Since macrophages play an important role in T cell activation, we investigated the role of CD14+ cells in T cell activation, proliferation and activation-induced cell death (AICD). Using PHA for activation, it was found that CD14+ cell depletion resulted in significantly greater AICD, decreased lymphocyte growth and increased IL-2 secretion. Lymphocyte activation was delayed as defined by CD69 and CD25 expression. ³H-TdR-incorporation was reduced in proportion to the percentage of AICD in the cultures. Addition of supernatants from activated CD14+ cells to CD14+ cell-depleted mononuclear cell cultures reversed the effects on AICD, IL-2 secretion and lymphocyte growth. Supernatants from TNF-α matured dendritic cells demonstrated the same activity as the CD14 cocktails. These data suggested a soluble survival signal from CD14+ cells that prevented apoptosis, maintaining an active immune response. Further studies demonstrated that depletion of CD14+ cells in rheumatoid arthritis (RA) patients resulted in greater AICD, decreased lymphocyte proliferation and even higher secretion of IL-2 compared with normal control subjects. While the CD14+ cell percentage in RA was not significantly higher than that in the age/sex-matched healthy controls, CD14 supernatants from RA showed a significantly greater protective effect on AICD. Data analysis revealed that CD14 supernatants supported the survival of activated lymphocytes in RA. These data underscored the importance of the soluble survival signal in the homeostasis of the immune system and in the pathogenesis of RA. The analysis of apoptotic cells showed that most of the apoptotic cells bore activation antigen CD69 and T cell antigen CD3, suggesting that most of the apoptotic cells were activated T lymphocytes. The soluble signal was also investigated. The CD14 cocktails contain IL-1β, TNF-α, TGF-β and IL-6, but not IL-12, or IL-15. Depletion assays using a panning method followed by blocking of residual IL-1β and TNF-α with corresponding monoclonal antibodies had no effect on the active AICD protection. As TGF-β was found in the culture medium containing 10% FBS, TGF-β might not be an active factor in the CD14 cocktails. Because the CD14 cocktails did not contain IL-12 or IL-15, these two cytokines were not the active factors either.

Sleep disordered-breathing (SDB) including obstructive sleep apnea syndrome (OSAS) is increasingly recognized as an important cause of morbidity in children. Clinical symptoms of OSAS in children include snoring, nocturnal arousals, restlessness during sleep, enuresis, daytime sleepiness and hyperactivity. Evidence also suggests that the adverse effects of SDB include behavioral, learning, and personality problems. No large epidemiological study using polysomnography has been conducted to determine the prevalence and correlates of SDB in young children. The Tucson Children's Assessment of Sleep Apnea study (TuCASA) is a prospective cohort study designed to determine the prevalence of objectively documented SDB in pre-adolescent children and to investigate its relationship to symptoms, performance on neurobehavioral measures, and physiologic and anatomic risk factors. Hispanic and Caucasian children were recruited to participate in TuCASA by soliciting the cooperation of elementary schools in the Tucson Unified School District (TUSD). Through the use of a screening survey completed by parents, the TuCASA study has shown that children between 4-11 years of age with learning problems (LP) are more likely to have habitual snoring (SN) and excessive daytime sleepiness (EDS). Additionally, Hispanic children in this age group are more likely to have parental report of EDS, witnessed apnea (WA), and SN. Similar to studies in adults, girls 4-11 years of age are more likely to have parental report of daytime sleepiness than boys. Furthermore, the TuCASA study has demonstrated the feasibility of collecting high quality unattended multi-channel polysomnography in children ages 5 to 12 years. More importantly, the TuCASA study has documented the relationships between respiratory disturbance indices based on polysomnography and parental report of clinical symptoms of SDB in children ages 6-11. There are threshold values of respiratory disturbance index (RDI) associated with an increase in the prevalence of clinical symptoms of SDB. Until now, data linking objective indices of RDI severity to the presence of clinical symptoms have been lacking. Additionally, these findings contribute much needed information for determining clinically significant levels of RDI based on differing definitions of respiratory events. Therefore, these results represent an important step towards examining the natural history of SDB and the relationship between SDB severity and specific clinical outcomes in pre-adolescent children.

