Induction of germline (GL) ε transcripts, an essential step preceding immunoglobulin (Ig) isotype switching to IgE, requires activation of transcription factors by IL-4 and a B cell activator, e.g. CD40 ligand (CD40L). AP-1 (Fos and Jun), induced transiently by CD40L, binds a DNA element in the mouse GL ε promoter. AP-1 synergizes with Stat6 to activate both the intact GL ε promoter and a minimal heterologous promoter driven by the AP-1 and Stat6 sites of the mouse GL ε promoter. By contrast, C/EBPβ, which transactivates the human GL ε promoter, inhibits IL-4 induction of the mouse promoter, probably by attenuating the synergistic interaction between AP-1 and Stat6. Furthermore, AP-1 does not transactivate the human GL ε promoter. Thus, due to selective binding of either AP-1 or C/EBP proteins, induction of GL ε transcripts in mouse and human may be regulated differently. In addition to AP-1, NF-кB activity is also induced by CD40L stimulation in normal B cells. Using GST pulldown assays and coimmunoprecipitation techniques I show that NF-кB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. When Stat6 and NF-кB proteins are co-expressed in human embryonic kidney 293 (HEK 293) cells, an IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4. Furthermore, the same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-кB proteins in IL-4-stimulated B lymphoma cells I.29μ. Consistently, by using nuclear extracts from transfected HEK 293 cells and from I.29μ B cells in electrophoretic mobility shift assays (EMSAs), I show that Stat6 and NF-кB bind cooperatively to a DNA probe containing both sites, and the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-кB. I conclude that the direct interaction between Stat6 and NF-кB may provide the basis for synergistic activation of the GL ε promoter. Finally, although mouse GL ε transcripts have a half-life of approximately 100 min, the RNA level continues to increase for up to 24 h and the promoter appears to be active for at least 2 days after B cell activation. These data suggest that induction of AP-1 and NF-кB activities by CD40L, although transient, is required for activation of the mouse GL ε promoter by IL-4-induced Stat6.

Investigators have been attempting to identify the receptor for ribosomes on the rough endoplasmic reticulum (RER) for almost 20 years, yet the ribosome receptor has remained elusive. Rough microsomal membranes contain endogenous ribosomes bound in at least two types of interactions. Loosely associated ribosomes can be removed by extraction with a high ionic strength solution, but ribosomes that were actively engaged in translocation when the membranes were isolated remain tethered to the membrane by a nascent polypeptide (Adelman et al., 1973). The original assay for the ribosome receptor involved stripping all of the endogenous ribosomes off of intact membranes before adding back a quantitated amount of ribosomes. More recent assays have employed detergent solubilization of the membrane and then reconstitution of the membrane proteins into lipid vesicles before adding back ribosomes. In both cases ribosome binding to its receptor is measured in an assay that does not involve translation or translocation. We utilized a crosslinking assay to attempt to identify membrane proteins that function as a binding site for ribosomes engaged in protein translocation across the endoplasmic reticulum. In vivo bound ribosomes that remain associated with the membrane after extraction with a high ionic strength solution are likely to be bound to a functional translocation site. The water soluble, membrane impermeable, thiol-cleavable crosslinker 3,3'-dithiobis (sulfosuccinimidylpropionate) was selected to limit reaction to protein domains located on the cytoplasmic face of salt extracted microsomal membrane vesicles. A specific subset of RER proteins was reproducibly crosslinked to the endogenous ribosomes. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35 kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180 kD protein that had recently been proposed to be a ribosome receptor (Savitz and Meyer, 1990). RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes in an in vitro binding assay to determine whether ribosome binding activity could be ascribed to the 180 kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180 kD protein, as determined by Coomassie blue staining of a polyacrylamide gel. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments and functional assays with proteoliposomes demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180 kD protein by chromatography. Taken together, the evidence indicates that the 180 kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor. To continue the investigation of ribosome binding, an assay was designed to characterize ribosome-nascent chain complexes bound to the microsomal membrane during translocation. A series of translocation intermediates consisting of discrete sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. Proteinase K was then used as a probe to detect cytoplasmically and lumenally exposed segments of nascent polypeptides undergoing transport. Only those partially translocated nascent chains of 100 amino acids or less were insensitive to protease digestion by externally added protease. It was concluded that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, shorter nascent polypeptides appear not to have lumenally exposed segments. Ultimately, a functional assay for the ribosome receptor should include binding studies conducted under physiological conditions. For this purpose, an assay was developed that allowed translation, translocation, and termination of a secretory protein to be monitored with probes designed to independently quantitate translating and non-translating ribosomes. A synchronized wheat-germ translation system was programmed with bovine preprolactin mRNA and aliquots were taken at various time points before and after adding membranes. The samples were then separated into membrane bound and soluble species by centrifugation. RNA was isolated from each supernatant and pellet sample and blotted onto nylon sheets. By probing the dot blots with probes that hybridize with either the 5S RNA of wheat germ ribosomes or the preprolactin transcript, the translating ribosomes could be monitored without the interference of the endogenous canine ribosomes on the membrane. By comparing the total amount of preprolactin transcript that bound to the membrane versus the total amount of wheat germ ribosomes bound to the membrane, it was discovered that the vast majority (>99%) of wheat germ ribosomes that bound to the microsomal membrane were non-translating ribosomes. In later experiments it was found that the non-translating ribosomes did not compete with the translating ribosomes; under all conditions tested, the translating ribosomes had access to translocation sites on the microsomal membrane. One interpretation of this data is that all ribosome binding sites are not identical. It may be that functional sites for translocation are a distinct subclass of total ribosome binding sites. Another interpretation is that a ribosome in a nascent chain-SRP complex has a much higher affinity for the ribosome receptor than nontranslating ribosomes or 60S subunits. Perhaps the non-translating ribosome can not compete with ribosomes engaged in translocation. As stated earlier, ribosomes do make at least two kinds of interactions with the microsomal membrane surface. This data may be indicative of those types of interactions.

