The preputial glands of male nude (nu/nu) mice were analyzed by a combination of electron microscopy and gas chromatography to determine whether or not they are affected, like developing hairs and nails, by the nu/nu genotype. Results of the analyses revealed no differences between the glands of nude and normal male mice in either their ultrastructural characteristics or lipid secretory products.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Athymic nude mouse models have been extensively used to study biological behaviour of normal and diseased human tissues. In such models, immune-deficient mice act as hosts for cysts constructed from human material. A unique biocyst model that entails transplantation of human vaginal cysts into athymic nude mice has been implemented to study diseases of oral mucosa. To date, only one immunohistochemical study of this biocyst model has been reported. Nevertheless, conclusions made in that study were only based on the observed expression patterns of human and murine markers. Statistical assessment of immunohistochemical data had been omitted by the investigator. Therefore, the objective of this study was to further delineate the immunohistochemical profile of normal human vaginal tissue and human vaginal tissue that had been xenografted into nude mice.Experimental cysts constructed from human vaginal mucosa were xenografted into athymic nude mice and harvested 9-weeks post transplantation. Immunohistochemical analysis of normal human vaginal tissue and human vaginal tissue that had been xenografted into nude mice was performed using a panel of human and murine markers. Expression patterns of human and murine markers were assessed. Human markers included cytokeratin 1,cytokeratin 5, cytokeratin 13, cytokeratin 14, collagen type IV, laminin, elastin, fibronectin,Langerhans cells and VEGFR-3. Murine markers included collagen type IV, laminin,fibronectin, Langerhans cells and VEGFR-2. Staining intensities were quantified and statistically analysed using one-way ANOVA with subsequent Friedman’s test for multiple comparisons. Since the sample size was small, the power of the test statistic was enhanced by including Dunn’s post-test for further multiple comparisons. A strong positive expression of all cytokeratins was detected in both normal and xenografted vaginal tissues. Human markers that exhibited weak to moderate positive expression were collagen IV, laminin, fibronectin and VEGFR-3. Human elastin and human Langerhans cells exhibited strong and varying expression patterns respectively. Weak expression patterns for all murine markers were reported, with an exception of VEGFR-2 which was negatively expressed in all xenografted vaginal tissues. Significant differences (P<0.05) in the mean staining intensities between normal and xenografted vaginal tissues were reported for cytokeratin 1, fibronectin and Langerhans cells. There were no statistical differences (P>0.05) in the mean staining intensities for other markers.In conclusion, immunohistochemical studies proved that human vaginal tissue could not only survive in nude mice, but could also become active and develop structures necessary for survival, in this case, a newly formed stromal layer. The epithelium and stromal layer exhibited a human ecosystem.

No description available.

Breast cancer is the most common malignancy diagnosed among women and is the first cause of neoplastic death in women globally. In the last decade our understanding of breast cancer biology has increased and led to the development of a number of targeted therapies, one of which is targeting the cell apoptosis pathway. One of the new targeting pathways under investigation, which was found to be involved in both cell apoptosis and cell proliferation processes, is the sphingolipid signalling pathway. The sphingolipid pathway represents a group of intracellular and extracellular bioactive lipid molecules, including ceramide, ceramide- 1-phosphate, sphingosine, and sphingosine-1-phosphate (S1P). In my research, I focused on the role S1P plays in breast cancer and its potential application as a therapeutic agent. I examined the effects of S1P on the apoptosis, proliferation, and cytotoxicity of different types of breast cancer cell lines in vitro. In addition, I evaluated the effect of both low and high doses of S1P when co-administrated with anticancer drugs commonly used in breast cancer treatment in vitro and in vivo. Moreover, I studied the S1P cellular distribution following exogenous administration. My results demonstrate that S1P can selectively induce apoptosis in breast cancer cells without harming normal breast cells and that S1P is more effective against aggressive breast cancer cells. Another major finding of my study is that S1P can increase the efficacy of chemotherapies against human breast cancer cells. Although S1P cannot directly substitute the current chemotherapies, S1P may function as a good candidate for combination therapy. Furthermore, my work showed that the pro-apoptotic and anti-proliferative effect of S1P is correlated with its intracellular action and that chronic exposure of exogenous S1P in vivo is not toxic to the major organs. Certainly, S1P inclusion in breast cancer treatment modalities may decrease the morbidity and mortality of breast cancer patients and improve clinical outcomes. Further investigations are required to understand the mechanism by which S1P induces apoptosis and inhibits cell proliferation.

<p>Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. </p><p>In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate.</p><p>In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36.</p><p>In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice.</p><p>In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.</p>

Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate. In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36. In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice. In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.

IFN-α has been applied in medical treatment of various neuroendocrine (NE) tumors, either alone or combination with somatostatin analogues. They can improve clinical symptoms in 50-70% of patients but a significant tumor reduction is only observed in 5-15% patients. Vasostatin (vaso) is believed to be an angiogenic inhibitor. The aim of this study is to evaluate the feasibility to use IFN-α and vasostatin gene therapy in NE tumors. We constructed plasmid vectors carrying human IFN-α2 (hIFN-α2) gene and human vasostatin gene, which were transfected into BON I cell to obtain stable gene-expressing cell lines. We found that in animal tumor model and cell experiments gene transfer of vasostatin caused a faster cell growth and tumor development via down-regulation of the tumor suppressor gene and p27. Cell adhesion, spreading, migration and invasion ability were increased in vaso-expressing BON I cells. Transfecting chicken vinculin could reverse the malignant behavior and restored expression of tumor suppressor genes. Moreover, vinculin knockdown could result in a faster cell growth and an increased colony formation. Condition medium taken from hIFN-α2-expressing BON I cells showed significant antiproliferative effects both on the NE tumor cells, BON I and LCC18, and the endothelial cells, PAE. It also suppressed cell adhesion and cell invasion and inhibited angiogenesis on CAM assay. Mice implanted with a mixture of WT BON cells and hIFN-α2-expressing BON cells (1:1) demonstrated significantly lower tumor incidence and longer tumor doubling time. Furthermore, long-acting IFN-α2b (PEGIntron®) demonstrated a better anti-tumor effect in contrast with IFN-α2b (IntronA®). Intratumoral injection of hIFN-α2 plasmids significantly inhibited NE tumor growth and caused tumor regression. We concluded that gene transfer of vasostatin into BON I cells might cause an enhanced malignant tumor behavior. Therefore, vasostatin therapy can not be recommended for patients with NE tumors. Vinculin might play an important role in NE tumor development and growth regulation. Gene therapy by using plasmid DNA carrying hIFN-α2 gene is feasible and promising in NE tumors.

