Hematopoietic precursor cells express cell surface receptors for a variety of cytokines and growth factors. These hematopoietic secreted humoral factors can influence the proliferation, differentiation, survival and mobilisation of blood stem cells. Moreover, simultaneous exposure to different combinations of hematopoietic hormones can synergistically affect one or more of these cellular responses. Our laboratory has been involved in studying the role played by insulin-like growth factors (IGFs) in hematopoiesis. IGFs are known to stimulate the expansion of precursor cells from various hematopoietic lineages when administered in vivo. However, difficulties in producing recombinant IGFs with conventional bacterial or yeast expression systems make the use of IGFs as therapeutic hematopoietic agents a cost prohibitive concept. A baculovirus based expression system was developed for the production of large amounts of properly folded and biologically active secreted IGF analogues (BOMIGFs). This system was later adapted for the synthesis of a fusion protein consisting of BOMIGF and intedeukin-3 (BOMIGF-IL-3). The in vitro and in vivo activities of this chimeric molecule were subsequently studied. / The BOMIGF-IL-3 chimera promoted greater thymidine incorporation activity into TF-1 and bovine fetal erythroid cells than observed with the combined administration of BOMIGF and IL-3. This chimera also stimulated the formation of BFU-Es, CFU-GMs and, in particular, the highly proliferative macroscopic colonies in peripheral blood cell hematopoietic colony formation assays. These effects were reproduced in colony formation assays from bone marrow- and spleen-derived colony-forming cells from mice treated with BOMIGF-IL-3. This chimera also helped in the recovery of weight loss, anemia and neutropenia in an AZT-mediated myelossuppression mouse model. The chimera was shown to be significantly better than the corresponding equimolar mixture of the single factors at promoting the survival of TF-1 cells. This effect is associated with a sustained activation of PI-3 kinase, STAT5 phosphorylation and BclxL expression. The chimera was also better than the co-addition of BOMIGF and IL-3 at stimulating the migration of TF-1 cells across Transwell plates. The chimera-dependent potentiation of migration appears to be mediated by at the very least, an enhancement of PI-3 kinase activity. / This recombinant BOMIGF-IL-3 fusion molecule could prove useful for the therapeutic treatment of conditions with decreased production of blood cells such as in AZT- or chemotherapy-induced anemia. Other useful applications may include the mobilisation of stem cells and their ex vivo expansion for the purpose of stem cell transplantation.

Toxic effects have been reported for positively-charged liposomes containing stearylamine (SA-liposomes). We have observed that hemolysis and turbidity changes in native plasma were induced by the presence of SA-liposomes. In order to study the mechanism of the SA-liposomes toxicity to blood, we have examined the interaction of SA-liposomes with erythrocyte ghosts (EG) by resonance energy transfer for the lipid mixing and by Terbium/Dipicolinate (Tb/DPA) assay for the mixing of aqueous contents. The results demonstrate that SA-liposomes and EG, after aggregation, have a tendency to mix their lipid before mixing of their internal contents. This interaction was reduced by the presence of 10$ sp{-3}$ or 10$ sp{-2}$% (w/v) carboxymethyl chitin (CMC). / We have also explored the properties of the association of SA-liposomes with CMC, using fluorescein labeled CMC (Flu-CMC). The polarization value of Flu-CMC increased upon SA-liposomes binding, the amount of Flu-CMC on the liposome surfaces increased with increase in the concentration of phospholipid and SA, and it decreased by 20-40% in the presence of 10 mM Ca$ sp{2+}.$ These results indicate that association of SA-liposomes to CMC reduces the propensity of SA-liposomes to interact with blood components and this association is due to predominantly electrostatic interactions and possibly some hydrophobic interactions between SA-liposomes and CMC. / Finally, using the resonance energy transfer assay, preliminary results indicate that there is lipid mixing between SA-liposomes and platelets. This is not inhibited by phagocytosis inhibitors such as EDTA, 2,4-dinitrophenol with iodoacetate and cytochalasin B, suggesting that the lipid mixing is not accompanied by phagocytosis of SA-liposomes by platelets.

