This thesis explores the role of an adhesion protein, CD44, in the biology of colon cancer. CD44 glycoprotein is a main extracellular receptor for hyaluronic acid. A single CD44 gene composed of 20 exons encodes a variety of isoforms due to alternative splicing of 10 middle exons. Overexpression of CD44 and appearance of abnormal isoforms containing products of variant exons have been implicated in the origin and progression of cancer, including human colon carcinoma. / Of two main parts comprising the dissertation, the first one ("CD44 and the adhesion of neoplastic cells" by Z. Rudzki and S. Jothy) reviews the current state of knowledge on CD44, emphasizing its role in neoplastic processes. The second part ("Changes in CD44 expression during carcinogenesis of the mouse colon" by Z. Rudzki, L. LeDuy and S. Jothy) reports an experimental study of CD44 expression in a murine model of colon cancer. The tumors were induced by subcutaneous injections of a colon--specific carcinogen (1,2-dimethylhydrazine). CD44 expression was studied by RT-PCR/Southern blot and immunohistochemistry. The CD44 transcripts were generally strongly overexpressed in tumors compared with normal colon. Both neoplastic and normal colon samples exhibited the same complex array of transcript bands representing the standard molecule (CD44s) and its variant isoforms. Immunohistochemistry revealed marked heterogeneity of tumor staining, contrasting with a rather uniform mRNA overexpression. There was a significant tendency towards the progressive loss of CD44 immunoreactivity in larger and invading tumors. It is concluded that CD44 isoforms are globally overexpressed at an early, premacroscopic stage of colonic carcinogenesis. Expression of CD44 in the murine model of colon cancer shows some similarities to its human counterpart.

Intermediate filaments (IFs) are highly regulated and conserved during cell transformation and tumor development. The co-expression of keratin 8, 18 and vimentin has been shown to be related to recurrence and metastasis in both cutaneous and uveal melanomas. This thesis provides the first immunohistochemical evidence that the co-expression of keratin 8, 18 and vimentin is present in one third of conjunctival melanomas. The results show that conjunctival melanomas co-expressing IFs are mixed cell tumors with diffuse growth patterns. It is also shown that melanoma cells co-expressing IFs are mainly located around the peripheral or marginal area of the tumors. In addition, this thesis indicates that the initial tumor thickness significantly increases in conjunctival melanomas co-expressing IFs. Therefore, it is demonstrated that the co-expression of IFs plays an important role in influencing the malignant progression of conjunctival melanomas, and the co-expression of IFs can be used as a prognostic marker for conjunctival melanomas.

The human insulin-like growth factor-II is an important fetal mitogen with demonstrated effects on growth, proliferation and survival of a wide spectrum of cells and tissues in mice and humans. The transcriptional regulation of the gene (IGF2) is under developmental control and is subject to epigenetic and genetic effects. At the epigenetic level, the mouse gene is parentally imprinted with exclusive expression of the paternally-inherited gene copy in most of the tissues examined. In this work, we demonstrate that the human gene is also subject to genomic imprinting and that the trait is tissue-specific and polymorphic whose basis may be genotype-dependent. Additionally, we show that in culture, primary placental fibroblasts lose the ability to functionally imprint IGF2. In instances of biallelic IGF2 gene expression, both in human tissue as well as in culture, we observe that the adjacent, reciprocally-imprinted H19 gene, which is normally expressed from the maternally-transmitted allele, is undetectable. / The imprinted IGF2 gene is located in close physical proximity to a variable number of tandem repeats (VNTR) sequence polymorphism that is found upstream of the insulin gene promoter, in humans. Alleles of the VNTR have demonstrated transcriptional effects on insulin gene expression in human fetal and adult pancreas in vivo and in pancreatic beta cells in vitro. In this work, we show that the VNTR also has allelic effects on IGF2 expression in human placenta in vivo and on INS-IGF2 reporter gene activity in human lymphoblasts, in vitro. / Alleles of the 5' INS (VNTR) are associated with susceptibility to type I diabetes mellitus. The preferential transmission of paternal susceptibility haplotypes at this locus suggests the functional involvement of a nearby imprinted gene in the susceptibility to type I diabetes. The demonstration of allelic effects of the VNTR on IGF2 mRNA levels, in vivo and in vitro, makes IGF2 an attractive functional candidate.

