Melanoma-associated antigen (MAA) MART-1/Melan-A has been described in human cutaneous melanoma. The first objective of this study was to investigate the expressivity of MART-1/Melan-A in primary uveal melanomas. 56 formalin-fixed, paraffin-embedded human uveal melanomas were analyzed by immunohistochemistry. MART-1/Melan-A was found to be positive in 73% of cases, with almost 50% of tumours showing a diffuse involvement. The second objective was to investigate the expression of MAA Mart-1/Melan-A and gp100 in primary and metastatic human uveal melanoma lesions developed in albino rabbits. MART-1/Melan-A and gp100 were expressed in all primary lesions, with mainly focal distribution. Conversely, MART-1/Melan-A and gp100 exhibited a homogeneous pattern in the metastatic lesions, which implies upregulation of these antigens during disease progression. It may be postulated that MART-1/Melan-A and gp100 would be effective immunomarkers for immunotherapeutic strategies in human uveal melanoma.

<p> Ischemic heart disease impacts millions worldwide and can progress to heart failure. Percutaneous coronary intervention (PCI) is first-line therapy for patients presenting with an acute ischemic event or acute coronary syndrome (ACS). However, PCI can also worsen cardiomyocyte death, cardiac dysfunction and adverse remodeling via reperfusion injury, largely an oxidative stress-mediated insult. Novel alternative therapies for ACS have proven elusive, with no new classes of agents in years. We investigated cardiac troponin I-interacting kinase (TNNI3K), a cardiomyocyte-specific kinase, as a potential modulator of ischemia/reperfusion (I/R) injury and chronic left ventricular (LV remodeling). We found TNNI3K enhances production of mitochondrial reactive oxygen species (mROS) and induces mitochondrial dysfunction, thus increasing cardiomyocyte death and I/R injury. Moreover, TNNI3K-mediated injury is largely dependent on p38 MAPK activation. We developed a series of small-molecule TNNI3K inhibitors that reduce mitochondrial-derived superoxide generation, p38 activation, and infarct size when delivered at reperfusion to mimic ACS intervention. Moreover, although TNNI3K inhibition does not modulate the adverse remodeling that occurs after a non-reperfused myocardial infarction (MI), TNNI3K inhibition preserves cardiac function and limits chronic adverse remodeling in a model of MI with reperfusion. Taken together, TNNI3K plays an adverse role in the cardiomyocyte response to I/R, in part by driving mROS production and augmenting p38-mediated cell death specifically via reperfusion injury. Our findings reveal a previously unexplored role for TNNI3K in regulating the oxidative stress response in the heart, and support the potential for TNNI3K as a novel therapeutic target for ACS.</p>

Atherosclerosis, a vascular disease which may lead to coronary artery occlusion and consequent myocardial infarction is primarily caused by dyslipidemia. Ischemic cardiomyopathy due to atherosclerosis is the leading cause of morbidity and mortality in the western world today. Vasoactive factors are increasingly being recognized not only as contributors to atherosclerotic plaque formation, but also to cardiac function and remodeling following ischemic cardiac injury. Urotensin II (UII) is one such vasoactive factor. UII possesses a wide range of cardiovascular effects, from contraction of the rat aorta to complete cardiovascular collapse in cynomolgus monkeys. UII binds a seven transmembrane spanning G-protein coupled receptor termed UT. Expression of UII is significantly elevated in the hearts of patients with congestive heart failure (CHF). Recent reports have also shown increased plasma levels of the peptide in patients with CHF, and these levels correlated with the severity of the disease. This project was designed to investigate the role of UII and UT in both atherosclerosis and CHF. To this end, UII expression was evaluated both in a model of CHF in the rat, and in human atherosclerosis of the carotid arteries and aortae. Furthermore, the pathophysiological role of urotensin-I1 in CHF was investigated, with the use of a selective UII antagonist, SB-611812. Finally, genetically modified mice deficient in either ApoE, UT, or both genes, were evaluated to study the role of UII/UT signaling in a model of atherosclerosis. We found that UII and its receptor, UT, were both significantly elevated in a model of CHF induced by coronary artery ligation. UII antagonism significantly attenuated mortality, cardiac dysfunction, and hypertrophy. This was associated with a significant decrease in cardiac fibrosis. We next went on to demonstrate that UII and UT were significantly elevated in human atherosclerotic carotid arteries and aortae. Finally, we demonstrated that deletion of the UT gene in mice deficient for ApoE exacerbates atherosclerosis of the aorta. Furthermore, this was associated with significantly increased hyperlipidemia and organ hypertrophy as well as significantly reduced body mass, liver mass, and hepatic steatosis. / In conclusion we were the first to demonstrate a pathophysiological role for UII in cardiovascular diseases which may lead to a breakthrough in the management of CHF and may also give more insight into the pathogenesis of atherosclerosis.

