<0.05) upon incubation with 100 units/ml of TNF-α for 18 hrs. TNF-α had little effect on the antioxidant enzymes of both 3R.1 and 6R.1 cells. However, the constitutive levels of most antioxidant enzymes were found to be higher in 3R.1 cells than in the 6R.1 cells. Therefore, the susceptibility of 6R.1 to TNF-α may, in part, be due to a low level of antioxidant enzymes present in this clone. In conclusion we found that the differential cytotoxicity of TNF-a may, in part, due to: (1) differential EGF-R expression, (2) differential mitochondrial cytotoxicity, and (3) differential ability to modulate the tyrosine phosphorylation in untransformed and A-MuLV transformed cells and (4) differential antioxidant status of these cells to handle oxidative stress imposed by TNF-α. / Ph. D.

Yes / Extracts of Erythrina addisoniae are frequently used in the traditional medicine of Western Africa, but insufficient information about active compounds is available. From the stem bark of E. addisoniae, three (1, 2, 4) and three known (3, 5, 6) flavanones were isolated: addisoniaflavanones I and II, containing either a 2″,3″-epoxyprenyl moiety (1) or a 2″,3″-dihydroxyprenyl moiety (2) were shown to be highly toxic (MTT assay: EC50 values of 5.25 ± 0.7 and 8.5 ± 1.3 μM, respectively) to H4IIE hepatoma cells. The cytotoxic potential of the other isolated flavanones was weaker (range of EC50 values between 15 and >100 μM). Toxic effects of addisoniaflavanone I and II were detectable after 3 h (MTT assay). Both compounds induced an apoptotic cell death (caspase-3/7 activation, nuclear fragmentation) in the hepatoma cells and, at high concentrations, also necrosis (membrane disruption: ethidium bromide staining). Formation of DNA strand breaks was not detectable after incubation with these compounds (comet assay). In conclusion, the prenylated flavanones addisoniaflavanones I and II may be of interest for pharmacological purposes due to their high cytotoxic and pro-apoptotic potential against hepatoma cells.