Pericentrin and γ-tubulin are two resident centrosome proteins that are involved in microtubule nucleation and organization. When cytosolic extracts of Xenopus eggs were analyzed on sucrose gradients and gel filtration, the two proteins comigrated on gradients and co-eluted from the column. Immunodepletion of γ-tubulin removed all of the soluble pericentrin. The complex of the two proteins was estimated to be ~3-5 megaDaltons (MD), consisting of a pericentrin complex of ~20S and a γ-tubulin complex of ~25S, presumably the γ-TURC (~2 MD). When analyzed at the centrosome by enhanced deconvolution immunofluorescence the two proteins colocalized within a novel ring-like lattice structure, unlike other centrosome proteins analyzed, and were sufficiently close to generate FRET. The levels of the two proteins increased through the cell cycle, peaking at metaphase, and these changes were accompanied by structural changes in the lattice. Nucleated microtubules appeared to contact lattice elements throughout the centrosome. Inhibition of pericentrin function diminished assembly of γ-tubulin onto centrosomes, as did microtubule depolymerization and inhibition of dynein funciton. Separate fractions of the two proteins showed that pericentrin was required in the form a ~20S complex to bind γ-tubulin and for γ-tubulin assembly and microtubule nucleation. Overexpressed and purified pericentrin from cells eluted as a single polypeptide and was not competent to bind γ-tubulin. These results show that pericentrin in the context of a ~20S complex functions to assemble γ-tubulin into the centrosome lattice, and suggests that the pericentrin complex associated with the γ-TURC consists of an essential unit for centrosome formation.

This dissertation was intended to identify potential roles for phosphatidylinositol-3 kinase (PI-3 kinase) in the responses of lymphocytes to activation. To understand what functions PI-3 kinase is performing in lymphocytes, experiments were performed to identify proteins that will stably associate with the p85 subunit of PI-3 kinase. Co-precipitation revealed an activation dependent association of p85 with two different phosphotyrosine containing proteins. One protein, pp36-38, is a membrane protein that interacts with PI-3 kinase, PLCγ1, and Grb2/S0S. The other associated protein was identified as the proto-oncogene c-Cbl. The interaction of p85 with cbl was shown to be mediated through the SH2 domains of p85. More importantly, the interactions of p85 with p36-38 and cbl were found to be specific for p85 isoforms. Although the SH2 domains of the α and β isoforms are highly similar in amino acid sequence, they are shown to establish distinct protein interactions in intact cells. Experiments on the cbl/PI-3 kinase complex revealed a stimulation dependent translocation into membrane and insoluble/cytoskeletal fractions of wild type, but not mutant cells. The movement of cbl did not require tyrosine phosphorylation or PI-3 kinase activity. The cbl/PI-3 kinase complex was greatly enhanced in the membrane fraction in contrast to the cytosol, where the largest concentration of cbl can be found. In addition, these complexes were found to form at the membrane in the absence of the tyrosine kinase, p56lck.