The present experiments were carried out to test the hypothesis that NC is involved in nicotinic receptor mediated muscle cell degeneration. As a model to study nicotinic receptor mediated myopathy, neonatal skeletal muscle cultures prepared from one day old Sprague-Dawley pups were used. Skeletal muscle cultures were exposed to different concentrations of nicotine. The results demonstrate a significant dose dependent decrease in the number of muscle branch points with increasing nicotine concentrations. The degenerative effect(s) of nicotine were prevented by preincubating the muscle cultures with d-tubocurarine, a nicotinic receptor blocker, suggesting that the effects of nicotine are receptor mediated. Experiments were then done to assess an involvement of the NO generating system in the nicotine induced degeneration. NO synthase (NOS) inhibitors prevented/inhibited the nicotine induced degenerative effects on myotube size and branching, while exposure of the cells to sodium nitroprusside (SNP), an agent which releases NO spontaneously, resulted in myotube degeneration. As another approach to determine an involvement of NO in the myopathy, the effect of nicotine on NOS activity was determined. NOS activity increased in a time and tissue dependent manner in neonatal rat skeletal muscle cultures. NOS activity was then determined in the absence or presence of nicotine. (Abstract shortened by UMI.)

In contrast to normal cells, most neoplastic cells are resistant to the growth inhibitory effect of the transforming growth factor-$ beta$(TGF-$ beta$). The neoplastic transformation of cultured rat liver epithelial cells induced spontaneously or by chemical carcinogens and oncogenes is consistently associated with the development of resistance to the mito-inhibitory effect of TGF-$ beta$, suggesting that such phenotype plays an essential role during the transformation of these cells. We have studied the development of this "TGF-$ beta$, resistant" phenotype in a clonal strain of early passage normal cultured rat liver epithelial cells whose proliferation is markedly inhibited by TGF-$ beta$. A continuous control culture of these cells results in a gradual but spontaneous development of TGF-$ beta$ resistance. When the same cells were exposed to step-wise increases of the TGF-$ beta$ concentration in the medium, spontaneous transformation occurred significantly earlier than that in the control cells. After the same number of cumulative population doublings, the TGF-$ beta$ treated cells were much more resistant to the mito-inhibitory effect of TGF-$ beta$ than the control cells, and they exhibited an enhanced constitutive expression of the c-myc mRNA. The results suggest that TGF-$ beta$ facilitates or "promotes" spontaneous transformation. The TGF-$ beta$ resistant cells also showed increased resistance to the cytotoxins Adriamycin and Melphalan. This drug resistance was accompanied by an increase in the MDR-1 mRNA level, the cellular content of the glutathione and the activity of glutathione-S-transferase, thus suggesting a close association between the development of the TGF-$ beta$ resistance and the multidrug resistant phenotypes.

