A novel test system using isolated erythrocytes (red blood cells, RBCs) and arsine (AsH₃ gas dissolved in aqueous solution was characterized which allows for the quantitation of RBC exposure to AsH₃ in vitro and for in vitro study of the toxicity of AsH₃. AsH₃ was found to be rapidly and strongly associated with RBCs. Toxicity, measured as hemolysis, was time- and dose-dependent and exhibited a lag phase of about 30 minutes in both dog and rat RBCs. Hemolysis of dog RBCs was completely blocked by carbon monoxide preincubation and was reduced by pure oxygen. Sodium nitrite induction of methemoglobin (metHb) in intact rat RBCs decreased hemolysis, confirming the importance of hemoglobin in the mechanism of AsH₃-induced hemolysis. Spectrophotometric studies of the reaction of AsH₃ with purified dog hemoglobin revealed that AsH₃ reacted with reduced and oxygenated hemoglobin (HbO₂) to produce metHb and degraded Hb, characterized by gross precipitation of the protein. AsH₃ did not alter the spectrum of deoxyHb and did not cause degradation of metHb in oxygen, but bound to and reduced metHb in the absence of oxygen. Oxidative reactions as the putative cause of hemolysis were investigated. AsH₃ caused a slight decrease in cellular glutathione levels in dog RBCs, but only after hemolysis had reached maximum levels. Hydrogen peroxide (H₂O₂) was detected in aqueous solutions containing AsH₃ and HbO₂ or AsH₃ alone but not in intact red blood cells or lysates suggesting that cellular defenses were adequate to detoxify the amount formed. Additionally, high activity catalase or glutathione peroxidase added to solutions of HbO₂ and AsH₃ had little effect against AsH₃-induced damage. The differences between the visible spectra of HbO₂ treated with either AsH₃ or H₂O₂ suggest that two different degradative processes occur. Superoxide anion was not detected in AsH₃ mixtures and superoxide dismutase did not affect AsH₃-induced HbO₂ damage. Other antioxidants, mannitol, DMSO, ascorbate, and glutathione, had no effect against HbO₂ damage. These results indicate that the superoxide anion, the hydroxyl radical, and H₂O₂ are not responsible for AsH₃-induced HbO₂ damage and the subsequent hemolysis. A mechanism by which an arsenic species is the hemolytic agent is proposed.

Hepatic granulomas are induced by a single intraperitoneal injection of streptococcal cell wall (SCW) into female Lewis rats. This inflammatory response has a biphasic course with an early accumulation of polymorphonuclear leukocytes in the liver followed by infiltration of mononuclear phagocytes. The expression of adhesion molecules may contribute to the recruitment of leukocytes to the liver. Therefore, in this study I investigated the expression and regulation of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) on liver endothelial cells in a SCW rat model and in primary endothelial cell cultures treated with cytokines IL-1β, IL-6, IFN-γ, TNF-α and lipopolysaccharide (LPS). Immunohistochemical staining demonstrated that ICAM-1 was constitutively expressed on sinusoidal lining cells. Enhanced ICAM-1 expression was observed 3, 14 and 28 days after SCW injection. There was a gradation of ICAM-1 staining intensity with the strongest staining in periportal area. In primary endothelial cell culture, cells up-regulated surface ICAM-1 expression in response to cytokines in the following order: IL-1β + IFN-γ + TNF-α > IFN-γ + TNF-α > TNF-α + IL-1β or IFN-γ + IL-1β > TNF-α >IFN-γ > IL-1β. ICAM-1 was also up-regulated by LPS. Cytokine and LPS activated liver endothelial cells also increased ICAM-1 mRNA expression. The induction profile of ICAM-1 mRNA was identical to the profile of ICAM-1 expression on the cell surface. This parallel suggests that the increased surface ICAM-1 expression is associated with the increased ICAM-1 mRNA level. Interestingly, IL-6 had no effect on either surface ICAM-1 or mRNA production. Primary endothelial cell culture constitutively expressed both 7 domain (3.7 kb) and 3 domain (1.6 kb) VCAM-1 mRNAs. IL-1β, IL-6 and IFN-γ did not significantly affect VCAM-1 mRNA expression. TNF-α and LPS up-regulated 7 domain mRNA level 6 and 10 fold respectively with no effects on 3 domain mRNA expression. The most striking affects were induced by IFN-γ + TNF-α. This study suggests that the expression of ICAM-1 and VCAM-1 on liver endothelial cells could play an important role in the recruitment of leukocytes into the liver during SCW-induced hepatic inflammation. TNF-α was found to be produced by endothelial cells and may be one of the reasons for the chronicity of this lesion.