Part 1: Monitoring Response to Single-Dose (1000cGy) Radiotherapy in a RIF-1 Tumor Model The current standard of measure for monitoring chemotherapeutic and radiotherapeutic treatment response is tumor volume. Unfortunately, changes in tumor volume are generally slow and tumor volume does not necessarily indicate the degree of tumor viability. The development of marker(s) with the ability to detect an early therapeutic response would greatly aid in patient management, opening the possibility for both rapid dose optimization and replacement of ineffective therapies with alternative treatment. Previous studies have shown that diffusion measurements using magnetic resonance imaging (MRI) techniques are sensitive to therapy-induced changes in cellular structure, allowing demarcation between regions of necrosis and viable tumor tissue. This sensitivity, based on the correlation between water apparent diffusion coefficient (ADC) values and tumor cellular density, may allow diffusion measurements to be employed in non-invasive monitoring of treatment response. Therapy-induced increases in tumor ADC preceding tumor regression have been reported in a variety of experimental tumor models and several human brain tumors. Despite the demonstrated diffusion sensitivity to therapeutic response in these particular studies, shortcomings still remain that hinder the efficacy of clinical application in oncology. Earlier studies have concentrated on the mean ADC present within the tumor, either within the entire tumor volume or a region of-interest (ROI) defined by the user, and their evolution pre-treatment and post-treatment. Because of inter- and intra-tumor heterogeneity, volume-averaged ADC measurements suffer from poor correlation with treatment efficacy. In addition, most studies make little or no attempt to characterize the entire tumor volume (necrotic, viable, edema). The identification of regions of differing tissue viability should aid in the staging of treatment, therefore making accurate and reproducible tissue segmentation an important goal. The results of earlier, single-parameter studies indicate that a multi-parametric approach in which several MR parameters are monitored (ADC, T2, M0) may provide greater power than that of the single parameter approach. A multi-parametric or multi-spectral (MS) analysis uses pattern-recognition techniques, such as clustering, for image segmentation. Clustering algorithms use characteristics of the multiple MR-parameter dataset to group tissue of similar type, e.g., fat, muscle, viable tumor, necrosis. Specifically, k-means (KM) is an unsupervised segmentation algorithm that groups together similar tissue based on the difference in MR parameter space between the image voxel of interest and the mean parameter values of the voxels in that cluster. In the first step of the classification algorithm, it is applied to separate the data into two clusters (k = 2), tissue and background noise voxels. All voxels classified as background noise are set to zero and removed from further processing. In the second step, KM is applied to the remaining tissue voxels to segment the data into multiple tissue types. In the case of tumors, it is not clear in advance how many different types of tissue exist. The number of clusters, k, should be varied to ensure that all relationships between tissues are found. In the final step, the resulting KM maps may be compared to histological slices taken from the same tissue as the imaging slices in order to identify the tissue type of each cluster. In line with the studies and analyses described above, quantitative MRI was performed to investigate the spatial correlation between ADC, spin-spin (T2) relaxation times, and proton density (M0) in murine radiation-induced fibrosarcoma (RIF-1) tumors following single-dose (l000cGy) radiotherapy using the KM algorithm (Chapters 3 and 4) and different combinations of features and/or clusters. For all cluster/feature combinations, an in-depth comparison between KM-derived volume estimates and conventional histology via the hematoxylin-eosin (H&E) staining procedure (for identification of viable tumor versus necrosis), as well as via hypoxic-inducible factor-lα. (HIF-1α) immunohistochemistry (for identification of regions of hypoxia versus well-oxygenated tissue) was performed (Chapter 3). The optimal cluster/feature combination was determined by minimizing the sum-of-squared-differences (SSD) between the actual datapoints and the ideal one-to-one correlation that should exist between KM-derived volume estimates and histology-derived volume estimates. The optimal cluster/feature combination was determined to be a 2-feature (ADC, T2) and 4-c1uster (2 regions each of viable tissue and necrosis) segmentation. This KM method was then applied in analysis of the radiotherapeutic response: first, to gain insight into the various processes whose combination yield the total ADC response over time; second, to identify the contribution of tissue heterogeneity to the treatment response and changes in tumor growth kinetics and cell kill (Chapter 4). Comparisons between control and various time-points out to 14 days post-radiotherapy permitted more accurate tissue characterization and prediction of therapeutic outcome over analysis using ADC alone. The results based on histological validation demonstrated: (1) MS analysis provides an improved tissue segmentation method over results obtained from conventional methods employing ADC alone; (2) MS analysis permits subdivision based on the degree of necrosis, as well as delineation between well-oxygenated and hypoxic viable tissue; and (3) Individual KM volumes corresponded well with both H&E volumes and regions with increased HIF-1α expression. The results based on the radiotherapeutic