Characterization of the cytochrome P-450 drug metabolism system of LS174T human colon tumor cell line

The human colon tumor cell line, LS174T, has been shown to have four major components of the drug metabolizing system; cytochrome b$\sb5$ reductase, cytochrome b$\sb5$, cytochrome P450 reductase and cytochrome P450, by activity measurements, spectral studies and antibody cross-reactivity. Cytochrome P450IA1 is induced by benzanthracene in these cells as shown by activity with the specific substrate, ethoxyresorufin, cross-reactivity with rabbit antibodies to rat IA1, and by a hybridizing band on a Northern blot to a rat IA1 probe.^ Further, this system has proven responsive to various inducers and conditions of growth. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 $\mu$mol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b$\sb5$ per milligram and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone treatment showed a consistent, but not always significant increase in the NADPH and NADH cyt c reducing activity and benzanthracene treatment an increase in the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5mM) caused a significant decrease in the specific activity of all enzyme contents and activities tested.^ Finally, the cytochrome b$\sb5$ to cytochrome P450, by the coordinate induction of the cytochrome b$\sb5$ pathway by P450 inducers, by the high ratio of NADH to NADPH ethoxycoumarin deethylase activity in uninduced cell microsomes, and by the increase in NADH and NADPH ethoxycoumarin deethylase activity when the microsomes were treated with potassium cyanide, a desaturase inhibitor. ^

Evidence that lymphokine -activated killer (LAK) cell function is regulated by both serine and tyrosine kinase pathways

Signal transduction pathways operative in lymphokine activated killer (LAK) cells during execution of cytolytic function have never been characterized. Based on ubiquitous involvement of protein phosphorylation in activation of cytolytic mechanisms used by CTL and NK cells, it was hypothesized that changes in protein phosphorylation should occur when LAK encounter tumor targets. It was further hypothesized that protein kinases would regulate LAK-mediated cytotoxicity. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets consistently led to increased phosphorylation of two 65-kD LAK proteins pp65a and -b, with isoelectric points (pI) of 5.1 and 5.2 respectively. Increased p65 phosphorylation was initiated between 1 and 5 min after tumor coincubation, occurred on Ser residues, required physical contact between LAK and tumors, correlated with target recognition, and also occurred after crosslinking Fc$\gamma$RIIIA in the absence of tumors. Both pp65a and -b were tentatively identified as phosphorylated forms of the actin-bundling protein L-plastin, based on pI, molecular weight, and cross-reactivity with specific antiserum. The known biochemical properties of L-plastin suggest it may be involved in regulating adhesion of LAK to tumor targets. The protein tyrosine kinase-specific inhibitor Herb A did not block p65 phosphorylation, but blocked LAK killing of multiple tumor targets at a post-binding stage. Greater than 50% inhibition of cytotoxicity was observed after a 2.5-h pretreatment with 0.125 $\mu$g/ml Herb A. Inhibition occurred over a period in pretreatment which LAK were not dependent upon IL-2 for maintenance of killing activity, supporting the conclusion that the drug interfered with mobilization of cytotoxic function. Granule exocytosis measured by BLT-esterase release from LAK occurred after coincubation with tumors, and was inhibited by Herb A LAK cytotoxicity was dependent upon extracellular calcium, suggesting that granule exocytosis rather than Fas ligand was the principal pathway leading to target cell death. The data indicate that protein tyrosine kinases play a pivotal role in LAK cytolytic function by regulating granule exocytosis, and that tumor targets can activate an adhesion dependent Ser kinase pathway in LAK resulting in phosphorylation of L-plastin. ^

Immunologic characteristics of immunogenic variants generated by {\it in vitro\/} treatment of a methylcholanthrene-induced murine fibrosarcoma MCA-F with 1 methyl-3-nitro-1-nitrosoguanidine, 5 -aza-2$\sp\prime$-deoxycytidine, or ultraviolet radiation