LP-BM5 murine leukemia retrovirus infection in C57BL/6 mice rapidly induces murine AIDS with many functional similarities to human AIDS, including progressive lymphoproliferation and severe immunodeficiency. The present studies indicate that retrovirus infection induces immune dysfunctions mostly via modulating T and B cell proliferation, natural killer cell toxicity, and cytokine secretion. In addition, retrovirus infection causes oxidative damage via nuclear factor κB (NF-κB) activation, excessive free radicals production, and antioxidant deficiency. Such oxidative damage has the theoretical potential to accelerate the development of AIDS via immunosuppression secondary to antioxidant deficiency. Synthesized T cell receptor (TCR) peptides treatment largely blocks the excessive stimulation of a T cell subset by retroviral superantigens, normalizes the retrovirus-induced aberrant cytokines production, thereby significantly ameliorates immune dysfunction, and prevents excessive lipid peroxidation and antioxidant deficiency in murine AIDS. Results from the current studies suggest that dietary vitamin E supplementation significantly prevents the dysregulation of cytokines production, excessive tissue lipid peroxidation, and vitamin E deficiency. Furthermore, vitamin E may play an important role in inhibiting the NF-κB activation which results from the retrovirus infection. In conclusion, TCR peptide treatment and vitamin E supplementation effectively prevents immunodysfunction, excessive lipid peroxidation and free radical production, and antioxidant deficiency during murine AIDS. This may provide additional therapeutic approaches for treatment of human AIDS without additional immunotoxicity.

This study was an investigation of early changes in long latency event-related potentials with an emphasis on the N200/P300 complex in a group of adults with mild traumatic brain injury (TBI). Subjects with mild TBI and a matched group of non-injured subjects were presented three auditory oddball tasks differing in degree of difficulty. Subjects with TBI were tested within 100 hours of the injury and again at 20 days post-injury. Non-injured subjects also underwent two test sessions, with visit two occurring 18 days after visit one. Event-related potentials were recorded from three midline sites (Fz, Cz, Pz) during the three oddball tasks, two tone-frequency discrimination tasks and one tone-duration discrimination task. The amplitude and latency of both the N200 and the P300 were compared between the two groups. The amplitudes of the two components did not differ significantly between the two groups. However, the latencies of both the N200 and the P300 were prolonged in the mild TBI group. This delay interacted significantly with recording site, with the maximal between-group difference occurring at Fz for both components. The group effect did not interact significantly with the timing of the test session or the difficulty of the oddball task. Taking the latencies of the two components as indices of information processing speed, the data suggest the presence of reduced processing speed in the mild TBI group that persists for at least three weeks post-injury. Increasing task difficulty, at least to the level used in the present study, did not enhance the observed difference between the two groups. The findings related to recording site are consistent with neurobehavioral, neuroimaging, and pathophysiological data which indicate greater effects of injury on frontal regions of the brain.

AIDS, or acquired immunodeficiency syndrome, is caused by the human immunodeficiency virus (HIV). HIV is a retrovirus that is capable of rapid genetic mutation, which makes the virus and the disease difficult to treat. Several drug therapies are currently available, in the form of viral enzyme inhibitors. Other inhibitors of the viral entry and replication process are being investigated to enhance the drug therapy arsenal. Our research has focused on the development of HIV entry inhibitors. We are working towards the development of novel carbohydrate-based agents that are capable of binding the gp120 protein on the viral surface, such that viral entry into an uninfected host cell is prevented. In order for our research to progress, a qualitative method by which our synthetic compounds could be evaluated for gp120 binding was sought. We have developed a unique ELISA (enzyme-linked immunosorbent assay) that indicates whether or not a compound has binding affinity for the viral protein. A TIRF (total internal reflection fluorescence) microscopy method, has been developed as part of a collaborative effort with the laboratories of Professors Saavedra and O'Brien, to assess active compounds for quantitative equilibrium binding constants to gp120. We have synthesized several carbohydrate-based molecules targeted to one or more of the binding sites on the surface of gp120; the galactosylceramide site, the V3 loop, and the CD4 binding site. Utilizing both the ELISA and TIRF methods, we have succeeded in probing the binding profile of gp120. Circular dichroism studies have also been employed to evaluate the secondary structural characteristics of oligomeric carbohydrate materials. Molecules with helical properties have potential as CD4 binding site inhibitors. The long term goals of this project involve the synthesis and gp120 binding evaluation of novel carbohydrate-based materials to serve as entry inhibitors of the HIV replication process. A possible application of this project lies in the development of compounds capable of binding to more than one site on the protein. A variation of this goal involves the tethering of various compounds with specificities to different sites on gp120, for the purpose of inhibiting multiple binding sites on the protein.