response demonstrated: (1) MS analysis provides a method for monitoring the range of tissue viability as a function of time post-treatment; (2) MS analysis permits assessment of the various contributions to the total ADC response post-treatment; (3) The relative fractions of well-oxygenated (i.e., radiosensitive) versus hypoxic (i.e., radioresistant) tissue pretreatment may be predictive of treatment response; and (4) The early ADC increase did not seem to be a result of radiation-induced vasogenic edema, but instead was most likely due to a slight reduction in cellular density following therapy. These studies provide a non-invasive method of tissue characterization that may be used in monitoring treatment response and optimizing drug dose-timing schemes, with the potential for predicting treatment efficacy. Part 2: Role of Spreading Depression in Ischemic Stroke Stroke is a prevalent disease that ranks as the 3rd leading cause of death and disability in the United States, according to NIH statistics, costing millions of dollars in medical costs and lost wages. At present, the mechanism by which focal ischemia evolves into infarction remains poorly understood. By determining the patho-physiological mechanisms involved in the evolution of focal brain ischemia, therapeutic strategies may be designed for instances of acute ischemic stroke. In the late 1980s, researchers discovered MRI techniques that allow the detection of stroke very early after onset. Such techniques as diffusion-weighted imaging and perfusion-weighted imaging (DWI and PWI) have been applied both clinically and experimentally. Previous studies employing these techniques suggest that cortical spreading depression plays a detrimental role in the evolution of focal brain ischemia. Spreading depression (SD) is characterized by a spontaneous and reversible depression of cortical electrical activity that spreads from the site of onset as a wave with a speed of 2-5 mm/min. It is accompanied by an ionic redistribution, with efflux of potassium ions (K+) and influx of sodium, chloride, and calcium (Na+, Cl-, Ca2+) ions, as well as water. This results in cellular swelling and a decreased extracellular space (ES), yielding a decline in ADC. A positive correlation between the number of both spontaneous and induced SDs and infarct volume has well been documented, supporting the idea that SD inhibition might be neuroprotective if initiated early after ischemic onset. Even though these studies show promise in their ability to track SD using diffusion mapping, changes in ADCs reflect cytotoxic edema and do not necessarily correspond to SD or SD-like depolarizations or calcium (Ca2+) influx, leading to cell death. Recent studies have reported the use of manganese ions (Mn2+) as a depolarization-dependent contrast agent in monitoring brain activation through the application of glutamate, as well as in the study focal ischemia. Since extracellular accumulation of potassium (K+) ions or glutamate in ischemic tissue is believed to play a central role in the initiation and propagation of SDs, and knowing that Mn2+, having an ionic radius similar to that of Ca2+, is handled in a manner similar to Ca2+, these studies suggest the possible use of manganese ions (Mn2+) in tracking SD or SD-like depolarizations in the evolution of focal brain ischemia. In order to determine the utility of Mn2+ as a marker for SD, two sets of T1-weighted MRI experiments were performed before applying Mn2+ in an experimental stroke model (Chapter 6). First, for verification purposes, a glutamate administration group was evaluated to validate our use of the manganese-enhanced MRI (MEMRI) method previously developed by Aoki et al, a modification of the original by Lin and Koretsky. When satisfied that the contrast enhancement was specific to glutamate only, a second set of experiments was performed. Here, experimental SD was elicited by chemical stimulation (direct application of concentrated potassium chloride [KC1] on the exposed cortical surface) and compared with control conditions (perfusion of sodium chloride [NaC1] on exposed brain cortex). This study demonstrated: (1) Mn2+, specific to Ca2+ channel activity, is a more accurate marker for SD than DWI or T2 methods; (2) Cortical restriction of MEMRI enhancement supports the contention that apical dendrites are necessary for SD propagation; (3) Subcortical enhancement is a result of corticalsubcortical neuronal connectivity; and (4) Because of the relatively slow clearance of Mn2+, MEMRI permits higher spatial resolution and signal-to-noise ratios (SNRs) via increased signal averaging. Based on these results, preliminary experiments involving the study of SD in focal ischemia using Mn2+ were performed (Chapter 7). Initial results indicate: (1) MEMRI of ischemia, when compared with standard DWI/PWI methods, may provide a method for estimating the likelihood of progression to infarct at acute time points post onset of stroke. These studies provide a foundation for further investigation into the role of SD in stroke, and the application of Mn2+ towards the design of therapeutic strategies targeting SD inhibition. Conclusions and Medical Significance The research within this dissertation employed magnetic resonance imaging techniques for monitoring the temporal evolution of pathological disease states such as focal ischemia and cancer, with and without therapeutic intervention. Optimization of these techniques in experimental models will open the possibility for future application in a clinical setting. Clinical availability of these non-invasive methods, with the ability to detect an early therapeutic response or to provide staging and prediction of tissue fate, would greatly aid in patient management of both cancer and stroke.