Target-dependent cell death is an important embryogenic mechanism for regulating and sculpting the developing motor system. Efficient characterization of apoptosis has been more difficult in the nervous system than in other systems due to the use of several different primary culture systems as well as with heterogeneity of neuronal cell populations. We have developed a simple in vitro model of apoptosis with the motor neuron hybrid NSC34, a cell line which expresses much of the motor neuron phenotype (Cashman et al. 1992). Serum-deprived NSC 34 cells in bulk culture undergo cell death, likely from the withdrawal of the growth factors and/or hormones present in fetal calf serum medium supplements. This cell death is accompanied by fragmentation of chromatin into nucleosome multimers, heterochromatization of the nucleus, and other ultrastructural changes reminiscent of apoptotic death. Cell death is inhibitable by addition of agents which block new gene expression ( e.g. cycloheximide) or inhibit endonuclease activity (e.g. aurintricarboxylic acid). / We report similar findings with primary embryonic rat motor neurons identified by surface immunoreactivity for p75 LA NGFR, the low-affinity neurotrophin receptor (Bloch-Gallego et al. 1991; Camu and Henderson 1992; Chao and Hempstead 1995). The p75+ motor neuron population could be maintained for more than 48 hours in mixed suspension cultures supplemented with 10% fetal calf serum. However, the p75+ cell population was rapidly depleted in serum-deprived cultures, a phenomenon accompanied by the appearance of oligonucleosomal ladders. Serum-deprived p75+ cells were supported by the motor neuron-relevant factors BDNF, CNTF, GDNF and IGF-1, but not the non-relevant factor NGF. Serum-deprived p75 + cells were also protected by cycloheximide, suggesting a role for apoptosis in the cell death. / We have investigated the role of reactive oxygen species in acquired and genetic motor neuron diseases. Interestingly, a rapid burst of reactive oxygen species is observable within one hour of serum deprivation in both NSC34 and rat motor neuron systems. This burst precedes measurable cell death by at least one day, indicating that oxygen species generation may be an initial hallmark of target-dependent death. The amplitude and temporal nature of this burst may be altered by manipulating various cellular ROS defence mechanisms. Such manipulations also alter cell death progression, suggesting that the apoptotic cascade may be dependent upon this early ROS burst. The identity, source and activity of the relevant ROS may provide insight into the etiology and treatment of human motor neuron diseases.

The intervertebral disc has been identified as a possible contributor to two very common back disorders: degenerative disc disease and scoliotic deformity. Little is known about the composition and turnover of extracellular matrix in the human intervertebral disc and end-plate with these two pathologies. / We studied 121 intervertebral discs representing all age groups and degeneration grades. In addition, fifteen scoliotic discs and seventeen control discs were analyzed. / Synthesis in intervertebral discs, end-plates, and scoliotic tissues were measured by immunoassay measuring the content of a putative aggrecan biosynthesis marker (846) and contents of types I and II procollagen markers (CPI & CPII). The percentage of denatured type II collagen was assessed by the presence of an epitope that is exposed with cleavage of type II collagen. / We identified three matrix turnover phases in the intervertebral disc and end-plate. Phase I (growth) is characterized by active synthesis of matrix molecules and active denaturation of type II collagen. Phase II (maturation and ageing phase) is distinguished by progressive drops in synthetic activity and denaturation of type II collagen. Phase III (degeneration and fibrotic phase) is illustrated by decreased synthesis of aggrecan and type II procollagen and an increase in collagen type II denaturation and type I procollagen synthesis, the later dependent on age and grade of tissue degeneration. / The results of the scoliotic study indicate that synthesis of type II procollagen in the end-plate and annulus was higher in scoliotic discs. Aggrecan synthesis showed a similar trend in the nucleus pulposus of scoliotic disc. By contrast, the contents of hydroxyproline, glycosaminoglycan, and water were significantly lower in scoliotic discs while total protein content was higher in scoliotic discs, indicating that non-collagenous protein content was increased in scoliotic discs. This elevated synthetic activity without an associated collagen denaturation reflects an overall synthetic response without an increased matrix turnover, suggesting that scoliotic changes are due to an altered and ineffective synthetic response to a pathologic mechanical environment. / This thesis represents the first in situ quantitation of human lumbar intervertebral disc matrix composition and turnover with ageing, degeneration, and scoliotic change. These observations are an important advancement in our biochemical understanding of disc ageing, disc degeneration, and adolescent idiopathic scoliosis.