Sex steroid exposure constitutes a risk factor for breast cancer, but little is known about the effects of sex steroids on factors mediating angiogenesis, the development of new blood vessels, in normal and malignant breast tissue. In this thesis we have investigated the effects of estradiol, progesterone, and the nonsteroidal anti-estrogen tamoxifen on vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2) in normal human breast tissue, endothelial cells, and breast cancer. We have applied the technique of microdialysis to provide in situ sampling of estradiol and VEGF in tumors and normal breast tissue of breast cancer patients in vivo. Furthermore, we present a novel method of culturing normal human breast tissue ex vivo. Our results suggest a pro-angiogenic effect of estradiol and an anti-angiogenic effect of tamoxifen in the breast. Estradiol increased extracellular levels of VEGF in normal human breast tissue and breast cancer cells in vitro. In addition, estradiol decreased sVEGFR-1 in breast cancer cells and indirectly increased VEGFR-2 in endothelial cells. Compared to estradiol treatment alone, estradiol + tamoxifen increased sVEGFR-1 and decreased VEGF in breast cancer cells in vitro. Furthermore, estradiol + tamoxifen decreased tumor VEGF levels and tumor vasculature in human breast cancer xenografts in vivo. In breast cancer patients, a significant correlation was found between in vivo levels of estradiol and VEGF sampled by microdialysis in normal human breast tissue, suggesting that estradiol may be a potent regulator of VEGF in the breast in vivo. Tumor levels of VEGF were significantly higher than in normal breast tissue in vivo, supporting the role of VEGF in tumor angiogenesis. For studies of normal human breast, whole breast tissue may be cultured in vitro for up to one week with preserved morphology. Using this method, estradiol, and not progesterone, appears to be the main sex steroid regulator of extracellular VEGF in normal breast tissue. In conclusion, the data suggest that sex steroids and tamoxifen exert pro- and anti-angiogenic effects in normal breast tissue and breast cancer.

No description available.

A major effort to develop anticancer drugs through both empiric screening and rational design of new compounds has been under way for over 30 years (Katzung, 2004). In recent years, research and development in the field of sitespecific drug therapy has progressed significantly. Safe and non-toxic formulations of cytotoxic drugs based on polymers with their improved sitespecific delivery and effective activation to biologically active cytotoxic compounds at the targeted tumours have become a promising approach to cancer therapy. Drug delivery systems based on polymer micelles, coated microand nanoparticles, liposomes and various pro-drug systems including watersoluble polymer–drug conjugates and immunoconjugates have been prepared and extensively studied as novel drug delivery systems designed for cancer chemotherapy. Amongst these drug delivery systems that enable specific drug delivery and release, water-soluble polymer–drug conjugates rank among the most promising, versatile and efficient systems. This dissertation reviews the preclinical testing and pharmacokinetic study of D85, a novel water-soluble macromolecular pro-drug that is a polymer with pHcontrolled methotrexate (MTX) release with potential for treatment of cancer in humans (Ulbrich&Subr, 2004). As MTX is also indicated in low doses for the treatment of chronic inflammatory conditions, the polymer was further tested in an acute inflammatory model to determine whether the polymer would be more effective than MTX in controlling inflammation. The objective of this study was to compare the potency and efficacy of D85 to MTX. D85, a MTX conjugated polymeric lead compound, was designed and synthesised as a potential anti-neoplastic and anti-inflammatory agent. It was initially tested in vitro on three different cancer cell lines where selective toxicity towards the cancer cell cultures compared to primary cell cultures and greater toxicity than MTX was observed. The initial in vitro tests showed very promising results with D85 demonstrating approximately 300 times greater cytotoxicity than MTX against a colon cancer cell line (COLO 320 DM). This high cytotoxic effect warranted further investigation in an in vivo colon cancer tumour model. An induced murine tumour model of COLO 320 DM was successfully developed in nude mice, and the anti-tumour efficacy of D85 tested in this model. The maximum tolerated dose of D85 was established by carrying out an in vivo dose ranging toxicity test in BALB/c mice. The anti-inflammatory effects of D85 were also determined using the carrageenan-induced paw oedema model in rats where carrageenan was injected into a footpad of a rat causing acute oedema, which was measured using a water displacement plethysmometer. D85 was found to exacerbate the inflammatory response. Finally, the pharmacokinetic parameters of MTX and D85 were assessed using a LC/MS/MS method specifically developed and validated to determine low concentrations of MTX in small volumes of plasma. This new method made use of online solid phase extraction and sample cleanup on 2μl injections of diluted plasma allowing an entire pharmacokinetics study to be completed on an individual rat. Fairly similar pharmacokinetics were determined from both compounds. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Pharmacology / unrestricted