In an attempt to more fully understand the generation of antibody diversity to carbohydrate antigens, we produced and characterized a panel of hybridoma cell lines specific for group A streptococcal carbohydrate from mice injected with the intact bacteria (minus the hyaluronic acid capsule and cell wall protein antigens). We have analyzed the use of heavy and light chain variable region genes in the early (day 7) and late response (hyperimmune) and have determined the nucleotide sequence of the dominant VH gene used in several of our hybridomas. Our data allowed us to assess the extent to which the recombination of various V, D, and J gene segments and somatic mutation contribute to antibody diversification in this system. In this report we confirm that a minimum of two VH and four VK gene segments are used to encode this response. We extend this analysis to show that multiple D and J gene segments are used and that a significant amount of junctional variability is tolerated in CDR 3. Our results also suggest that there is a positive selection for somatic mutation in CDR 1 during the hyperimmune response to group A streptococcal carbohydrate.

Insulin stimulates glucose uptake in adipose and muscle cells via translocation of the intracellular vesicles containing GLUT4. It was largely unknown whether and/or how the signaling molecules such as PI 3-kinase and Akt regulate the mechanical movements of the GLUT4-containing vesicles. Hence, this study was performed to test the hypothesis that actin and microtubules function in translocating GLUT4 vesicles. Treatments of insulin as well as endothelin-1 (ET-1), an insulin-mimicking peptide which does not act through PI 3-kinase, induced polymerization of actin without affecting the microtubular network. By mass spectrometry, the tyrosine kinase PYK2 was identified to be tyrosine phosphorylated specifically by ET-1 but not by insulin. Expression of the carboxyl-terminal fragment (CRNK) PYK2, but not wild type nor kinase-deficient PYK2 mutants, inhibited ET-1-stimulated actin polymerization while expression of all three PYK2 constructs had no effect on insulin-stimulated actin polymerization. More importantly, expression of CRNK, but not wild type nor kinase-deficient PYK2 constructs, blocked ET-1- but not insulin-stimulated GLUT4 translocation to the plasma membrane. These suggest that ET-1 and insulin stimulate actin polymerization via distinct signaling pathways, and that the actin polymerization is required for GLUT4 vesicle translocation. In order to test the possible involvement of microtubule in GLUT4 vesicle translocation, time lapse imaging of 3T3-L1 adipocytes expressing GLUT4-YFP and tubulin-CFP was performed. GLUT4-YFP vesicles move long-range bi-directionally on microtubules, which suggests the presence of molecular motors on the vesicles. Moreover, insulin increased the number of vesicle movements on microtubules without changing the velocities. Interestingly, the stimulatory action of insulin appears to be independent of PI 3-kinase activation. Conventional kinesin was identified as a highly expressed kinesin isotype in adipocytes. Notably, expression of dominant negative mutants but not wild type kinesin inhibited insulin-stimulated long-range GLUT4 vesicle movements and GLUT4 translocation to the plasma membrane in live and fixed cells, respectively. These data indicate that insulin signaling induces the movement of GLUT4 vesicles on microtubule which is mediated by conventional kinesin. Overall, the data presented here provide evidence supporting the hypothesis that actin and microtubule cytoskeletons are required for insulin to mobilize GLUT4 vesicles in adipocytes.

The purpose of this work was to determine whether the previously identified fatty acid activation of large conductance Ca2+-activated K+ (BK) channels from rabbit pulmonary artery smooth muscle cells was due to the direct interaction of the fatty acid with a site on the channel protein. If this was found to be the case, this study would also attempt to identify the site of fatty acid-protein interaction. Fatty acids released from membrane phospholipids by cellular phospholipases or available to the cell from the extracellular environment are important signaling molecules. Fatty acids can modulate the activity of a large number of molecules including protein kinases, phospholipases, adenylate and guanylate cyclases, G-proteins and ion channels. Fatty acids have also been shown to activate transcription of genes belonging to the steroid/thyroid superfamily of receptors. The actions of fatty acids on signal transduction pathways can be direct, whereby the fatty acid molecule itself is responsible for changes in the activity of enzymes, ion channels and other proteins. Alternatively, the effects of fatty acids may be indirect. In this case, biologically active lipids, produced from the metabolism of arachidonic acid are responsible for changes in cellular signaling. A previous study on the fatty acid modulation of rabbit pulmonary artery smooth muscle BK channel activity concluded that channel activation by fatty acids did not involve cycloxygenase, lip oxygenase and P450 metabolites (122), eliminating this indirect action of fatty acids as a possible mechanism. When dealing with the effects of fatty acids on membrane bound ion channel proteins, other mechanisms of action are also possible. For example, fatty acids are capable of entering the cell membrane and can thus affect properties of the lipid bilayer, such as membrane fluidity or membrane surface charge, that may consequently alter the activity of ion channel proteins. In addition, fatty acid mediated alterations of ion channel activity could result from the effect of fatty acids on ion channel associated proteins. To determine the