Uveal melanoma is the most common intraocular tumor in adults. Despite advances in the accuracy of diagnosis, the ten-year mortality rate for patients has remained constant at approximately fifty percent. This indicates that a further understanding of the biological mechanisms behind this malignancy is required. / Our laboratory utilizes five human uveal melanoma cell lines (92.1, MKT-BR, OCM-1, SP-6.5, UW-1) that have a previously described metastatic potential from an animal model (MP). We used four methods to characterize the proliferation rate of these five cell lines. We also used a Matrigel invasion assay to assess the invasive ability of the same cell lines in response to the potential chemo-attractants: interleukin-6, Vascular Endothelial Growth Factor, and Fetal Bovine Serum. / From these results we were able to propose a novel classification system for our cell lines: high proliferation and high invasion/high MP, low proliferation and low invasion/low MP, and high proliferation and invasion/no MP. / From these results we were able to propose a novel classification system for our cell lines: high proliferation and high invasion/high MP, low proliferation and low invasion/low MP, and high proliferation and invasion/no MP.

The c-met proto-oncogene encodes a tyrosine kinase receptor for hepatocyte growth factor/scatter factor. Losses of c-met alleles have been documented in human carcinomas. In breast carcinoma c-met has been suggested to be a potential inactivated tumour suppressor gene (TSG). In non-small cell lung carcinoma (NSCLC) a general reduction of c-met expression in squamous cell carcinomas (SQCCs), as well as overexpression in adenocarcinomas (ADCs) have been revealed. Furthermore, many SQCCs and ADCs had undetectable c-met mRNA and protein. These results prompted us to explore c-met allelic alterations in NSCLC. Our main goal was to address the possible involvement of inactivated c-met alleles in the development and progression of NSCLC. In this LOH analysis two polymorphic-sensitive probes corresponding to the c-met gene, p-metH and p-metD, were used to analyze normal and tumour DNA samples from patients with primary NSCLC. Of 110 cases examined with p-metH, 56 (50.9%) were informative and 4 (7.1%) exhibited LOH. Among 109 patients examined with p-metD, 28 (25.7%) were informative and 1 (3.6%) was LOH-positive. The combined LOH incidence for c-met was 7.4% (5/68 informative cases). Lastly, since a locus adjacent (distally) to c-met on chromosome 7, D7S95, was shown to exhibit a significant LOH frequency in gastric carcinoma (43.3%), we also sought to determine if loss of this locus was common in NSCLC. Of 104 patients examined at D7S95, 45 (43.3%) were informative and 1 (2.2%) exhibited LOH. These findings suggest, firstly, that c-met does not have a possible role as an inactivated TSG in the tumourigenesis of NSCLC nor is it closely linked to a putative TSG, and secondly, D7S95 does not contain a detectable inactivated TSG or a closely-mapping TSG in NSCLC.

Allergen-induced rhinitis is associated with the recruitment and activation of inflammatory cells, particularly eosinophils and CD4 + T cells into the nasal mucosa. Monocyte chemotactic proteins (MCPs) have been shown to induce chemotactic activity in these particular cell types under in vitro assay conditions. To assess the contribution of MCPs in the recruitment of inflammatory cells in vivo, we investigated the allergen-induced late response in subjects with allergic rhinitis. Using immunocytochemistry and in situ hybridization, we demonstrated a constitutive expression of MCP-1, -3 and -4, of which MCP-3 and -4 were significantly increased in the nasal mucosa following allergen provocation. This upregulation of MCP-3 and 4 immunoreactivity in response to allergen, was reduced in patients pretreated with topical corticosteroids. Colocalization experiments revealed that the majority of MCP-positive cells were macrophages. The results of this study suggest that allergen-induced rhinitis is associated with an increased expression of MCP-3 and -4, which may be closely related to the influx of inflammatory cells and may thus contribute to the pathogenesis of allergic rhinitis.