Cardiovascular complications are prevalent in patients with AIDS. Cardiovascular complications, particularly ischemia-reperfusion injury, may be severe in AIDS patients. The pathology underlying cardiovascular complications in AIDS patients is unclear. Perhaps interplay of several pathologic factors amplifies the response to ischemia. Murine retrovirus (LP-BM5) induced murine AIDS is the best model of human AIDS research because LP-BM5 causes similar immune changes. Ethanol consumption has the advantage and disadvantage to health. The aim of this study was to determine if chronic ethanol consumption influences pathological changes caused by murine AIDS, specifically in cardiovascular complications, and if vitamin E supplementation could attenuate cardiovascular injury by murine AIDS. In our present study, we found that retrovirus infection enhanced neutrophil CD11b expression and ROS production, increased platelet CD62p and platelet microparticle formation, exaggerated coronary permeability to macromolecules and caused a severe myocardial ischemia-reperfusion injury. Chronic ethanol consumption down-regulated neutrophil CD11b expression, but neutrophil ROS production, platelet CD62p expression and platelet microparticle formation were enhanced. Chronic moderate ethanol consumption improved coronary microcirculation and attenuated ischemia-reperfusion injury. Our results indicate that neutrophil and platelet adhesion molecule expression increases in murine AIDS. Neutrophil and platelet-mediated severe ischemia-reperfusion injury may contribute to increased incidence of cardiomyopathy in AIDS. The cardiovascular protective effects of moderate ethanol consumption may be related to modulation of neutrophil CD11b expression and improve coronary microcirculation. However, chronic ethanol consumption did not preserve myocardial damage by retrovirus infection. In this study, we also demonstrated that vitamin E attenuated AIDS-induced myocardial injury. Vitamin E may be a therapeutic adjuvant agent for preventing and treatment AIDS-induced cardiovascular diseases.

Alzheimer's disease (AD) is the most important cause of dementia, but the mechanism of pathogenesis remains unknown. The epsilon4 allele of apolipoprotein E (apoE) has been confirmed as the most important genetic risk factor for most cases of AD and is associated with Abeta-containing senile plaques, one of the neuropathological hallmarks of AD. We determined the neuronal binding, internalization and degradation of human apoE isoforms apoE3 and apoE4 in the presence and absence of Abeta peptides using primary hippocampal neurons from rat. The findings demonstrate apoE binding and internalization in neurons without endogenous synthesis. Increased apoE3 degradation was observed relative to the apoE4 isoform. Lipophilic Abeta peptides increased apoE binding to hippocampal neurons; the increase was greater for the apoE4 isoform than for the apoE3 isoform. Abeta uptake by hippocampal neurons was increased in the presence of apoE, more so in the presence of the apoE4 than apoE3 isoform. Further, apoE was shown to reduce the extracellular level of Abeta in hippocampal cultures. We also find that all three major apoE isoforms were equally potent inhibitors of Abeta fibril formation, which is thought to be necessary for in vitro neurotoxicity and possibly the neuronal degeneration observed in AD. Further, when apoE was associated with cholesterol-containing liposomes or very low density lipoproteins, its ability to prevent Abeta fibril formation was diminished. In two brain regions affected by AD neuropathology, the hippocampus and frontal cortex, apoE levels were found to be decreased while Abeta levels were increased. Levels of apoE and Abeta1--40 were found to be apoE genotype dependent, with lowest levels of apoE and highest levels of Abeta occurring in epsilon4 allele carriers, suggesting that a reduction in apoE levels may give rise to increased deposition of amyloid peptides in AD brain. The low density lipoprotein receptor-related protein, an important neuronal ap

