A model of $\beta$-adrenergic and muscarinic cholinergic stimulation of the bullfrog atrial myocyte has been developed that mimics the dose-dependent effects of isoprenaline (ISO) on the action potential duration (APD); i.e., low doses of ISO lengthen the APD, while high doses shorten the ADP. This reduction in APD is modeled as the result of (1) calcium-dependent inactivation of $I\sb{Ca}$ resulting from the enhancement of $I\sb{Ca}$ by ISO and (2) an enhancement of $I\sb{K}$ due to both an ISO-induced increase in the rate of activation of $I\sb{K}$ and an increase in peak action potential height. The effect of acetylcholine (ACh) is to reduce the ISO-induced increase in $I\sb{Ca}$ and $I\sb{K}$ through a reduction in relative (cAMP) as well as to stimulate the ACh-sensitive $K\sp{+}$ current $I\sb{K,ACh}.$ At low (ISO) levels or high (ACh) levels, the muscarinic cholinergic effect dominates over the $\beta$-adrenergic effect. However, for a large (ISO) and a small (ACh), this pattern of changes in transmembrane currents is different; in this case the model predicts that ACh can actually increase APD. A distributed parameter model of an idealized bullfrog atrial trabeculum is developed. Individual cardiac cells are resistively coupled end to end via intercalated discs to form an idealized cylindrical cardiac strand encased in a resistive-capacitative trabecular sheath which, in turn, is located in a finite cylindrical volume conductor. A second-order implicit finite numerical integration method is used to calculate the time-varying potentials within the intracellular $(V\sp{i}),$ interstitial $(V\sp{e}),$ and the outer volume conductor $(V\sp{o})$ media of the concentric cylindrical structure. 'Reduced' cell membrane models which lack the complete complement of transmembrane currents are compared with regard to their accuracy in representing the foot, upstroke, and plateau regions of the propagated action potential in the complete model. A reduced cell membrane model should contain the sodium current $I\sb{Na},$ the calcium current $I\sb{Ca}$ and the background rectifying $K\sp{+}$ current $I\sb{K1}.$ A cell membrane model which contains a linear background $K\sp{+}$ current $I\sb{L}$ instead of $I\sb{K1}$ produces a poor approximation to the upstroke, plateau and conduction velocity of an action potential. The trabecular sheath reduces the extracellular resistance seen by the cell by shunting current away from highly resistive interstitial medium into the volume conductor medium which is of low resistance, and thereby increases conduction velocity. Finally, the effects of the cholinergic neurotransmitter, acetylcholine (ACh), on both the passive and active properties of the trabeculum are investigated. The addition of ACh to the extracellular medium reduces the space constant and input resistance of the trabeculum, as well as the conduction velocity of electrical activity propagating through it.

Basic fibroblast growth factor (bFGF) plays an instrumental role in the cascade of events leading to restenosis, vascular re-occlusion due to excessive smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) deposition following arterial intervention procedures such as balloon angioplasty. The mechanism of bFGF activation following vascular injury has remained elusive. bFGF is stored bound to heparan sulfate proteoglycans in the ECM of the arterial media; release from extracellular sequestration may activate bFGF and initiate SMC proliferation and migration. bFGF mobilization at injured sites may be induced by platelet degranulation products. We have carried out in vitro studies demonstrating that platelet-derived heparanase and platelet factor-4 (PF4) liberate bFGF from the ECM of vascular SMCs, resulting in the induction of SMC proliferation and migration. Increases in proliferation and migration were inhibited by treatment with a bFGF-neutralizing antibody, suggesting that proliferation and migration in response to heparanase or PF4 are mediated by bFGF activation. When platelets were seeded on top of SMCs, degranulation products were found to release bFGF from the ECM, increasing cell proliferation and cell migration. These increases in SMC proliferation and migration were completely inhibited by the addition of a bFGF-neutralizing antibody. In order to investigate the role of heparanase and PF4 in vivo, each was delivered to the uninjured rat carotid artery. Heparanase and PF4 were both found to release bFGF, induce substantial SMC proliferation and increase the expression of several growth factor receptors thought to promote restenosis. An antibody that neutralizes platelet-derived heparanase was developed and evaluated in a rat carotid balloon injury model. Perivascular delivery of anti-heparanase IgG was found to inhibit bFGF depletion from the arterial wall by approximately 60% (p < 0.001) at 4 days. This correlated with the reduction in intimal thickening observed at 14 days. Platelet degranulation products, such as heparanase and PF4, may liberate bFGF from extracellular sequestration, activating the growth factor and inducing the SMC proliferation and migration that contribute to luminal narrowing following vascular injury. In addition, platelet-derived heparanase is likely to play a key role in initiating events leading to restenosis via bFGF mobilization.