Initiating a computerized population-based registry of Alzheimer's disease (AD), the IMAGE Project has developed a multimatrix model to investigate the disease. Part of the IMAGE Project 1, the neuropathological study, is designed to correlate clinical, neuropsychological and neuropathological features of AD for characterization of subtypes. This thesis reports mainly the morphometrical studies associated with project 1. / The study, based on (a) brain autopsy, (b) standardized histopathology, and (c) quantitative morphometry, shows heterogeneity in pathophenotypes of AD. Four morphological subgroups have been presently recognizes, by their characteristic histological abnormalities, and the densities, the distribution, and progression patterns of their lesions. The heterogeneity in pathophenotypes indicates that AD is not a disease with a single cause, but rather a syndrome with multiple elements involved in etiology and pathogenesis. These lead to different pathological features, and correspondingly, similar, but distinguishable clinical expressions.

The effect of a heat-killed, unencapsulated strain of type III group B streptococcus (GBS), COH 1-13, on cerebral blood flow (CBF) and prostanoid production was investigated in two separate piglet models. In the first model, we examined the temporal profile of changes in CBF and cerebrospinal fluid cytochemistry in response to GBS challenge during a 6 hour study period. Despite marked leukocytosis at 6 hours, GBS did not significantly affect CBF and other cardiovascular parameters, hematology and cerebral prostanoid production. In the second model, we evaluated the early effect of GBS on CBF autoregulatory function and subsequently employed pharmacologic agents to inhibit the effect of GBS. Cerebral perfusion pressure (CPP) was altered by inflating balloon-tipped catheters placed at the aortic root to produce cerebral hypotension and at the descending aorta to produce cerebral hypertension. Piglets inoculated with GBS exhibited a narrow range of CBF autoregulation due to an impairment at the upper limit of CPP, which was prevented by treatment with dexamethasone or ibuprofen. Cerebral prostanoid production did not change significantly with adjustments in CPP between the various treatment groups. We conclude that GBS may impair CBF control without affecting cerebral prostanoid production and may increase the risk of brain damage in neonates with bacterial meningitis.

Patients with allergic rhinitis exhibit an exaggerated nasal response to allergen, in large part a consequence of IgE-mediated inflammatory cell activation. IgE within the tissue is considered to derive from IgE-bound inflammatory and/or IgE+ plasma cells. Resting B lymphocytes and cells expressing IL-4 and IL-13, cytokines that induce B cells to undergo epsilon germline transcription and isotype switching to IgE, are present within allergic nasal mucosa. As such, it has been hypothesized here that local isotype switching to IgE may occur at this site. Probes to detect the epsilon germline transcript (Iepsilon RNA), a pre-requisite for isotype switch recombination, the mature epsilon transcript (Cepsilon RNA) and IL-4 and IL-13 mRNA were employed. Since steroids reduce the number of cells expressing these cytokines, the effect of pre-treating patients with fluticasone propionate (FP) was also investigated. / Nasal biopsies were obtained from seasonal allergic rhinitis patients before and following in vivo allergen challenge or natural exposure during the pollen season. To confirm local RNA synthesis, nasal tissue was challenged with allergen ex vivo. Immunocytochemistry (ICC) confirmed the presence of B cells within the nasal tissue. In situ hybidization (ISH) demonstrated increases in the number of Cepsilon and IL-4 mRNA and the appearance of Iepsilon RNA+ cells in nasal tissue from placebo-treated patients following in vivo allergen exposure, but not those pre-treated with FP. Ex vivo allergen challenge also resulted in higher numbers of Cepsilon, Iepsilon, IL-4 and IL-13 RNA+ cells in allergen-stimulated compared to unstimulated tissue. With simultaneous ICC/ISH, CD20+/Cepsilon+ (37%) and CD20+/Iepsilon+ (32%) were observed, while the presence of mature epsilon mRNA (Cepsilon+/Iepsilon-) was confirmed using double ISH. Furthermore, IL-4 was associated primarily with T cells (&cong;70%) and mast cells (&cong;32%), while these cell types also produced IL-13 (&cong;44%, &cong;18%). / It is demonstrated here that the epsilon germline transcript, mature epsilon mRNA and IL-4 and IL-13 mRNA are synthesized locally within allergic nasal mucosa and that steroids inhibit their production. These results indicate that events within the nasal mucosa itself play a primary role in regulating the allergic response at this site and emphasize the importance of focusing on this local regulation when designing future diagnostic and therapeutic strategies.