A series of studies were undertaken with the aim of assessing the static and dynamic profiles of the most common types of epileptogenic lesions: hippocampal sclerosis and cortical developmental malformations. Neuronal metabolic dysfunction measured by proton magnetic resonance spectroscopic imaging (1H-MRSI) overlaps the structural lesion displayed by magnetic resonance imaging (MRI), The extent of neuronal metabolic dysfunction, however, tends to be wider than the MRI-visible lesion and may reflect the intrinsic nature and extent of the original epileptogenic damage. In addition, neuronal metabolic dysfunction and synchronized neuronal firing often coincide spatially and vary together in intensity possibly reflecting the severity of the epileptogenic process. / Non-foreign tissue lesional, temporal lobe epilepsy (TLE) syndrome is a heterogeneous condition, which displays a spectrum of neuronal damage. The different patterns of neuronal damage measured by MRI volumetry (MRIVol) and 1H-MRSI enable accurate probabilistic prediction of TLE lateralization and discrimination of TLE from extra-TLE. Furthermore, both MRIVol and 1H-MRSI have a prognostic value in surgical TLE patients, which can be used to streamline surgical candidates. / The neuronal damage is present in the early stages of the epileptogenic process in patients with localization related epilepsy. This process is dynamic and shows a slow progressive neuronal loss and dysfunction in TLE patients, which is not related to seizure burden. Neuronal metabolic dysfunction, lesions, spikes, cognitive decline, and psychiatric disorders are part of the epileptogenic process. These different domains parallel each other in a given time, although their pathophysiological processes are distinct. Thus seizures and neuronal damage co-exist but are not causally related. / Normalization of neuronal metabolic function is seen in post-operative seizure-free patients, with a recovery half time of six months. However, the process of neuronal recovery does not occur in patients who are seizure-free due to antiepileptic medication. The epileptogenic process causes disruption of normal neuronal network and in order to reverse this disruption the epileptogenic area must be isolated or resected surgically. The epileptic state is a translation in time of the activity of the epileptogenic process. Seizures, stereotyped behavioral manifestations, are the hallmark of the epileptogenic process, Absence of seizures, however, does not reflect inactivity of the epileptogenic process, Neuronal damage as measured by NAA/Cr can serve as a surrogate marker of the epileptogenic state.

The phosphodiesterase (PDE) inhibitors Pentoxifylline (PTX), a general inhibitor, and Rolipram (ROL), a type 4 inhibitor, have been shown to have anti-inflammatory effects. They elevate intracellular CAMP levels and suppress the production of inflammatory cytokines such as tumor necrosis factor (TNF)alpha, interferon (IFN)gamma, and interleukin-12 (IL-12). These drugs have also been reported to modulate the immune response in favor of Th2 responses and to be therapeutically effective in various models of autoimmune and/or inflammatory disorders. Their effects on nitric oxide (NO) production are not well studied. Inflammatory cytokines and NO are important mediators implicated in islet beta-cell destruction. / In the first part of the study, we examined the effect of PTX and ROL in preventing insulitis and diabetes in non-obese diabetes-prone (NOD) mice as a spontaneous model of insulin-dependent diabetes (IDDM). We found that a 4 week treatment with either PTX or ROL had a strong protective effect, that was still apparent 11 weeks after withdrawing the drugs. Both drugs were equally effective at optimal doses in preventing mstiulitis and diabetes in NOD mice. / In the second part of the study, we examined the effects of PDE inhibitors on NO production by peritoneal macrophages and RAW 246.7 cells. We also correlated these effects with elevated cAMP levels. We found that both PTX and ROL suppress NO production by IFNgamma and lipopolysaccharide (LPS)-activated macrophages. The inhibitory effects correlated with elevated CAMP levels and were mimicked by other agents which elevate CAMP levels such as dibutyryl cAMP, 8-bromo cAMP, and Forskolin. This suppression was found to be at the transcriptional level. In vivo, ROL treatment prevented macrophage activation by staphylococcal enterotoxin B (SEB) and suppressed NO production by these macrophages in ex vivo culture. / In the third part of the study, we examined the effects of PDE inhibitors on NO production by insulin-producing NIT-1 insulinoma cells and normal islet cells. It has been reported that islet beta-cells express PDE3 and PDE4. We found that inhibitors of PDE4 (ROL), PDE3 (Cilostamide; CIL), or a general inhibitor (PTX), suppressed NO production by islet cells. A combination of ROL and CIL appeared to have more than an additive effect, suggesting synergism. Like in macrophages, the suppression was at the transcriptional level and mimicked by other agents which elevate cAMP levels. In vivo, ROL treatment suppress iNOS expression in the islets of NOD mice with cyclophosphamide-accelerated disease, as determined by immunohistochemistry. / These studies establish for the first time that PDE inhibitors have a therapeutic potential in IDDM and other NO-and/or cytokine-mediated inflammatory disorders.