A Lagrangian approach is used to perform an energy analysis on a nonlinear model of the human lung. Energy functions associated with the mechanics of breathing are obtained. Analytical expressions are derived for the work of breathing (WOB) and are subsequently evaluated for a typical pulmonary function laboratory test that includes tidal breathing, panting and the forced vital capacity maneuvers. The analysis successfully mimics information presented in the conventional Campbell diagram, which is often used to graphically estimate the work of breathing. In addition, it reveals dynamic aspects of muscular effort during a breathing cycle that are not immediately apparent in the Campbell diagram. Additional simulations, based on an expanded model that accounts for gas compressibility, reveal that WOB is underestimated during forceful breathing maneuvers when compressibility effects are ignored. The energy analysis provides valuable insight into the mechanics of respiration, and, in particular, the significance of individual model components.

The brain is the organ at greatest risk of injury during periods of reduced blood flow; to prevent tissue death and protect organ function in such conditions, hypothermia is used. The primary goal of this research is to use a thermal model as a tool to develop and optimize hypothermic protection methods for the brain tissue given the fact that the cooling time, as well as the degree of temperature reduction required for successful outcome are still uncertain, and the knowledge of brain temperature is desirable during clinical treatment, but highly destructive. To improve the existing thermal models of brain, the effect of the temperature over the metabolic heat generation, and the regulatory processes that control the cerebral blood perfusion were incorporated in this project. The proposed thermal model was validated using data obtained from experiments of perinatal asphyxia, and different cooling strategies on swine. The temperature calculations show the same behavior and tendencies observed experimentally, and the importance of accurate thermal properties and anatomy in the temperature prediction is observed. Based on these observations, a realistic geometric model of the human head obtained from tomographic data was created to study cooling and rewarming during brain ischemia produced by circulatory arrest and stroke. The calculations performed have helped to understand the importance of the tissue temperature gradients in the success of hypothermic therapies. The calculations show that hypothermic cardiopulmonary bypass (CPB) together with external head cooling help reduce the temperature gradients within the head during periods of reduced blood flow, and reduce the temperature increase in the deep tissue produced by the residual cerebral metabolic activity. The results of this research can be used in the refinement of cooling techniques used in the treatment of brain injury, ischemia, asphyxia and to improve organ protection during surgery.

An astonishing number of important physiological processes are regulated by the small alkaline earth metal, calcium. Regulatory Ca2+-binding proteins must be able to distinguish Ca2+ ions in the presence of greater concentrations of other metal cations, such as Mg2+, Na+ and K+. The EF-hand family is a large class of Ca2+-binding proteins that displays this sort of preferential Ca2+-binding. The secondary and tertiary structure of the EF-hand metal ion binding site is highly conserved from one member of the family to the next. Because of this conservation, we can use the small, amenable, EF-hand protein, parvalbumin, as a model system to study the mechanisms that define the metal ion affinities and specificities of EF-hand Ca2+-binding sites in general. Our collaborator, Dr. James Potter, has designed a mutant to test directly the role of the last coordinating residue in the EF-hand binding site, the PVEF-E101D parvalbumin mutant. The crystal structures of both the Ca 2+- and Mg2+-bound complexes of PVEF-E101D have been determined. The PVEF-E101D mutant displayed a 100-fold decrease in the binding affinity for Ca2+, and the Mg2+-binding affinity was increased 10-fold. Moreover, the Ca2+ off-rate escalated from 1 s--1 in wild-type parvalbumin to 600 s--1 in the PVEF-E101D mutant. The conformation of the mutated EF-hand in the PVEF-E101D/Mg2+ structure was typical of a Mg 2+-bound EF-hand, with the exception of an F helix movement of ∼1 A toward the bound cation that allowed the shorter aspartate residue to coordinate the Mg2+ ion. The PVEF-E101D/Ca2+ structure showed that the aspartate residue is unable to bind Ca2+ in the bidentate mode normally adopted by the wild type glutamate. The resulting sixfold Ca2+ coordination in the mutant is usually characteristic of Mg2+-bound EF-hands, and this finding indicates that the binding loop is not sufficiently flexible to allow the aspartate residue to move in far enough to offer bidentate ligation of the Ca2+ ion. Two MD simulations were used to further investigate the relationship between the last coordinating residue of the EF-hand binding loop and the overall plasticity and flexibility of the loop region. The first simulation, called Alchemy, simulated the transition from Ca2+ to Mg 2+ coordination through varying the van der Waals parameters for the bound metal ions. The glutamate at position 12 was accurately and reversibly predicted to be the source of bidentate ligation of Ca2+ in our simulations. A second simulation, the Aspartate simulation, produced results that correlated well with the experimental result that an E101D substitution at EF loop position 12 resulted in monodentate Ca2+ coordination. The F helix was able to move in to the binding cavity during the simulation to allow one aspartate oxygen to bind the Ca2+ ion, but the aspartate was unable to achieve a favorable orientation for bidentate Ca 2+ coordination. The findings indicate that the interplay between the last coordinating residue of the loop, and the plasticity, or flexibility, of the binding loop, to a great extent determines the species of cations that are allowed to bind in a particular EF-hand site.