$ beta$-Hexosaminidase is a lysosomal enzyme which exists as two isoenzymes: Hex A ($ alpha beta$) and Hex B ($ beta beta$). Its presence in the testis and epididymis suggest important roles for this enzyme and their substrates in male fertility and reproductive functions. The main focus of this investigation was to analyze the effects of a deficiency of these isoenzymes within the male murine reproductive tract. Disruption of the Hexa gene encoding the $ alpha$-subunit of $ beta$-hexosaminidase led to the generation of a mouse model of human Tay-Sachs while disruption of the Hexb gene encoding the $ beta$-subunit of $ beta$-hexosaminidase led to a mouse model of human Sandhoff disease, thus providing the opportunity to analyze the effects of Hex A and Hex B deficiency on epithelial cellular morphology of the male reproductive tract. Analysis of the testis, efferent ducts and epididymidis of 5 weeks, 3, 5 and 12 month old Tay-Sachs (Hexa $-/-$) and 1 and 3 month old Sandhoff (Hexb, $-/-$) was performed; the wild-type (Hexa +/+, Hexb +/+) mice at all ages were also examined as controls. (Abstract shortened by UMI.)

In this thesis, we investigated the progression of cellular injury using two markers of neuronal degeneration, in a model of hippocampal epileptogenesis. Pilocarpine was used to induce a controlled period of status epilepticus (SE) in six groups of rats. Following a 1--2 week period of apparent recovery, the animals developed spontaneous recurrent seizures. A silver impregnation technique for neurons subjected to cytoskeletal stress, the Dark Neuron stain, was used to label hippocampal neurons at each of six selected time points (from 3h--3weeks) following SE. The proportion of labelled cells was then compared to that obtained with the anionic fluorochrome Fluoro-Jade, reported to be specific for degenerating neurons. / Results revealed a differential temporal pattern of labelling. The Dark Neuron stain preferentially revealed cells at earlier time points, while with Fluoro-Jade staining was found at a later time point during epileptogenesis. Interestingly, maximal Fluoro-Jade labelling at 1 week post-pilocarpine injection coincided with significant cell loss and gliosis in the CA3 principal cell layer. / The two stains, therefore, appear to detect separate processes of neuronal damage during epileptogenesis. This finding highlights the fact that distinct cellular events take place at different stages of epileptogenesis, which may differ significantly from the permanent changes observed in chronically epileptic tissue. Understanding the progression of cellular injury may be important for the development of antiepileptogenic therapy, a potentially valuable complementary approach to anticonvulsant therapy for the treatment of chronic epilepsy.

Introduction. Human papillomavirus (HPV) has been detected with varying frequency in oral cancers and in normal oral tissues. The main objective of the present study was to examine the association between HPV infection and risk of developing oral cancer. / Methodology. This investigation, as a component of an international multi-centre study coordinated by the IARC, followed a hospital-based case-control design. Cases consisted of newly diagnosed patients with primary squamous cell carcinoma of the oral cavity, including mouth and oropharynx. Controls were frequency matched to cases by sex, age, and hospital. All subjects were interviewed to elicit detail information on known and putative risk factors. / Oral exfoliated cells were collected from all subjects for detection of HPV DNA using the PGMY09/11 PCR protocol. Antibodies against HPV 16, 18, and 31 capsids were detected in patients' plasma using an immunoassay technique. Logistic regression was used for estimation of odds ratios (ORs) and 95% confidence intervals (CI) of oral cancer for HPV and other candidate risk factors. / Results. A total of 72 cases and 129 controls were recruited. HPV DNA was detected in 19% of cases (14 out of 72), and in 5% of controls (6 out of 129). Analysis for cancers related to Waldeyer's ring (palatine tonsil and base of tongue) showed that the OR of disease for detection of high risk HPV types was 19.32 (95%CI:2.3--159.5), after adjustment for socio-demographic characteristics, tobacco and alcohol consumption. The adjusted OR of disease for HPV 16 seropositivity was 31.51 (95%CI:4.5--219.7). Analysis for non tonsillar oral cancers showed that the OR for detection of high risk HPV DNA in oral cells and for seropositivity were 2.14 (95%CI:0.4--13.0) and 3.16 (95%CI:0.8--13.0), respectively. / Discussion. The results from this study provide evidence supporting a strong association between HPV infection and cancers of the oropharynx, especially those arising from Waldeyer's ring. On the other hand, the association with non tonsillar oral cancers was of much lower magnitude. The biological evidence establishing a firm etiologic link remains to be established for the latter subsites, whereas the association between HPV and Waldeyer's ring carcinomas is consistent with a causal link.