The studies presented in this thesis investigated the mechanism responsible for glucococorticoid secretion during graft-versus-host disease (GVHD), and the role of endogenous glucocorticoids on the outcome of the disease. GVHD was induced in unirradiated F1 hybrid mice by an intravenous injection of parental lymphoid cells. Our results demonstrated that the secretion of glucocorticoids during GVHD was independent of pituitary adrenocorticotropin hormone (ACTH). However, adrenal hyperactivity was associated with increased expression of proopiomelanocortin (POMC) mRNA in the adrenal glands of GVHD mice. Expression of adrenal POMC transcripts was not due to mononuclear infiltrates. The transcripts for interleukin-12, a cytokine produced by activated macrophages, were also upregulated in GVHD adrenals. Since macrophages have been shown to reside in the adrenal glands and produce ACTH, it appeared that resident adrenal macrophages were activated during GVHD to produce local ACTH that stimulated the secretion of glucocorticoids, independent of pituitary ACTH. / We next investigated the role of endogenous glucocorticoids on the outcome of GVHD by adrenalectomizing the F1 recipient mice before GVHD induction in order to deplete the source of glucocorticoids. Our results showed that adrenalectomized (ADX), but not non-ADX, F1 recipients injected with parental lymphoid cells recovered rapidly from symptoms characteristic of GVHD, after a two week manifestation of the disease. Recovery from GVHD was attributed to the induction of a glucocorticoid sensitive, asialoGM1$ sp+$ and/or CD8$ sp+$, but not NK1.1$ sp+$, F1-anti-parental effector cell that rejected or eliminated the parental graft, after an initial period of engraftment. In addition, the effector was not dependent on a mature thymus and was not renewed after anti-asialoGM1 treatment, but was renewed after glucocorticoid treatment. / We further demonstrated that high levels of glucocorticoids during GVHD caused severe deficiency of host T cell populations in the lymph nodes and contributed to the suppression of lymph nodes T cells. Taken together these studies suggest that endogenous glucocorticoids play a central role in the pathogenesis of GVHD.

The Bcr-abl translocation arises from a reciprocal translocation between chromosomes 9 and 22 and results in the augmentation of the tyrosine kinase activity of c-Abl. Chronic myelogenous leukemia (CML) is one of several hematological malignancies associated with Bcr-abl expression. The pathogenesis of CML, associated with P210Bcr-abl, is bi-phasic consisting of an initial chronic phase followed by a severe terminal phase referred to as acute blast crisis. The chronic phase of the disease is characterized by granulocytic hyperplasia but in which normal hematologic maturation is still intact. Patients ultimately enter the terminal blast crisis where hematologic maturation is lost, resulting in the accumulation of immature blast cells and a severe immuno-compromised state. Progression to the blast terminal phase is associated with genomic instability demonstrated by the accumulation of genetic and cytogenetic abnormalities. Results from in vitro cell line systems expressing Bcr-abl have suggested that the loss of cell-cycle arrest and induction of apoptosis, as a result of genotoxic stress, might be responsible for this phenotype. In this study, I utilized a transgenic mouse model which expresses P190Bcr-abl to extend those observations to an in vivo model for leukemia. I observed normal cell-cycle arrest and induction of apoptosis following the induction of DNA damage. However, using the Big Blue in vivo mutagenesis mouse assay system, I evaluated genomic instability in P190Bcr-abl mice by measuring mutation frequencies in vivo. I observed an increase in mutation frequencies in spleens and kidneys from P190Bcr-abl mice. This Bcr-abl-induced mutator phenotype may explain the inherent genomic instability associated with the progression of CML and other diseases associated with the expression of activated tyrosine kinases.