This study addresses the questions of whether the frequency of generation and in vivo cross-reactivity of highly immunogenic tumor clones induced in a single parental murine fibrosarcoma cell line MCA-F is more closely related to the agent used to induce the Imm$\sp{+}$ clone or whether these characteristics are independent of the agents used. These questions were addressed by treating the parental tumor cell line MCA-F with UV-B radiation (UV-B), 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), or 5-aza-2$\sp\prime$-deoxycytidine (5-azaCdR). The frequency of Imm$\sp{+}$ variant generation was similarly high for the three different agents, suggesting that the frequency of Imm$\sp{+}$ generation was related more closely to the cell line than to the inducing agent used. Cross-reactivity was tested with two Imm$\sp{+}$ clones from each treatment group in a modified immunoprotection assay that selectively engendered antivariant, but not antiparental immunity. Under these conditions each clone, except one, immunized against itself. The MNNG-induced clones engendered stronger antivariant immunity but a weaker variant cross-reactive immunity could also be detected.^ This study also characterized the lymphocyte populations responsible for antivariant and antiparental immunity in vivo. Using the local adoptive transfer assay (LATA) and antibody plus complement depletion of T-cell subsets, we showed that immunity induced by the Imm$\sp{+}$ variants against the parent MCA-F was transferred by the Thy1.2$\sp{+}$, L3T4a$\sp{+}$, Lyt2.1$\sp{-}$ (CD4$\sp{+}$) population, without an apparent contribution by Thy1.2$\sp{+}$, L3T4a$\sp{-}$, Lyt2.1$\sp{+}$ (CD8$\sp{+}$) cells. A role for Lyt2.1$\sp{+}$T lymphocytes in antivariant, but not antiparent immunity was supported by the results of LATA and CTL assays. Immunization with low numbers of viable Imm$\sp{+}$ cells, or with high numbers of non viable Imm$\sp{+}$ cells engendered only antivariant immunity without parental cross-protection. The associative recognition of parental antigens and variant neoantigens resulting in strong antiparent immunity was investigated using somatic cells hybrids of Imm$\sp{+}$ variants of MCA-F and an antigenically distinct tumor MCA-D. An unexpected result of these latter experiments was the expression of a unique tumor-specific antigen by the hybrid cells. These studies demonstrate that the parental tumor-specific antigen and the variant neoantigen must be coexpressed on the cell surface to engender parental cross-protective immunity. (Abstract shortened with permission of author.) ^

Characterization of sea urchin yolk glycoproteins

To study the fate of the yolk glycoproteins found in eggs and embryos of the sea urchin, S. purpuratus, a polyclonal antibody to a 90-kDa polymannose glycoprotein was prepared. lmmunoblot analysis of total proteins over the course of development showed that this antibody recognized a family of glycoproteins. Concomitant with the disappearance of the major 160-kDa egg yolk glycoprotein during embryogenesis, glycoproteins with a lower molecular mass appeared. These glycoproteins (115, 108, 90, 83, and 68 kDa) were purified and peptide mapping revealed that they were cleavage products derived from the major yolk glycoprotein. The antibody identified a homologous set of yolk glycoproteins with similar molecular masses in the embryos of three other species in the class Echinoidea: L. pictus, A. punctulata, and D. excentricus. However, eggs from other echinoderm classes and from chicken, frog, fruit fly, and nematode did not contain any cross-reactive molecules. Cross-reactivity within the class Echinoidea was not due to a common carbohydrate epitope, because the antibody recognized the glycoproteins even after the N-linked, polymannose carbohydrate side chains were enzymatically removed. The major yolk glycoprotein (160-170 kDa) from each of the three sea urchin species was purified and analyzed, revealing striking similarities in pI and in amino acid and monosaccharide composition. Peptide mapping showed that the 160-kDa glycoprotein from the four echinoids are structurally homologous. The major yolk glycoprotein appeared to be proteolyzed by a thiol protease, which could be activated in yolk particles prepared from unfertilized eggs by low pH. Immunolocalization by electron microscopy in S. purpuratus showed that the yolk glycoproteins remained within the yolk platelet throughout embryonic development, and that externalization of the glycoproteins was not detectable. The yolk glycoprotein precursor began to be synthesized in premetamorphosis larvae, and continued in adult males and females. Both the yolk glycoproteins and the yolk platelets disappeared during larval development. This disappearance has special significance because there were no yolk proteins in the direct developing sea urchin, H. erthryogramma, which bypasses larval development and metamorphoses directly into an adult. ^

Cytochrome P450 2D18 in the mammalian brain: Recombinant expression, purification, and characterization of xenobiotic and endobiotic metabolism