This Ph.D. dissertation presents results that shed light into the fundamental question of whether there is an epidemiological association between asthma and the subsequent development of chronic obstructive pulmonary disease (COPD). This question is investigated in the context of a longitudinal study that establishes asthma as a predictor for the future development of characteristics consistent with COPD. Asthmatic subjects without chronic bronchitis or emphysema diagnoses were identified at the initial survey and followed longitudinally to assess the development of COPD. Longstanding asthma has been proposed as a possible contributing factor to the risk of developing COPD. To evaluate this hypothesis, a question concerning the association between asthma duration and asthma persistence with lower lung function and odds of cough and sputum in the elderly is also investigated. This second question is addressed by means of comparative analyses of immunologic and pulmonary characteristics of elderly versus younger non-smoking asthmatics. In addition, this work investigates the role of genetic factors that may predispose to the development of COPD, in particular, the association between alpha 1-antitrypsin deficiency (PiMZ and PiMS) and rapid decline in pulmonary function. This work utilizes data derived from the Tucson Epidemiologic Study of Airways Obstructive Diseases (TESAOD), initiated in 1971. Results show a significant association between an active asthma diagnosis at initial survey and subsequent development of signs and symptoms consistent with COPD. Results show as well, that longstanding asthma is associated with lower pulmonary function, and that this association is stronger for older persistent asthmatics compared to younger asthmatics. In addition, subjects with shorter asthma duration have higher risk for cough and sputum than non-asthmatics or those with longer asthma duration. Furthermore, no association between rapid decline in pulmonary function and the PiMZ or PiMS phenotypes is seen. These results support the implied relationship of asthma with COPD proposed in the Dutch hypothesis, where despite the known differences between asthma and COPD at initial diagnosis, over time the two diseases develop signs and symptoms that overlap. However, the proposed involvement in this association of heterozygous forms of alpha1-antitrypsin by other studies was not supported in the present research.

Matrix metalloproteinases (MMPs) are involved in many normal and pathological processes that require remodeling of the extracellular matrix. In this dissertation, the distribution of MMPs in human prostate tissue was determined. Matrilysin localized to epithelial cells in prostate ducts surrounded by inflammatory cells, and was focally expressed in carcinoma and prostatic intraepithelial neoplasia, but not in normal glands. Gelatinase A was detected in both benign and malignant prostate tissue in similar amounts. MT-MMP1, an activator of progelatinase A, was present in 100% of the carcinomas, in 88% of the cases with PIN lesions, but only in 34% of the normal glands. Matrilysin converted gelatinase/TIMP-complexes and free gelatinase B into polypeptides with gelatinolytic activity. In contrast, matrilysin was unable to proteolytically cleave gelatinase A/TIMP2 complex, but led to a transient increase in gelatinolytic activity of the proenzyme. Active matrilysin did not enhance the autocatalytic conversion of its own proform. Using indirect immunofluorescence microscopy, degradation of the fibronectin fibrils produced fibroblasts by matrilysin was demonstrated. Fibronectin fibrils represent a major component encountered by tumor cells during invasion. Removal of matrilysin resulted in regrowth of the fibrils, suggesting that matrilysin was not cytotoxic. Stable fragments derived from the gelatin-binding, the heparin-binding, and the cell attachment domains, respectively, of fibronectin, were identified. Their isolation may allow further studies on their influence on cell migration, attachment and signal transduction which are expected to be different from the effects of undegraded fibronectin. Effects of matrilysin on integrins were also investigated. Incubation of beta4, but not of alpha6 or beta1, with matrilysin, resulted in complete degradation in vitro. Thereby a specific fragment of 90 kD was generated, which was not observed with calpain or trypsin. Two putative cleavage sites for matrilysin at residues 107 (isoleucine) and 417 (leucine) located within the extracellular domain of the beta4 were identified by sequence comparisons with known substrates. Degradation of beta4 by matrilysin may partly explain the loss of beta4 integrin in prostate carcinoma. Taken together, the data presented here demonstrate effects of matrilysin on a variety of processes important in carcinogenesis.