Introduction. Human papillomavirus (HPV) infection is now believed to be the central cause of cervical cancer. However, most of the epidemiological evidence has come from retrospective, case-control studies, which do not provide information on the dynamics of a cumulative or persistent HPV infection. / Objectives. (1) To measure the risk of incident neoplastic cervical lesions over time related to prior cumulative and persistent HPV infections. (2) To evaluate the influence of HPV viral burden on lesion risk longitudinally. (3) To estimate the progression rates and sojourn time for precursor squamous intraepithelial lesions (SILs) and how they relate to HPV infection status. / Design and methods. In 1993, the Ludwig-McGill study team began a large longitudinal study of the natural history of HPV infection and cervical neoplasia in the city of Sao Paulo, Brazil. Follow-up involved repeated measurements on individual subjects over time. 2462 women were enrolled into the study and were seen every 4 months in the first year (0, 4, 8 and 12 months), and twice yearly thereafter for a period of up to eight years. In addition to obtaining risk factor information via questionnaire, cervical specimens were taken for Pap cytology and HPV testing at every visit. Statistical analyses entailed: (1) using different modalities for defining HPV persistence by type and intensity; (2) using modeling approaches that take into account the repeated measurements of HPV and SIL over time within individuals; (3) analyzing changes in transition states between different cervical lesion grades and the rate of progression from one state to the next. / Rationale. A longitudinal, repeated measurement cohort investigation, such as this one, permits an accurate and unbiased assessment of the relationship between cumulative HPV exposure and lesion incidence. An elevated relationship between persistent HPV infections and SIL incidence supports the proposal for the application of type-specific molecular HPV DNA testing as a screening tool for the detection of cervical neoplasia. Better understanding of the natural history of disease can help in developing effective and efficient public health programs in prevention for cervical cancer.

We report that the chitinase-like protein, human cartilage glycoprotein 39 (HC-gp39) is a growth and survival factor. It promotes connective tissue cell survival and growth, and counteracts catabolic processes. Dose-dependent growth stimulation was observed when human synoviocytes and fibroblasts were exposed to HC-gp39. Both the ERK1/2 MAP kinase and the PI3 kinase pathways were activated by HC-gp39. Thus HC-gp39 elicits a signaling cascade leading to increased connective tissue cell proliferation, suggesting that HC-gp39 may play a major role in the pathological conditions leading to tissue fibrosis. In addition to acting as a mitogen, HC-gp39 also reduces stress-induced apoptosis in human connective tissue cells. HC-gp39 significantly inhibited H2 O2 activation of SAPK/JNK and p38 in chondrocytes. A reduction of caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage and apoptotic cell number was also seen. These results suggest that HC-gp39 plays an important role in the survival of connective tissue cells. The protective effect is further illustrated by the ability of HC-gp39 to reduce cellular responses to inflammatory cytokines. IL-1 or TNF-alpha stimulated phosphorylation of SAPK/JNK and p38 were greatly reduced by HC-gp39. Signalling through the NF-kappab pathway was unaffected. These actions resulted in a significant reduction of MMP1, MMP3, MMP13 and IL-8 production, suggesting that HC-gp39 can participate in the catabolic aspects of tissue remodelling as a repair or protecting factor counteracting the catabolic processes. Although HC-gp39 affected the response to IL-1 and TNF-alpha, these cytokines had no effect on the high expression level of HC-gp39 in chondrocytes cultures. However, inhibition of the NF-B signalling pathway, which has been implicated as a major effectory mechanism for TNF-alpha, significantly decreased HC-gp39 protein and mRNA levels in a concentration and time dependent manner. Increased NF-kappaB / Therefore, this work demonstrated that expression of HC-gp39 at inflammation sites is part of the reaction chain in the pathogenesis of joint degeneration and/or inflammation. The activity of the elements regulating the expression of HC-gp39 suggests that it could serve as a negative feedback regulator for the inflammatory cytokines.