Although nitric oxide (NO) from inducible nitric oxide synthase (NOSII) is proposed to be the major factor in the vascular abnormalities of sepsis in rats, its role in higher order species is not well established. This thesis thus, addresses the role of NO in septic pigs. / I first hypothesized that induction of NOSII in pigs is the cause of sepsis-induced hypotension as occurs in rodents. To test this, I treated pigs with lipopolysaccharide (LPS) to simulate sepsis. In contrast to what is observed in rats and mice, plasma nitrite did not change during the 4 hours of the LPS infusion and there was no increase in calcium (Ca)-independent nitric oxide synthase (NOS) activity in lung tissue from endotoxemic pigs compared to control animals. Furthermore, there was only minimal induction of NOSII mRNA. / My second hypothesis was that NOS (NOSIII) is not down regulated in septic pigs, and NO from NOSIII, could contribute to peroxynitrite formation. In support of this, western blot analysis of samples from aorta and vena cava showed no down regulation of NOSIII. There also was an increase in Ca ++-dependent NOS activity of aorta and vena cava after 2-hour of endotoxemia, which indicates increased constitutive NOS activity. Moreover, there was an increase in NOSI in the vena cava. LPS produced a leftward shift in the dose-response curves of SVR and RVR in response to the NOS inhibitor, L-NAME (NG -nitro-L-arginine-methyl-ester). / Peroxynitrite is formed from NO and superoxide (O2 -). Since NO was not increased in endotoxemic pigs, I next hypothesized that O2- is increased in endotoxemic pigs and this reacts with NO from constitutive NOS to form peroxynitrite. To test this hypothesis, I first studied rats to confirm that O2 - is increased in arterial vessels of endotoxic rats. There was a small increase in basal O2- in endotoxic rats. Stimulation of NAD(P)H oxidase by giving the substrate, NADH, did not result in different O2- production in control and endotoxic rats. However, inhibition of NOS increased O2 - in endotoxic rats. / I demonstrated by immunohistochemistry and western blot analysis, that the components of NAD(P)H oxidase, gp91phox, p22phox , and p47phox are present in skeletal muscle and it has similar behavior to other non-phagocytic NADPH oxidases. The addition of L-NAME to NADH-treated diaphragm strips of endotoxic rats, increased O 2- production in endotoxic rats indicating that O2- production counters the increased NO formation as we observed in rat aorta. These observations indicate that O2 - generation from NADPH oxidase in skeletal muscles can play a role in the pathophysiology of sepsis. (Abstract shortened by UMI.)

This study consists of evaluating the effects of zidovudine or ribavirin treatment on the humoral response to human immunodeficiency syndrome (HIV-1) in a cohort of 36 HIV-1 seropositive patients. Viral neutralization antibodies were demonstrated against HTLV$ sb{ rm IIIb}$ virus while titers of circulating antibody-dependent cellular cytotoxicity (ADCC) antibodies were measured against the HIV-1 envelope protein, gp120, using the vaccinia virus expression system which has been successfully used to express foreign viral proteins in target cells. Virologic (viral isolation) and immunologic (CD4$ sp+$ cells) parameters were also monitored pre and post antiviral therapy. / The results indicate that patients receiving zidovudine for 36 weeks, have a diminished anti-HIV-1-ADCC directing antibody response, while the levels of these antibodies in patients receiving ribavirin or placebo remain constant. The titers of neutralizing antibodies and CD4 counts remain stable regardless of the treatment except for patients receiving ribavirin, where a decline in CD4 cells is observed. The decrease in the HIV-1 specific ADCC during zidovudine treatment parallels with the decrease in the amount of viral burden. This suggests that the two effects are somehow correlated. The impact of a washout period was also assessed. An increase in viral burden during cessation of AZT was reported which may reflect the inability of the treated host to mount a rapid immune response. As a consequence, this may lead to the deterioration of the immune status and the progression of the disease. The implications of these findings will be discussed in this study.

No description available.

Uveal melanoma is the most common primary malignant intraocular tumor in adults. The management of uveal melanoma remains a clinical dilemma, which reflects our poor understanding of this life-threatening disease. A major challenge facing researchers investigating this malignancy has been to develop a suitable animal model. The purpose of this work is to characterize, in detail, the processes of tumor development, malignant cell dissemination and metastasis in a 10-week albino rabbit model of uveal melanoma. Intraocular tumors successfully developed, and metastatic disease was present in all animals at the end of the experiment. For the first time using an animal model of uveal melanoma, the presence of circulating malignant cells in the bloodstream was demonstrated. Knowledge gained from this study has led to a better overall understanding of the progression of the disease in this experimental model and may facilitate the development of methods for the prevention, early detection and treatment of metastatic uveal melanoma.

Obliterative bronchiolitis (OB) is the leading cause of chronic irreversible lung graft failure. It is assumed by many to be caused by chronic rejection. We hypothesized that non-specific airway inflammation induced by factors such as foreign airway deposited particles, antigens and microbes is essential for the development of OB after lung transplantation (LTx). In this study, we examined the histological and immunohistological changes in allografted airways following transtracheal installation of foreign irritant particles. / Inadequate immunosuppression of lung allografts leads to severe vascular changes consistent with chronic vascular rejection but fails to induce airway inflammation and OB. The addition of a nonspecific foreign irritant was necessary to induce diffuse airway inflammation and OB in our animal model. This suggests that a co-factor inducing airway inflammation, such as deposited foreign particles or infections, is likely necessary for the development of OB after lung transplantation. Cytokine expression reveals a link between the observed influx of airway inflammatory cells and IL-8. While bFGF is associated more with the proliferative phases of airway and vascular injury. These markers however are not predictive of either rejection or OB, due to the expression of both these markers in all three study groups. (Abstract shortened by UMI.)