mechanism of action of fatty acids on the activity of BK channels from rabbit pulmonary artery smooth muscle cells, all of the above mentioned mechanisms were considered. Most of the experiments described here were carried out using the patch-clamp technique and current recordings were performed in cell free, excised inside-out or outside-out membrane patches, in the absence of any added nucleotides and calcium. As a first step towards understanding how fatty acids modulate BK channel activity, as well as the type of protein site with which fatty acids may be interacting, we determined the structural features of the fatty acid molecule that are required for channel modulation. To do this the effects of a range of fatty acids and other lipids on BK channel activity were examined. The features required for BK channel activation were found to be the negatively charged head group and a carbon chain of greater than eight carbons. We also found that positively charged lipids produced the opposite effect of negatively charged lipids, a decrease in BK channel activity. A similar chain length requirement was also necessary for channel inhibition by positively charged lipids; short chain compounds did not alter activity while those with fourteen carbons or greater decreased activity. The identification of these required structural features suggested that a specific interaction between the charge on the lipid head group is required for channel modulation by these lipids. The requirement for a chain length of greater than eight carbons also suggests that a hydrophobic interaction is necessary for these lipids to be effective modulators of this channel. In addition, the identification of these required structural features makes it unlikely that modulation of BK channel activity by these lipid compounds is a consequence of a perturbation of the lipid environment in which the channel resides. Experiments were then carried out to determine whether modulation of BK channel activity by fatty acids and other charged lipids involved any of the following indirect mechanisms of action: 1) alterations in the concentration of calcium in the vicinity of the channel due to changes in membrane surface charge, or due to calcium stores attached to excised membrane patches, 2) alterations in the membrane electric field that the channel perceives due to changes in membrane surface charge and 3) changes in the activity of membrane bound protein kinases or protein phosphatases. In experiments where high ionic strength solutions were used to shield membrane surface charge, fatty acids and other charged lipids were still able to modulate BK channel activity suggesting that fatty acids do not act through a mechanism involving surface charge. Experiments carried out in high concentrations of EGTA (20 mM) make it unlikely that calcium is involved in the modulation of BK channels by fatty acids and other lipids. The involvement of membrane bound kinases or phosphatases is also unlikely as fatty acids effectively modulated BK channel activity in the presence of staurosporin, a kinase inhibitor, and okadaic acid, a phosphatase inhibitor. The elimination of these indirect and non-specific suggests that fatty acids and charged lipids modulate BK channel activity by directly interacting with, either the channel protein itself, or some other channel associated protein. To obtain further evidence that this indeed is the mechanism by which these lipids modulate BK channel activity; experiments were carried out to identify the site of action (i.e. side of the membrane) of both negatively and positively charged lipids. The negatively charged palmitoyl coenzyme A (PCoA) and a myristoylated positively charged peptide (myr-KPRPK), two compounds that are incapable of flipping across the bilayer, were used to identify the site of action of negatively and positively charged lipids. PCoA and myr-KPRPK produced their predicted effects of BK channel activation and suppression, respectively, only when they were applied to outside-out membrane patches. These experiments, therefore, support the contention that fatty acids and other charged lipids modulate BK channel activity by interacting with a site on the channel protein or a channel associated protein and that this site is found on the external membrane surface. If the site responsible for channel modulation by fatty acids and other charged lipids is contained within the BK channel protein itself, other members of this family may also possess this site, and thus be modulated by fatty acids. Experiments were performed, therefore, to determine whether the BK cloned channels, mslo, hslo and bslo could also be modulated by fatty acids. These cloned channels were expressed in the Xenopus oocytes, and whole-cell currents were recorded using the two-electrode voltage clamp technique. The fatty acids myristic and arachidonic acid were able to increase whole-cell current of oocytes expressing all clone types. The modulation of these cloned channels by fatty acids did not appear to involve calcium, the BK β-subunit or a bioactive metabolite of arachidonic acid. Although all possible mechanisms of action were not addressed in this study, the results support the idea that the site of fatty acid interaction resides in the channel protein itself. Taken together, therefore, these studies suggest that it is very likely that fatty acids and charged lipids modulate the activity of BK channels from smooth muscle cells of the rabbit pulmonary artery by directly interacting with an externally located site on the channel protein itself. The BK clones, mslo, hslo and bslo, are also modulated by fatty acids and it is likely that they share the same mechanism of action seen for BK channels from rabbit pulmonary artery smooth muscle cells.