Cardiac action potential (AP) properties are not uniform because of functional specialization in different regions. The thesis of these studies was that important aspects of regional cardiac electrical function in health and disease are determined by heterogeneous ionic current expression. / The inward rectifier current, IK1, responsible for the resting membrane potential (RMP) and late repolarization, is greater in ventricle than atrium. Atrial-ventricular IKI differences result in important electrophysiological differences between atrium and ventricle. To gain insight into the mechanisms of this IK1 variation, we compared the expression and distribution of inward rectifier subunits Kir2.1/2.3, believed to underlie IK1, in both regions. Ventricles had greater Kir2.1 and lower Kir2.3 expression than atrium. The subcellular distributions of these subunits also differed between the two regions. Our findings suggest that, in addition to the intensity of subunit expression, cellular subunit location affects local channel function. / To further examine the effects of channel subunit expression and distribution on regional electrophysiology, we studied the pulmonary vein (PV) myocardial sleeve. PVs are important in atrial fibrillation (AF) initiation and maintenance and exhibit unique current profiles compared with adjacent atrial cardiomyocytes. Our findings demonstrate a greater PV expression of ERG and KvLQT1---the channel subunits that underlie the rapid and slow components of the delayed rectifier current, IK, respectively. KvLQT1 was diffusely distributed in atrial cardiomyocytes, in contrast to the transverse tubular (TT) and intercalated disk (ID) PV distribution. Kir2.3 abundance was similar in both regions, but differently distributed with a TT and ID LA distribution and ID localization in PVs. Observed expression and distribution differences between atrial and PV cardiomyocytes may account for the smaller Vmax, less negative RMP and shorter AP duration in PVs, which contribute to their arrhythmic diathesis. / The PVs are thought to act as fibrillatory foci in congestive heart failure (CHF). We compared channel subunit expression in PV cardiomyocytes from (rapid ventricular pacing induced) CHF and control hearts. CHF decreased KvLQT1 expression, increased expression of ERG and the sodium calcium exchange subunit, NCX, and altered the subcellular KvLQT1 distribution. (Abstract shortened by UMI.)

Two classes of antimigraine drugs specifically target serotonin (5-HT) receptors: agonists of the 5-HT1B/1D receptors and antagonists of the 5-HT2B/2C receptors. They are respectively used in symptomatic and prophylactic treatments. The study of their mechanisms of action can assist in better uncovering the mechanisms underlying migraine. / Using molecular, pharmacological, immunocytochemical and histochemical approaches, we have studied the distribution and the role of the 5-HT receptors targeted by these medications in human trigeminal ganglion (TG) and extracerebral vessels (PV), two tissues of primary importance in migraine pathology. / We first focused our study of symptomatic treatment on sumatriptan, a drug which works with high efficacy but can trigger cardiac adverse-effects. Two mechanisms have been suggested to explain its therapeutic effect during migraine headache: the contraction of overly dilated PV and the inhibition of a neurogenic inflammation within meningeal tissues following the stimulation of TG. Sumatriptan's cardiac effects have been associated with the contraction of coronary artery (CA) in high-risk patients. / In order to help develop drugs which minimize such harmful side-effects, we have identified in human the type and distribution of sumatriptan-sensitive receptors in TG, PV and CA. As sumatriptan has a high affinity for the 5-HT1B, 5-HT1D and 5-HT1F receptor subclasses, we used reverse transcriptase-polymerase chain reaction (RTPCR) to distinguish between them. We have demonstrated the non-selective distribution of the RNA for the 5-HT1D and 5-HT1F receptors in human TG and some PV whereas the 5-HT1B receptor mRNA was mainly expressed in PV. 5-HT1B and 5-HT1F receptor RNA have also been detected in the CA. / We then used an in vitro vascular reactivity assay with receptor-selective agonists and showed that of these three receptor subclasses, only the 5-HT1B receptor elicits vasoconstriction in PV. Agonists for 5-HT1D and 5-HT1F could therefore potentially be anti-migraine compounds devoid of vascular adverse-effects. / We followed this study by examining the medications used in prophylaxis which are antagonists at either 5-HT2B or 5-HT2C receptors. It has been suggested that stimulation of such receptors in the endothelium of cerebral vessels could trigger migraine via the release of nitric oxide, a compound both nociceptive and vasodilator. We have successfully demonstrated using RT-PCR that the 5-HT2B but not the 5-HT2C receptor RNA is present in cerebral vessels. We further utilized RT-PCR and immunocytochemistry to precisely localize the receptor and show its presence not, only in the endothelium but also the smooth muscle of cerebral vessels. (Abstract shortened by UMI.)