The cytochromes P450 (P450) comprise a superfamily of hemoproteins that function in concert with NADPH-cytochrome P450 reductase (P450-reductase) to metabolize both endogenous and exogenous compounds. Many pharmacological agents undergo phase I metabolism by this P450 and P450-reductase monooxygenase system. Phase I metabolism ensures that these highly hydrophobic xenobiotics are made more hydrophilic, and hence easier to extrude from the body. While the majority of phase I metabolism occurs in the liver, metabolism in extrahepatic organ-systems like the intestine, kidney, and brain can have important roles in drug metabolism and/or efficacy. ^ While P450-mediated phase I metabolism has been well studied, investigators have only recently begun to elucidate what physiological roles P450 may have. One way to approach this question is to study P450s that are highly or specifically expressed in extrahepatic tissues. In this project I have studied the role of a recently cloned P450 family member, P450 2D18, that was previously shown to be expressed in the rat brain and kidney, but not in the liver. To this end, I have used the baculovirus expression system to over-express recombinant P450 2D18 and purified the functional enzyme using nickel and hydroxylapatite chromatography. SDS-PAGE analysis indicated that the enzyme was purified to electrophoretic homogeneity and Western analysis showed cross-reactivity with rabbit anti-human P450 2D6. Carbon monoxide difference spectra indicated that the purified protein contained no denatured P450 enzyme; this allowed for further characterization of the substrates and metabolites formed by P450 2D18-mediated metabolism. ^ Because P450 2D18 is expressed in brain, we characterized the activity toward several psychoactive drugs including the antidepressants imipramine and desipramine, and the anti-psychotic drugs chlorpromazine and haloperidol. P450 2D18 preferentially catalyzed the N-demethylation of imipramine, desipramine, and chlorpromazine. This is interesting given the fact that other P450 isoforms form multiple metabolites from such compounds. This limited metabolic profile might suggest that P450 2D18 has some unique function, or perhaps a role in endobiotic metabolism. ^ Further analysis of possible endogenous substrates for P450 2D18 led to the identification of dopamine and arachidonic acid as substrates. It was shown that P450 2D18 catalyzes the oxidation of dopamine to aminochrome, and that the enzyme binds dopamine with an apparent KS value of 678 μM, a value well within reported dopamine concentration in brain dopaminergic systems. Further, it was shown that P450 2D18 binds arachidonic acid with an apparent KS value of 148 μM, and catalyzes both the ω-hydroxylation and epoxygenation of arachidonic acid to metabolites that have been shown to have vasoactive properties in brain, kidney, and heart tissues. These data provide clues for endogenous roles of P450 within the brain, and possible involvement in the pathogenesis of Parkinson's disease. ^

A screening for anti-West Nile immunoglobulin-G and immunoglobulin-M in the Cameron County Hispanic cohort

This study is designed to be a cross-sectional, retrospective analysis of the seroprevalence of anti-WNV antibodies in 1,006 plasma samples collected from February 2, 2006 to June 18, 2007 originally for The Cameron County Hispanic Cohort: Extreme obesity and uncontrolled diabetes on the U.S.-Mexico border, major concerns for populations with health disparities. The aim of this thesis research is to give a more up-to-date picture of Flavivirus activity in south Texas, which can potentially contribute to the surveillance objective of arboviral control in this area. A West Nile virus (WNV) seroprevalence study in humans in this particular area has never before been completed. Plasma samples were tested using immunoglobulin-G (IgG) and immunoglobulin-M (IgM) WNV enzyme linked immunosorbent assays (ELISA). Estimated seroprevalence for this particular population was 0.98% or 9.8 cases of West Nile disease per 1,000 citizens. After IgG testing, seroprevalence in the study population was found to be 15.4%. Specimens tested for WNV IgG were compared with a subset of specimens (N=803) tested for history of primary dengue virus (DENV) infection. Of the 803 specimens tested for IgG to DENV, 308 were positive. Of the 132 positive WNV IgG specimens in the subset, 131 (99.2%) tested also tested positive for DENV IgG. It would be helpful to use standard plaque reduction neutralization testing to determine if the seroprevalence is in fact lower because of cross-reaction to DENV on testing. Regardless of the possibility of other Flavivirus activity occurring prior to the introduction of WNV into the United States and the potential for cross-reactivity, Texas has ranked in the top 5 states with the highest, laboratory confirmed incidence of infection with WNV since 2003. Indicating that climate factors and the presence of suitable vectors makes Texas a hotspot for WNV activity. ^ A description of the study population by age, gender, and income was done indicating a statistically significant income difference with a mean household income per year being $13,413.55 for a case and $20,268.80 for non-cases (p=0.001). Lower income neighborhoods should be targeted for education and prevention of vector-borne diseases during the summer months in Cameron County. With respect to gender, being male has been noted in the literature to be a risk factor for infection with WNV (25). In this study, females comprised approximately 68% of the study population, they also made up 66.5% of the positive IgG specimens. An odds ratio of 0.91 indicates that women are less likely to be IgG positive for WNV as compared to men; however, this was not found to be significant based on the 95% confidence interval, but is consistent with the literature. When looking at age difference between positive and negative/equivocal cases, there was no statistical difference found between the two groups. ^ We concluded that this study will enable us to understand the epidemiology of WNV transmission since its introduction into the United States and hopefully to maintain or improve the current measures we have in place to prevent infections that are seen annually with WNV.^