Chronic ligation of one pulmonary artery results in pulmonary vascular remodelling and bronchial angiogenesis, known as postobstructive pulmonary vasculopathy (POPV). In previous studies of POPV, we found that responses of pulmonary arteries to 5-HT and of veins to histamine were markedly increased, but the role of putative factors, such as structure, endothelial modulation and alterations in receptors remains unknown. First, we examined the role of these mechanisms in the differential responses of pulmonary arteries and veins of normal guinea pigs to histamine and 5-HT, using a novel lung explant technique. We found that veins contracted more to both agonists than arteries, and that H2 receptors were responsible for the differential contractile responses of pulmonary arteries and veins to histamine, whereas endothelium-derived vasoactive substances (nitric oxide and prostacyclin) were responsible for their differential contractile responses to 5-HT. We also investigated relaxation responses, and found that endothelial-dependent NO-mediated relaxation was greater in pulmonary arteries than veins, and that acetylcholine-induced NO-mediated relaxation was reduced by the simultaneous production of cyclooxygenase-derived vasoconstrictors. / Second, we did experiments in guinea pigs with POPV, and found that the maximal contractions of pulmonary arteries to 5-HT and of veins to histamine were increased compared with controls, and that the augmented responses were not due to endothelial dysfunction nor to structural alterations, but probably to changes in the smooth muscle proper. To ascertain responses to endothelin (ET), and the role of altered receptors, specifically ET receptors, in POPV, we produced the model in rats: we found that contractions to ET-1 and ET-3 were increased and that relaxation to ET-1 was reduced significantly only in the pulmonary arteries with POPV compared with controls; these findings were attributed, using receptor binding studies, to an augmented proportion of ETA over ETB receptors. / We conclude that the differential contractile responses of normal pulmonary arteries and veins to histamine and 5-HT, and the altered vascular responses to these amines and to ETs in POPV, are due primarily to differences in receptors or in endothelial modulation, rather than to disparities in vascular structure.

The spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders characterized by symptoms and signs of degeneration in the cerebellum, spinal cord and brainstem. To date, ten different loci causing autosomal dominant SCA have been mapped. In six types of SCA the disease is caused by the expansion of a trinucleotide CAG repeat which codes for a polyglutamine tract at the protein level. / The overall aim of this research project was to investigate various molecular genetic aspects of the autosomal dominant spinocerebellar ataxias: (1) to assess genetic heterogeneity with linkage studies in suitable families and to study the origin of the MJD/SCA3 mutation by linkage disequilibrium analysis; (2) to determine the relative frequency of the different types of SCA-(CAG)n mutations in a large group of patients from different ethnic origins; (3) to investigate molecular and clinico-pathological correlations in SCA patients in whom mutations were found and; (4) to investigate whether polyglutamine expansions are present in other forms of SCA using a monoclonal antibody. / Three large families segregating autosomal dominant SCA were tested for linkage to the SCA1, SCA2 and MJD/SCA3 loci by the maximum likelihood method. Two-point and multipoint linkage analysis showed that two families (SA and GK) were linked to the SCA2 locus. The third family (FC) was significantly excluded from the SCA1, SCA2 and SCA3/MJD loci. Additional markers were typed in the SCA2 candidate region in the two linked families. We were able to narrow down the SCA2 candidate region on ch12q and construct a fine genetic map of the region that provided important information for the positional cloning of the SCA2 gene. Linkage disequilibrium studies, using markers around the MJD/SCA3 gene found evidence of a founder effect in our MJD patients. However, distinct haplotypes were observed in patients originating from each of the two Azorean islands showing the highest disease prevalence; indicating that there are more than one founder mutation in the Azorean MJD population. / We studied a large group of unrelated SCA patients in order to determine the frequency of the SCA1, SCA2, MJD/SCA3 and DRPLA mutations. (Abstract shortened by UMI.)