The present study examines the role of liver macrophages (Kupffer cells), of C57BL/6 mice, as effector cells responsible for the killing of Entamoeba histolytica trophozoites in vitro. Interferon gamma (IFN-$ gamma$) and tumor necrosis factor alpha (TNF) were each shown to endow murine Kupffer cells with significant amoebicidal activity. Interferon gamma alone was not able to activate Kupffer cells to amoebicidal state. However, IFN-$ gamma$ and lipopolysaccharide (LPS) acted synergistically in this phenomenon. It seems that the acquisition of amoebicidal activity is associated with the involvement of hydrogen peroxide, because the addition of catalase partially decreases the killing of this parasite by Kupffer cells. In addition, it appears that the amoebicidal activity of IFN-$ gamma$-treated Kupffer cells is contact-dependent. / In our study, Kupffer cells were also shown to be activated by TNF in vitro to an amoebicidal state and this cytolytic effect depends upon the ratio of Kupffer cells to amoebae, the concentrations of TNF used, and the time of exposure of the cells and the parasites to TNF. / Our results indicate that the immunologic production of IFN-$ gamma$ and TNF is important in the activation of Kupffer cells for controlling this parasite and that Kupffer cells are strong effector cells against the amoebae.

The major part of this thesis is to obtain basic information in regard to changes of amino acids and middle molecular substances in fulminant hepatic failure (FHF) using a galactosamine induced rat model. The changes of these substances after hemoperfusion were also studied. / A generalized elevation of amino acid concentrations is demonstrated in the systemic blood plasma, portal plasma, CSF, cerebrum, liver, kidney and skeletal muscle tissues in GalN-FHF. The skeletal muscle is found to constitute the greatest source of accumulated amino acids in FHF. The other tissues also contribute to the increased amino acids in the body as a whole. The increase in aromatic amino acids (AAA) tyrosine, phenylalanine and free and total tryptophan were the most striking among all the amino acids, including branched chain amino acids (BCAA), in all the tissues studied. The molar ratio of BCAA/AAA was found significantly reduced in all the tissues studied. $ gamma$-amino butyric acid was found significantly increased in the cerebrum and the brain stem. Most of the increased tryptophan in plasma, and almost 100% of the increased tryptophan in the brain were in the free form (non-protein bound). The increase in tyrosine concentration in plasma was closely correlated with tyrosine in the brain. Hemoperfusion using collodion coated activated charcoal (CAC) significantly reduced a number of aromatic amino acids in the plasma. This was followed by a significant reduction of the AAA in CSF, but not in the brain, of GalN-FHF rats. Hemoperfusion using tyrosinase immobilized within artificial cells selectively reduced tyrosine in the plasma but did not influence the tyrosine level in the brain. Hemoperfusion procedures resulted in a high plasma clearance for the aromatic amino acids. The results also suggested the loss of the blood brain barrier for amino acids across the capillaries. However, transport mechanisms between brain cells themselves and interstitial fluid seems to be maintained. / The buildup of middle molecular metabolites (MW 500-2,000) was demonstrated as the elevation of middle molecule peak 7g in the plasma and brain extract samples from GalN-FHF rats using serial liquid chromatography. CAC hemoperfusion significantly reduced the levels of 7g fraction in both plasma and brain extract samples. The fraction of subpeak 7g was found to contain peptidic substances. SDS-polyacrylamide gel electrophoresis further revealed the peptide nature of some of the middle molecule substances. The estimated molecular weight of these peptides was in the range of 1,300 and 1,400. Radioimmunoassay study indicated the increase of substance P (MW 1,345) in the plasma of GalN-FHF rats.