Motile response patterns and ultrastructural observations of the isolated outer hair cell

The tonotopic organization of the mammalian cochlea is accompanied by structural gradients which include the somatic lengths of outer hair cells (OHCs). These receptors rest upon the vibrating portion of the basilar membrane and have been reported to exhibit motile responses following chemical and electrical stimulation. These movements were examined in detail in this dissertation. It was found that isolated OHCs cultured in vitro respond to chemical depolarization with slow tonic movements, and to electrical waveforms with bi-directional, frequency following movements extending from DC to at least 10 kHz.^ Slow contractions were also elicited following electrical stimulation, bath incubation in carbachol (a cholinergic agonist), and increases in extracellular K+ concentration as little as 50 mM.^ Isolated OHCs display anatomical features which are remarkable when contrasted with those prepared from intact receptor organs. A complex structure located between the cuticular plate and the nuclear membrane was consistently observed and was examined by serial cross-sections which revealed a network of non-membrane bound densities. This corresponded to a granular complex seen at the light microscope level. The complex was composed of dense regions of organelles, striated structures embedded within the core, and a circumferential network of microtubules residing in the peri-nuclear portion of the cell. In cells which had lost their nuclear attachment to the terminal synaptic body, the granular complex could be made to contract without effecting any change in cellular length, implying that the complex may be the driving force behind certain aspects of the motile response.^ Most cells displayed movements which revealed asymmetries analogous to those reported for OHC receptor potentials in vivo. The contraction phase (for longer cells) was shown to have a small time constant (approximately 400 microseconds) and saturated with limited displacements. The expansion phase had time constants as large as 1.3 milliseconds but yielded displacements as much as 60 percent larger than those seen for contractions.^ Additional waveform characteristics seen in the in vivo response could be emulated either by biasing the cell's resting length with either direct current, triggering contractions via large electrical displacements, or incubation with depolarizing compounds.^ Alternatively, short (20-30 um) cells revealed more linear response characteristics to the probe stimulus. Partial saturation was achieved and revealed a DC component which was opposite in polarity to that seen in longer cells. (Abstract shortened with permission of author.) ^

Cell surface galactosyltransferase structure and function during mesenchymal cell migration

In order to more fully understand the function of surface GalTase on mesenchymal cells, anti-GalTase IgG was used to (a) examine the role of surface GalTase during mouse mesenchymal cell migration on laminin and fibronectin; (b) define the plasma membrane distribution of GalTase by indirect immunofluorescence on migrating cells; (c) quantitate the level of surface GalTase on migrating cells; and (d) determine whether GalTase is associated with the cytoskeleton.^ Results show that anti-GalTase IgG was able to inhibit migration (48-80% as compared to basal rate) when cells were migrating on laminin-containing matrices. Monovalent Fab fragments inhibited migration on laminin by 90% after 4 hours. On the other hand, anti-GalTase IgG had no effect on cells migrating on fibronectin. This illustrates the substrate specificity of GalTase mediated-migration. When anti-GalTase IgG was used to localize surface GalTase on cells migratory on laminin, the enzyme was restricted to the leading and trailing edges of the cell. Assays indicate that GalTase is elevated approximately 3-fold when cells are migrating on laminin-containing matrices as compared to migratory cells on plastic or fibronectin, or as compared to stationary cells on any substrate. Laminin appears to recruit GalTase from preexisting intracellular pools to the growing lamellipodia.^ Double-label indirect immunofluorescence studies indicate that there is an apparent co-localization between some of the surface GalTase and some actin filaments. This relationship was explored by extracting cells prelabeled with anti-GalTase IgG and quantitated by radiolabeled second antibodies. Results show that 79% of the surface GalTase is associated with the cytoskeleton (as judged by detergent insolubility) when monovalent antibodies (Fab) are used. However virtually all (80-100%) of the surface GalTase can be induced to associate with the cytoskeleton when cross-linked with bivalent antibodies. Furthermore, when cells in suspension are incubated with divalent antibodies, an additional 66% of the surface GalTase can be induced to associate with the cytoskeleton. The elevated levels of surface GalTase detectable on cells migrating on laminin also appear to be associated with the cytoskeleton.^ Several lines of evidence suggest that GalTase is associated with F-actin. Data suggest that laminin induces the expression of surface GalTase to the growing lamellipodia where it becomes associated with the cytoskeleton leading to cell spreading and migration. (Abstract shortened with permission of author.) ^