The immune system is responsible for defending a host animal from a wide variety of threats. Manipulation of the immune system can result in beneficial outcomes such as immunity to pathogens, or deleterious outcomes such as autoimmunity. Advances in our understanding of how the immune system develops and functions have benefited greatly from studies in animals, particularly in mice where the genetics are well known and a multitude of reagents are readily available for experimental use. Although much has been learned from animal experimentation, it must be cautioned that animals are not humans. Unforeseen outcomes and complications often arise when translating research obtained in animal models to treatment of human patients. A small animal model in which the human immune system can be established and manipulated experimentally in vivo would be valuable for the study of human immune responses in infectious diseases, transplantation, and autoimmunity, and ultimately for translation of these findings to the human patient. Contribution to this model is the overall goal of this thesis. The severe combined immunodeficiency (Prkdcscid, termed scid) mutation was discovered in 1983 in the C.B-17 strain of mice. Scid mice lack functional T and B lymphocytes and are unable to mount immune responses or reject allogeneic or xenogeneIc grafts. However, C.B-17-scid mice do develop normal or even elevated innate immune function. Based on the ability of scid mice to accept xenografts, human peripheral blood mononuclear cells have been injected to generate "Hu-PBL-scid mice." These Hu-PBL-scid mice have been proposed as an in vivo model of the human immune system. Although this model was described over 12 years ago, there are a number of obstacles that impede its ability to recapitulate human immunity. The Hu-PBL-scid mouse model established using C.B-17-scid mice as recipients is hindered by 1) low levels of human leukocyte engraftment, 2) engraftment of predominately memory/activated human T cells with specificity directed against host murine MHC antigens, 3) rapid transition of engrafted human lymphocytes to a functionally anergic state, and 4) a paucity of knowledge providing an understanding of the mechanisms that underlie engraftment and function of the human lymphocytes in the immunodeficient hosts. This thesis was initiated at the time the NOD-scid mouse became available for establishment of Hu-PBL-scid mice, and this model has subsequently been termed "Hu-PBL-NOD-scid." The NOD-scid mouse was designed as an improved recipient for human PBL based on its innate immune characteristics. Defects in innate immunity in wild-type NOD/Lt mice include reduced NK cell activity, defects in macrophage development and function, and a lack of hemolytic complement. NOD-scid mice retained these characteristics, and engraft human cells at higher levels compared to C.B-17-scid mice. However, the ratio of CD4+ to CD8+ T cells in Hu-PBL-NOD-scid mice remained skewed towards the CD8+ population, similar to that of the Hu-PBL-C.B-17-scid mouse. My thesis was based on the overall hypothesis that additional genetic manipulation of the recipient strain would enhance further the engraftment and function of human cells. Based on the engraftment results obtained in NOD-scid mice, we performed these genetic manipulations using the NOD-scid strain as the reference. We first hypothesized that removal of MHC expression would lower human anti-mouse xenoreactivity and enhance engraftment of naïve T cells, a cell population not readily detectable in Hu-PBL-C.B-17-scid or Hu-PBL-NOD-scid mice. Towards this goal, the NOD-scid mouse expressing a targeted mutation for beta-2 microglobulin (B2mnull) mouse was created by Dr. Leonard Shultz at the Jackson Laboratory. Because B2m is required for expression of MHC class I and for development of functional NK cells, we predicted that NOD-scid-B2mnull mice would first exhibit a normalized CD4:CD8 T cell ratio resulting from reduced CD8 engraftment due to decreased human anti-mouse MHC class I reactivity. Since MHC class I molecule expression is required for the development of NK cells, we further predicted that there would be a reduction of NK cell activity, permitting enhanced engraftment. Data presented in this thesis demonstrates that human PBL engraft in NOD-scid-B2mnull mice at levels higher than NOD-scid mice, and with an increased CD4:CD8 T cell ratio. The mechanism(s) responsible for the increased engraftment of human cells in these mice became a major focus of this thesis. This thesis is composed of three Specific Aims. Specific Aim 1 was to determine the mechanism(s) underlying human cell engraftment and function in Hu-PBL-scid mice. These data are contained in Part 1 of the Results. Specific Aim 2 was to elucidate the costimulation interactions between human T cells and murine host APCs that control the level of engraftment and activation state of the human lymphocytes. These data are contained in Part 2 of the Results. Specific Aim 3 was to utilize these fundamental observations to initiate studies into the induction of primary human immune responses in Hu-PBL-scid mice. Although the goal of these latter studies was not attained, the data from the experiments performed is provided in Part 3 of the Results, and is discussed relative to future directions for this arm of the project. In Part 1 of the Results, we utilized in vitro and in vivo techniques to study the underlying mechanisms regulating human cell engraftment and function in scid mice. We observed that the absence of mouse MHC class I antigens in NOD-scid-B2mnull mice does not reduce the stimulatory capacity of APCs toward human T cells in vitro. We further demonstrated that naïve human T cells persist for at least 2 weeks in the peritoneal