BB rats spontaneously develop an insulin-dependent diabetes mellitus strikingly similar to the syndrome observed in man. The disorder requires the presence of multiple susceptibility genes and unknown environmental factors. At least one susceptibility gene resides within the u haplotype of the rat major histocompatibility complex (RT1). Restriction fragment length polymorphism analysis of genomic DNA from rats generated from a series of intercrosses between diabetic BB rats and Buffalo rats (RT1$ sp{ rm b})$ demonstrated that animals heterozygous throughout the RT1 developed IDDM. A single dose of the high risk allele was thus shown to be sufficient for the development of IDDM if other susceptibility factors are present. RFLP analysis of DNA from rats generated in three other breeding studies involving the r8 and r4 recombinant haplotypes mapped the IDDM susceptibility genes between the RT1.A and RT1.C loci, the immune response region. As the u regions of the various haplotypes used in these studies were not derived from the BB rat, the development of IDDM in the progeny strongly suggested that the MHC requirement for IDDM is only for a "u" allele and not a particular or "diabetogenic" u allele. / Analysis of the expression of MHC genes in isolated islets of age-matched BB and Wistar-Furth rats demonstrated enhanced class I MHC gene expression in the islets of prediabetic BB rats. Immunohistochemical analysis confirmed that enhanced class I expression was an early event during the pathogenesis of IDDM, and did not detect aberrant expression of class II antigen on beta cells. Investigation of the inducibility of class I and II MHC genes on the rat insulinoma cell line RIN5F by crude lymphokine preparations or recombinant gamma-interferon indicated that although both classes of genes were inducible, their kinetics of induction are quite different. In vitro nuclear transcriptions demonstrated that induction of the genes had a transcriptional basis. Although class II genes were induced by gamma-interferon, class II antigen was not detected by flow cytometric analysis.

The accumulation of eosinophils at sites of inflammation is one of the hallmarks of asthma. The aim of the present study was to investigate the involvement of PGD2 and the DP1 and DP2 receptors in eosinophil recruitment in vivo. In this project, a group of Brown Norway rats were administered intratracheally with PGD2, which activates both DP1 and DP2 receptors, as well as selective agonists of DP1 (BW245C) and DP2 (15R-methyl-PGD 2 and 13,14-dihydro-15-oxo-PGD2) receptors. In addition, we have also tested the effect of 15-deoxy-Delta12,14-PGJ 2 which known to activate PPARgamma and NF-kappaB, and DP2 receptors. Negative control animals received saline alone while positive control animals received 5-oxo-ETE. In this work, we have shown for the first time that PGD2 and selective DP2 receptor agonists induce pulmonary eosinophilia in vivo in the following order of potency; (15R-methyl-PGD 2 > PGD2 ≈ 15-deoxy-Delta12,14-PGJ 2 > dhk-PGD2). This effect was time dependent with the maximal response being observed after 24 h. Interestingly; this response was somehow diminished when higher dose was tested (10mug). This effect is most likely to be mediated solely through the DP2 receptor since this effect was not shared by the DP1 specific agonist BW245C. These results are consistent with an important role for PGD2 and the DP 2 receptor/CRTH2 in allergic diseases such as asthma and suggest that this receptor may be an important therapeutic target for these conditions.

Objective. To examine the association between non-steroidal anti-inflammatory drug (NSAID) use and end stage renal disease (ESRD) among hypertensive subjects. / Study design. We conducted a nested case-control study within a cohort of 77,887 hypertensive adult subjects within the province of Saskatchewan, Canada. / Outcome. The primary outcome was ESRD, defined by chronic dialysis or renal transplantation. / Exposure. NSAID exposure was determined using prescription records, for various time windows up to 10 years preceding the onset of end stage renal disease. / Statistical analysis. Rate ratios (RR) were estimated with 95% confidence intervals using conditional logistic regression, adjusting for potential confounding variables and stratified for effect modifiers. / Results. We identified 397 cases and 7,399 controls. In subjects followed for at least 10 years continuous NSAID use was observed in 20.8% of cases and 17.9% of controls (RR = 1.18, 95% CI 0.68--2.05). Additionally, neither early (RR = 1.10, 95% CI 0.50--2.41) nor late (RR = 0.81, 95% CI 0.32--2.04) NSAID exposure was associated with ESRD during this time period. Evaluation of other time windows (0--2 years, 2--5 years and 5--10 years) and NSAID dosing provided similar results. Results were not modified by loop diuretic and angiotensin converting enzyme inhibitor use. / Conclusion. Up to 10 years of non-steroidal anti-inflammatory drug use does not appear to influence the development of end stage renal disease. These results however may be influenced by unmeasured co-morbidities and confounding by "contra-indication".