Analyses of pp60$\sp{{\rm c}-src}$ and epidermal growth factor receptor protein tyrosine kinases in a human colon adenocarcinoma cell line following induction of goblet cell-like characteristics by tumor necrosis factor-alpha

Regulation of colonic epithelial cell proliferation and differentiation remains poorly understood due to the inability to design a model system which recapitulates these processes. Currently, properties of "differentiation" are studied in colon adenocarcinoma cell lines which can be induced to express some, but not all of the phenotypes of normal cells. In this thesis, the DiFi human colon adenocarcinoma cell line is utilized as an in vitro model system in which to study mucin production. In response to treatment with tumor necrosis factor-alpha, DiFi cells acquire some properties of mucin-producing goblet cells including altered morphology, increased reactivity to wheat germ agglutinin, and increased mucin production as determined by RNA expression as well as reactivity with the MUC-1 antibodies, HMFG-1 and SM-3. Thus, TNF-treated DiFi cells represent one of the few in vitro systems in which mucin expression can be induced.^ DiFi cells express an activated pp60$\sp{{\rm c}-src},$ as do most colon adenocarcinomas and derived cell lines, as well as an amplified epidermal growth factor (EGF) receptor. To assess potential changes in these enzymes during induction of differentiation characteristics, potential changes in the levels and activities of these enzymes were examined. For pp60$\sp{{\rm c}-src},$ no changes were observed in protein levels, specific activity of the kinase, cellular localization, or phosphorylation pattern as determined by Staphylococcus aureus V8 protease partial proteolytic mapping after induction of goblet cell-like phenotypic changes. These results suggest that pp60$\sp{{\rm c}-src}$ is regulated differentially in goblet cells than in absorptive cells, as down-modulation of pp60$\sp{{\rm c}-src}$ kinase occurs in the latter. Therefore, effects on pp60$\sp{{\rm c}-src}$ may be critical in colon regulation, and may be important in generating the various colonic epithelial cell types.^ In contrast to pp60$\sp{{\rm c}-src},$ EGF receptor tyrosine kinase activity decreased ($<$5-fold) after TNF treatment and at the time in which morphologic changes were observed. Similar decreases in tyrosine phosphorylation of EGF receptor were observed as assessed by immunoblotting with an anti-phosphotyrosine antibody. In addition, ($\sp{125}$I) -EGF cell surface binding was reduced approximately 3-fold following TNF treatment with a concomitant reduction in receptor affinity ($<$2-fold). These results suggest that modulation of EGF receptor may be important in goblet cell differentiation. In contrast, other published studies have demonstrated that increases in EGF receptor mRNA and in ($\sp{125}$I) -EGF binding accompany differentiation toward the absorptive cell phenotype. Therefore, differential regulation of both EGF receptor and pp60$\sp{{\rm c}-src}$ occur along the goblet cell and absorptive cell differentiation pathways. Thus, my results suggest that TNF-treated DiFi cells represent a unique system in which to study distinct patterns of regulation of pp60$\sp{{\rm c}-src}$ and EGF receptor in colonic cells, and to determine if increased MUC-1 expression is an early event in goblet cell differentiation. ^

A FUNCTIONAL VARIANT OF HLA-DR1 DELINEATED BY T-LYMPHOCYTE CLONE REACTIVITY, PROTEIN CONFORMATION, AND GENOMIC RESTRICTION SITES