Haemophilus influenzae (Hi) is a Gram-negative bacteria that is the causative agent of bacterial meningitis. The outer membrane (OM) of Gram-negative bacteria functions as a selectively permeable barrier. The exchange of small hydrophilic solutes between the external environment and the periplasm is mediated by large water-filled channels, porins. Charged residues of the pore determine the functional properties of the protein, which include: ion conductance, ionic selectivity, and voltage gating. / To study the properties of the porin (341 amino acids; Mr 37,782) of Haemophilus influenzae type b (Hib), purified porin was subjected to chemical modification. The covalent modification of lysine residues with succinic anhydride (SA; Mr 100.08) results in charge reversal. The addition of up to 12 succinate groups per porin molecule was identified using electrospray ionization mass spectrometry (MS). Tryptic digestion of the modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight MS identified the sites of succinylation. The majority of modified lysines were positioned in surface-located loops, numbers 1 and 4 to 7. When the electrophysiological properties of SA-modified porin were analyzed in planar lipid bilayers (PLBs) and compared to Hib porin it was found that the single channel conductance was increased, while the threshold for voltage gating was decreased. The addition of extra negative charges increase the single channel conductance of Hib porin and function as voltage sensors. / Selected lysine residues that were found to be modified with SA were substituted with glutamic acid using site-directed mutagenesis. Single point mutations were made in a residue assigned to the barrel lumen and to three residues in each of loops 4 and 6. The mutant Hib porins had increased single channel conductances relative to wild-type Hib porin. Voltage gating of mutant Hib porins was altered by the introduction of negative charges into loops 4 and 6 and in the barrel lumen. Previous experiments had implicated surface-exposed loop 4 in voltage gating. This study ascribes a role for residues in loop 6 and a residue within the barrel lumen in the changes that accompany pore closure. / Hi strains causing infection in cystic fibrosis patients are capable of persistent infection despite prolonged antibiotic treatment with beta-lactam antibiotics. During the course of infection porin properties may be altered due to the changes in porin sequences that are attributed to antigenic drift. The electrophysiological properties of four porins from CF patient-derived Hi strains were characterized to examine changes in porin properties arising from persistent infection of the CF lung. The clinical Hi porins displayed altered channel properties that included increased voltage sensitivity and single channel conductances that were either greater or smaller than that of Hib porin. The decreased single channel conductance of one of the porins was associated with an increase in the minimal inhibitory concentration of the antibiotics novobiocin and streptomycin. These results demonstrate a porin-mediated decrease in OM permeability as an antibiotic resistance mechanism for Hi.

Anemia is an almost invariable manifestation of chronic renal failure (CRF) and it often contributes substantially to the morbidity of the condition. In its uncomplicated form, this anemia is due primarily to reduced production of erythropoietin (EPO) by the diseased kidney. The present study was carried out in order to determine the relative role in the anemia secondary to CRF of EPO and insulin-like growth factor-1 (IGF-1), a recently recognized important regulatory factor of erythropoiesis in the normal physiological state. / A mouse model of CRF was employed in this investigation. Six weeks after the surgical induction of renal failure, the mice were characterized in terms of biochemical and hematological parameters which included the response to a 3-week treatment with recombinant human EPO (r-HuEPO). Additionally the kidneys, liver and bone marrow were harvested for the determination of the mRNA expression of EPO and IGF-1 as assessed by the reverse transcription polymerase chain reaction followed by Southern blotting. Normal mice and mice rendered anemic by phlebotomy were included in all experiments. (Abstract shortened by UMI.)