Telepathology systems were developed to allow pathologists to perform remote diagnosis and consultations. These systems have the following essential components; a remote controlled microscope, a video processing system, a communication link, and a user interface and display. This thesis defines a telepathology communication system based on the Internet network. The basic telepathology system problems were addressed: (1) Determining available network bandwidth, (2) Investigating video image compression, (3) Developing a user interface and display. A software model of the telepathology system was tested. Results demonstrated that, depending on the location on the Internet, only about 40 Kbps and 1.1 Mbps of bandwidth can be obtained. Using an image compression algorithm that reduces images to one twentieth their original size does not reduce bandwidth requirements enough to have real-time video image updates. Current capabilities are about 5 frames per second. A 5 Mbps network is required for full motion video.

There is evidence that amyloid deposition within the brain is of major importance in the pathogenesis of Alzheimer's disease (AD), but the mechanism of this deposition is not known, and several theories are currently under great debate. The present study of intraneuronal changes in the human brain with age and disease suggests a new explanation for amyloid plaque formation in the pathogenesis of AD. / This work indicates that intraneuronal inclusions originally observed by Rees (1975), may contain amyloid peptide (Abeta), the beta-pleated form of which is the major component of the amyloid plaques in AD. We have therefore called them amylosomes. Using the techniques of histology, immunohistochemistry, computerized morphology, as well as protein isolation and analysis, we examined the distribution, size, density and chemical composition of amylosomes in the human brain throughout life. Amylosomes appear during early childhood and remain constant in size and number until advanced old age. Their protein content and immunostaining characteristics indicate they contain Abeta, and their dendritic location in regions where AD plaques form suggested a possible role in Abeta transport from the brain to the CSF. We therefore isolated and quantified human brain microtubule proteins (MT) and the dendritic microtubule-associated protein 2 (MAP2), assessed their ability to polymerize in vitro, quantified the MAP2 mRNA with age, and related these results to the apolipoprotein E (APOE) genotype, which is known to modulate the risk of AD. There was an age-related decrease in MT proteins and MAP2 and in their ability to polymerize, that was accelerated in individuals with APOE epsilon4 allele(s), the group at greatest risk of AD. These results are consistent with the proposal that amylosomes play a role in Abeta metabolism, and an age-related decrease in the dendritic MT transport system could lead to retention of amylosomes, within the brain, with deposition of the Abeta and plaque formation as a consequence. This theory can account for most of the important features of AD, and resolves a number of contradictory hypotheses, as well as suggesting relevant area for further investigation. A better understanding of the pathogenesis will hopefully reveal strategies for prevention and treatment of this common and presently incurable disease.

The proper regulation of Hox gene expression during development is crucial for normal patterning to occur along the anterior-posterior axis of the embryo. Misexpression of Hox genes often results in homeotic transformations whereby structures located in anterior regions of the embryo resemble posterior-derived structures. In vertebrate embryos, treatment with retinoic acid (RA) during specific gestation times can anteriorize the borders of Hox gene expression resulting in homeotic transformations. RA exerts transcriptional control through nuclear receptors of the RXR and RAR families by binding DNA as a heterodimer at an RA response element (RARE). Previously, our laboratory characterized an RARE in the neural enhancer of the murine Hoxd4 gene that is strictly required for its activity (Zhang et al., 2000). Cloning and sequencing of the zebrafish Hoxd4 gene reveals that the sequence and spacing of this RARE is highly conserved as well as the sequences that flank it. Other RAREs have been identified in Hox genes that are required for their expression in the developing hindbrain. The sequence and, in some cases, the spacing of these Hox RAREs are different. These differences may affect the affinity of the RXR•RAR heterodimer that bind to them which in turn may determine where their anterior borders of expression are set in the hindbrain. In order to test this hypothesis we alter the spacing and sequence of the RARE in the Hoxd4 neural enhancer. Changes that increase the heterodimer affinity for the RARE do not anteriorize transgene expression, whereas changes that decrease heterodimer affinity posteriorized transgene expression. Thus, the setting of the Hox anterior borders of expression is not a simple function of heterodimer affinity for their cognate elements. Since the sequences that flank the Hoxd4 RARE are highly conserved, they may also play a role in neural expression of Hoxd4 by recruiting factors that cooperatively interact with the RXR•RAR hete