This research characterized a serologically indistinguishable form of HLA-DR1 that: (1) cannot stimulate some DR1-restricted or specific T-lymphocyte clones; (2) displays an unusual electrophoretic pattern on two dimensional gels; and (3) is marked by a polymorphic restriction site of the alpha gene. Inefficient stimulation of some DR1-restricted clones was a property of DR1$\sp{+}$ cells that shared HLA-B14 on the same haplotype and/or were carriers of 21-hydroxylase (21-OH) deficiency. Nonclassical 21-OH deficiency frequently demonstrates genetic linkage with HLA-B14;DR1 haplotypes and associates with duplications of C4B and one 21-OH gene. Cells having both stimulatory (DR1$\sb{\rm n}$) and nonstimulatory (DR1$\sb{\rm x}$) parental haplotypes did not mediate proliferation of these clones. However, heterozygous DR1$\sb{\rm x}$, 2 and DR1$\sb{\rm x}$, 7 cells were efficient stimulators of DR2 and DR7 specific clones, respectively, suggesting that a trans acting factor may modify DR1 alleles or products to yield a dominant DR1$\sb{\rm x}$ phenotype. Incompetent stimulator populations did not secrete an intercellular soluble or contact dependent suppressor factor nor did they express interleukin-2 receptors competing for T-cell growth factors. Two dimensional gel analysis of anti-DR immunoprecipitates revealed, in addition to normal DR$\alpha$ and DR$\beta$ chains, a 50kD species from DR1$\sb{\rm x}$ but not from the majority of DR1$\sb{\rm n}$ or non-DR1 cells. The 50kD structure was stable under reducing conditions in SDS and urea, had antigenic homology with DR, and dissociated after boiling into 34kD and 28kD peptide chains apparently identical with DR$\alpha$ and DR$\beta$ as shown by limited digest peptide maps. N-linked glycosylation and sialation of DRgp50 appeared to be unchanged from normal DR$\alpha$ and DR$\beta$. Bg1II digestion and $DR\alpha$ probing of DR1$\sb{\rm x}$ genomic DNA revealed a 4.5kb fragment while DR1$\sb{\rm n}$ DNA yielded 3.8 and 0.76kb fragments; all restriction sites mapped to the 3$\sp\prime$ untranslated region of $DR\alpha$. Collectively, these data suggest that DRgp50 represents a novel combinatorial association between constitutive chains of DR that may interfere with or compete for normal T cell receptor recognition of DR1 as both an alloantigen and restricting element. Furthermore, extensive chromosomal abnormalities previously mapped to the class III region of B14;DR1 haplotypes may extend into the adjacent class II region with consequent intrusion on immune function. ^

ANALYSIS OF THE CORYNEBACTERIUM PARVUM-INDUCED DECLINE OF MURINE NATURAL KILLER CELL-MEDIATED CYTOTOXICITY

Treatment of mice with the immunomodulating agent, Corynebacterium parvum (C. parvum), was shown to result in a severe and long-lasting depression of splenic natural killer (NK) cell-mediated cytotoxicity 5-21 days post-inoculation. Because NK cells have been implicated in immunosurveillance against malignancy (due to their spontaneous occurrence and rapid reactivity to a variety of histological types of tumors), as well as in resistance to established tumors, this decreased activity was of particular concern, since this effect is contrary to that which would be considered therapeutically desirable in cancer treatment (i.e. a potentiation of antitumor effector functions, including NK cell activity, would be expected to lead to a more effective destruction of malignant cells). Therefore, an analysis of the mechanism of this decline of splenic NK cell activity in C.parvum treated mice was undertaken.^ From in vitro co-culturing experiments, it was found that low NK-responsive C. parvum splenocytes were capable of reducing the normally high-reactivity of cells from untreated syngeneic mice to YAC-1 lymphoma, suggesting the presence of NK-directed suppressor cells in C. parvum treated animals. This was further supported by the demonstration of normal levels of cytotoxicity in C. parvum splenocyte preparations following Ficoll-Hypaque separation, which coincided with removal of the NK-suppressive capabilities of these cells. The T cell nature of these regulatory cells was indicated by (1) the failure of C. parvum to cause a reduction of NK cell activity, or the generation of NK-directed suppressor cells in T cell-deficient athymic mice, (2) the removal of C. parvum-induced suppression by T cell-depleting fractionation procedures or treatments, and (3) demonstration of suppression of NK cell activity by T cell-enriched C. parvum splenocytes. These studies suggest, therefore, that the eventual reduction of suppression by T cell elimination and/or inhibition, may result in a promotion of the antitumor effectiveness of C. parvum due to the contribution of "freed" NK effector cell activity.^ However, the temporary suppression of NK cell activity induced by C. parvum (reactivity of treated mice returns to normal levels within 28 days after C. parvum injection), may in fact be favorable in some situations, e.g. in bone marrow transplantation cases, since NK cells have been suggested to play a role also in the process of bone marrow graft rejection.^ Therefore, the discriminate use of agents such as C. parvum may allow for the controlled regulation of NK cell activity suggested to be necessary for the optimalization of therapeutic regimens. ^