cavity of Hu-PBL-NOD-scid and Hu-PBL-NOD-scid-B2mnull mice. However, in both strains of recipients, the human cells progress to an anergic phenotype as documented by cell surface molecule expression and by functional activity. We further documented that the increased engraftment of human CD4+ T cells observed in NOD-scid-B2mnull mice is due predominantly to the ablation of host NK cell activity. Increased engraftment of human CD4+ cells in NOD-scid-B2mnull mice can be recapitulated in NOD-scid mice by antibody-mediated depletion of residual host NK cells. Finally, we demonstrated that expression of MHC class II molecules by recipient mice facilitated stimulation and engraftment of human cells. However, mouse MHC class II expression is not required for human cell engraftment into scid mice. In Part 2 of the Results, we addressed the cellular and costimulatory interactions of engrafted human cells in scid mice. Costimulatory interactions between T cells and APC are now known to be critical for the proper activation and functioning of cells in the immune system. Understanding the role of costimulatory interactions between human T cells, human APCs, and mouse APCs became a major focus of this thesis. We demonstrated that human CD4+ T cells are required for the engraftment of human CD8+ T cells. The mechanism by which human CD4+ cells mediate this "helper" activity requires expression of CD154. Antibody-mediated blockade of CD154 in vivo abrogates human cell engraftment in scid mice. The role of host APCs in the engraftment of human lymphocytes was demonstrated by blocking host CD40 with antibody. Preventing human T cell CD154 interaction with host CD40 on murine APCs blocked human cell engraftment in scid mice, demonstrating the importance of "trans-costimulation" in human T cell activation. This "trans-costimulation" appeared to be mediated by B7 expression on mouse APCs. We further demonstrated that in vivo depletion of human CD8+ T cells in Hu-PBL-NOD-scid mice leads to increased levels of human CD4+ T cells, elevated human immunoglobulin in the serum, and increased incidence of EBV-related lymphoproliferative disorders. These observations suggested that human CD8+ T cells are able to regulate human CD4+ T cell help and provide "surveillance" activity for EBV-related lymphoproliferative disorders. In Part 3 of the Results, we used the Hu-PBL-scid mouse to initiate experiments designed to generate primary human immune responses in vivo. These experiments were based on our observation that few if any human APCs survive in scid mice, and on reports that dendritic cells (DC) are required for activation of naïve T cells and initiation of a primary immune response. We used recombinant viruses expressing HIV-1 proteins that are being developed as potential vaccines for HIV as our immunizing reagent. For APCs, we used DCs from NOD-scid mice expressing human MHC class II molecules as the source of APCs presenting antigen to the engrafted human T cells in the scid mice. Our attempts to induce a primary immune response using DC from human MHC-transgenic NOD-scid mice were unsuccessful, as were direct immunization protocols. The results section, however, does highlight deficiencies that could be approached experimentally in future studies. In summary, the results presented in this thesis advance our understanding of the fundamental mechanisms controlling human cell engraftment in scid mice. This information supports the long-term goal of establishing a functional human immune system in a small animal model. We have identified many of the cell interactions and factors that regulate human cell engraftment and function in scid mice, and we have provided insights into host characteristics that will provide optimized engraftment of naïve human T cells. The studies led to the novel observations of the regulation of human CD4+ T cells by human CD8+ T cells, B cell activation, and progression of latent EBV infection to lymphoproliferative disorders in vivo. These studies further provide new information regarding "trans-costimulation", a previously unrecognized mechanism of T cell activation. These results provide data on the fundamental mechanisms that underlie obstacles to the goal of achieving engraftment of a functional human immune system in scid mice.

Signal transduction by G-proteins (a heterotrimer membrane protein composed of an α, β, and γ subunit) requires that the α-subunit undergoes a transition from a GDP-bound inactive state to an activated GTP-bound state. The exchange of GDP for GTP leads to a conformational change in the α-subunit that results in the loss of affinity for the βγ subunits. We predicted that appropriate genetic manipulation of key regions of the α-subunit could result in the induction of the active conformation that would mimic at least in part the activated GTP-bound state. We have demonstrated that the substitution of the 38 amino acid residue carboxyl termimus of Gαs with the last 36 amino acid residues of Gαi2 resulted in a chimeric Gα-subunit (C4) that exhibits a constitutively active Gαs-like activity. Similarly, the substitution of the amino terminal 61 amino acid residues of Gαs with the first 54 residues of Gαi2 also resulted in a chimeric Gα-subunit that is persistently active (Gs like). We have also generated point mutations in the Gαs subunit that are comparable to the activating mutations in the ras protein. Our results suggest that point mutations in the signature sequence of the A (Val 49) and C (Thr 225) homologous regions that are implicated in regulating the GTPase activity of the molecule also resulted in the activation of the subunit. The present study has identified four key regions of the α-subunit that are critical for the activity and regulation of the Gs protein.