Antibody chemical reactivity: Beneficial and pathogenic roles

Antibodies (Abs) to autoantigens and foreign antigens (Ags) mediate, respectively, various pathogenic and beneficial effects. Abs express enzyme-like nucleophiles that react covalently with electrophiles. A subpopulation of nucleophilic Abs expresses proteolytic activity, which can inactivate the Ag permanently. This thesis shows how the nucleophilicity can be exploited to inhibit harmful Abs or potentially protect against a virus. ^ Inactivation of pathogenic Abs from Hemophilia A (HA) patients by means of nucleophile-electrophile pairing was studied. Deficient factor VIII (FVIII) in HA subjects impairs blood coagulation. FVIII replacement therapy fails in 20-30% of HA patients due to production of anti-FVIII Abs. FVIII analogs containing electrophilic phosphonate group (E-FVIII and E-C2) were hypothesized to inactivate the Abs by reacting specifically and covalently with nucleophilic sites. Anti-FVIII IgGs from HA patients formed immune complexes with E-FVIII and E-C2 that remained irreversibly associated under conditions that disrupt noncovalent Ab-Ag complexes. The reaction induced irreversible loss of Ab anti-coagulant activity. E-FVIII alone displayed limited interference with coagulation. E-FVIII is a prototype reagent suitable for further development as a selective inactivator of pathogenic anti-FVIII Abs. ^ The beneficial function of Abs to human immunodeficiency virus type 1 (HIV-1) was analyzed. HIV-1 eludes the immune system by rapidly changing its coat protein structure. IgAs from noninfected subjects hydrolyzed gp120 and neutralized HIV-1 with modest potency by recognizing the gp120 421-433 epitope, a conserved B cell superantigenic region that is also essential for HIV-1 attachment to host cell CD4 receptors. An adaptive immune response to superantigens is generally prohibited due to their ability to downregulate B cells. IgAs from subjects with prolonged HIV-1 infection displayed improved catalytic hydrolysis of gp120 and exceptionally potent and broad neutralization of diverse CCR5-dependent primary HIV isolates attributable to recognition of the 421-433 epitope. This indicates that slow immunological bypass of the superantigenic character of gp120 is possible, opening the path to effective HIV vaccination. ^ My research reveals a novel route to inactivate pathogenic nucleophilic Abs using electrophilic antigens. Conversely, naturally occurring nucleophilic Abs may help impede HIV infection, and the Abs could be developed for passive immunotherapy of HIV infected subjects. ^

DIAGNOSTIC ANTIGENS FOR SCHISTOSOMIASIS MANSONI: PRODUCTION, CHARACTERIZATION AND PURIFICATION OF MONOCLONAL ANTIBODIES, AND AFFINITY PURIFICATION OF ANTIGENS

Attempts have been made in this dissertation to develop a purified antigen with high sensitivity and specificity for diagnosis of Schistosoma mansoni (Sm) infection by using the hybridoma technique.^ Spleen cells, obtained from mice immunized by infection with Sm and boosted by cercarial antigens, or by injection of circulating antigen (CA) in serum from infected mice, were fused with Sp2/0 myeloma cells. The active infection resulted a higher number of hybridomas (100%) than by CA (20%), and higher levels of antibody reactivity as measured by ELISA.^ The IgM and IgG monoclonal antibodies (MCAbs) were purified respectively by gel filtration, DE 52 ion exchange column and proteinase A affinity column. The cercarial and egg antigens were purified by affinity chromatography through MCAb/affi-gel column. The reactivity of the purified antigens were then monitored by ELISA, SDS-PAGE silver stain and EITB.^ The respective MCAbs recognized varying antigenic determinants (AD) present in adult, cercaria and egg stages. By EITB the MCAbs IgM and IgG, when reacted with nine antigens from the various stages, revealed identical bands, suggesting that the two MCAb classes originated from identical AD. By ELISA and COPT, the MCAbs from thirteen cell lines gave same results. But by CHR, two MCAbs showed negative results while eleven other MCAbs showed strong positive. It is assumed that the AD in the immunogen that ilicited the MCAbs were immunochemically closely related.^ One egg purified by immunoaffinity indicated that the epitopes recognized by MCAb were present on four antigenic components with molecular weights (Mr) of approximately 19, 25, 60 and >224 kd, respectively. By EITB the Mr 19 doublet appeared to be species specific; the Mr 25 kd genus specific. They reacted with mouse serum from 13-16 weeks after infection. In monkey serum Mr 19 doublet appeared 8-10 weeks after infection and disappeared at 8-12 weeks after Droncit treatment, paralleled to the disappearance of fecal egg. The Mr 60 and >224 kd bands were also demonstrated with S. japonicum, S. haematobium and Trichinella spiralis infection sera and may be the cause of cross-reaction in conventional serological test. ^

SYSTEMIC TOXICITY IN RODENTS FOLLOWING ACUTE AND SUBCHRONIC INHALATION OF FORMALDEHYDE (FLOW CYTOMETRY, CELL CYCLE KINETICS, ALKALINE ELUTION)

Systemic toxicity was evaluated in Sprague-Dawley (SD) rats and A-strain mice exposed to HCHO inhalation at 0, 0.5, 3, or 15 ppm for six hours/day, five days/week for up to 24 weeks. Toxicity was measured by flow cytometry to detect changes in cell cycle RNA and DNA content and by alkaline elution to detect DNA protein cross-link (DPC) formation.^ A G(,2)M block was detected in SD rat marrow following one week of exposure to 0.5, 3, or 15 ppm HCHO, but this block did not persist. No effect was noticed in mouse marrow. Only a minimal increase in RNA content was detected in rat or mouse marrow while exfoliated lung cells showed a significant increase in RNA activity after one week of exposure.^ Acute exposure in SD rats for four hours/day for one or three days at 150 ppm showed an increase in RNA activity in exfoliated lung cells but not in the marrow after one day. On the third day, dead cells were detected in exfoliated lung cells.^ In alkaline elution studies, no DPC were detected in marrow of SD rats after 24 weeks exposure up to 15 ppm. During acute exposures, a dose response relationship was detected in SD rat exfoliated lung cells which yielded cross-linking factors of 0.954, 1.237, and 1.417 following a four hour exposure to 15, 50, or 150 ppm, respectively. No DPC were detected in the marrow at 150 ppm. In vitro exposures to HCHO of CHO and SHE cells and rat marrow cells revealed the production of DPC and DNA-DNA cross-links.^ Cytoxan treatment of SD rats was used to provide positive controls for flow cytometry and alkaline elution. A drastic reduction in RNA content and cycling cells occurred one day following treatment. After four days, RNA content was greatly increased; and on day eleven the marrow had regenerated. DPCs were detected in both the marrow and the exfoliated lung cells.^ The lack of significant responses in SD rats and A-strain mice below 15 ppm HCHO is explainable by host defense mechanisms. Apparently, the mucociliary apparatus and enzymatic detoxification are sufficient to reduce systemic toxicity to low level concentrations of formaldehyde. ^

THE MOLECULAR INTERACTION BETWEEN TYPE II DIABETES AND ALZHEIMER’S DISEASE THROUGH CROSS-SEEDING OF PROTEIN MISFOLDING

With the population of the world aging, the prominence of diseases such as Type II Diabetes (T2D) and Alzheimer’s disease (AD) are on the rise. In addition, patients with T2D have an increased risk of developing AD compared to age-matched individuals, and the number of AD patients with T2D is higher than among aged-matched non-AD patients. AD is a chronic and progressive dementia characterized by amyloid-beta (Aβ) plaques, neurofibrillary tangles (NFTs), neuronal loss, brain inflammation, and cognitive impairment. T2D involves the dysfunctional use of pancreatic insulin by the body resulting in insulin resistance, hyperglycemia, hyperinsulinemia, pancreatic beta cell (β-cell) death, and other complications. T2D and AD are considered protein misfolding disorders (PMDs). PMDs are characterized by the presence of misfolded protein aggregates, such as in T2D pancreas (islet amyloid polypeptide - IAPP) and in AD brain (amyloid– Aβ) of affected individuals. The misfolding and accumulation of these proteins follows a seeding-nucleation model where misfolded soluble oligomers act as nuclei to propagate misfolding by recruiting other native proteins. Cross-seeding occurs when oligomers composed by one protein seed the aggregation of a different protein. Our hypothesis is that the pathological interactions between T2D and AD may in part occur through cross-seeding of protein misfolding. To test this hypothesis, we examined how each respective aggregate (Aβ or IAPP) affects the disparate disease pathology through in vitro and in vivo studies. Assaying Aβ aggregates influence on T2D pathology, IAPP+/+/APPSwe+/- double transgenic (DTg) mice exhibited exacerbated T2D-like pathology as seen in elevated hyperglycemia compared to controls; in addition, IAPP levels in the pancreas are highest compared to controls. Moreover, IAPP+/+/APPSwe+/- animals demonstrate abundant plaque formation and greater plaque density in cortical and hippocampal areas in comparison to controls. Indeed, IAPP+/+/APPSwe+/- exhibit a colocalization of both misfolded proteins in cerebral plaques suggesting IAPP may directly interact with Aβ and aggravate AD pathology. In conclusion, these studies suggest that cross-seeding between IAPP and Aβ may occur, and that these protein aggregates exacerbate and accelerate disease pathology, respectively. Further mechanistic studies are necessary to determine how these two proteins interact and aggravate both pancreatic and brain pathologies.