Chronic myelogenous leukemia (CML) is a neoplastic disorder of pluripotent hematopoietic stem cells which follows a biphasic clinical course consisting initially of a relatively benign chronic phase followed by progression to a fatal acute leukemia (CML blast crisis). A clonal cytogenetic abnormality (i.e. the Philadelphia or Ph chromosome), resulting from the reciprocal translocation t(9;22)(q34;q11), is found in over 95% of patients. A hybrid gene is created at the breakpoint of Ph, derived from the ABL proto-oncogene (9q34) and from the BCR gene (22q11), which results in the expression of a 210 KDa fusion protein tyrosine kinase called P210BCR/ABL (P210). Expression of P210 alone is generally believed to be sufficient to induce chronic phase CML but, the deregulation of additional genes appears to be required for progression to CML blast crisis, as inferred by the presence of secondary cytogenetic abnormalities in over 80% of patients. To investigate the potential significance of P210 expression in the induction of genetic instability associated with CML progression, I studied cytogenetic changes in a murine cell line (32D) which expressed P210BCR/ABL from a retroviral vector. Using Giemsa-trypsin banding technique, I found a common marker chromosome t(4;12) in 13 subclones, a second new marker t(2;17) in 3/13 subclones and, additional clonal marker chromosomes in all of the subclones examined. Six subclones consisted of two or three karyotypically distinct cell populations. This study demonstrates that BCR/ABL can directly induce both numerical and structural chromosomal abnormalities in hematopoietic cells. This murine cell model may provide a useful tool to further study the causal relationship of cytogenetic instability and cooperative molecular events involved in the initiation and progression of CML.

The determinants of repolarization in human atrium are poorly understood, and many discrepancies between basic research and clinical observations remain unexplained. This thesis presents a series of studies aimed: (1) to understand factors determining the occurrence of atrial arrhythmias; (2) to evaluate cellular mechanisms of drug actions on arrhythmias; (3) to determine ionic mechanisms controlling atrial repolarization. To achieve these goals we used approaches at three different levels: the whole animal level (with a mapping system), the cellular level (with microelectrode techniques), and the ionic level (with whole-cell patch-clamp techniques). Several antiarrhythmic agents have been used as pharmacological probes to study the mechanisms of drug action in arrhythmias, to assess the properties of desirable drug actions, and to explore the characteristics of drug-channel interactions. (Abstract shortened by UMI.)

The high vulnerability of the newborn brain to oxygen deficit strongly suggests a relative inadequacy in the adaptive cerebral vessel dilation. We hypothesized that this neonatal deficiency might be due to an insufficient brain adenosine production and/or a decreased sensitivity of the cerebral vessels to adenosine, a crucial metabolic regulator of adult cerebral blood flow. This hypothesis was tested on newborn (1-3 day old) and older pigs as the experimental model. Our findings are as follows: (1) Adenosine induced a concentration-dependent vasodilation of the newborn cerebral vessels, especially in the brain stem and periventricular area. (2) The cerebrospinal fluid concentration of adenosine and adenosine metabolites increased during both hypoxic-hypoxia and systemic hypotension. During hypoxia, these levels were inversely correlated to the arterial oxygen content and positively correlated to the increase in cerebral blood flow. (3) Adenosine receptor antagonism by 8-phenyltheophylline completely blocked the cerebral hyperemia during moderate hypoxia, partially reversed it during severe hypoxia, and abolished autoregulation (i.e. the maintenance of a constant cerebral blood flow over a wide range of systemic blood pressure). Moreover, adenosine receptor blockade altered the regional redistribution of cerebral blood flow during severe hypoxia and hypotension. (4) Cerebrospinal fluid concentrations of adenosine during normoxia and hypoxia were lower in the newborn than in the older animals. (5) Relative to adult internal carotid arteries (important cerebral resistance vessels), newborn vessels were less sensitive to adenosine. / These data demonstrate that adenosine is a potent dilator of the cerebral vessels and an important mediator of the regional cerebral adaptive response to brain oxygen deficit in the newborn. Brain interstitial concentrations of adenosine and the cerebral vessel sensitivity to adenosine are lower in the newborn than in the older individual. These findings may explain the relative deficiency in the adaptation of the newborn cerebral vasculature during brain oxygen deficit, leading to an unusual vulnerability to hypoxic-ischemic encephalopathy.

Heterogeneity is a ubiquitous finding in human tissues. Indeed, the cellular heterogeneity of a tumor may be viewed as a caricature of normal tissue that defines the tissue of tumor origin and prognosis. The heterogeneity of a simple tissue, bladder epithelium, has been studied with quantitative flow cytometry to measure light scattering properties and lectin binding to cells in normal and malignant tissue. It was confirmed that such quantitative assessments could be used to reveal relationships of normal tissue heterogeneity to malignant tissue heterogeneity which were relevant to both normal tissue development and tumor status. In addition, an indicator of DNA replication/cell proliferation (a monoclonal antibody for DNA cruciforms) was studied for future use in normal and tumor tissues to reveal various aspects of that functional heterogeneity. Fundamental methods for quantitative assessment and qualitative distribution of the initiation sites of DNA replication were established and applied to normal and malignant cell lines. Such functional analyses of heterogeneity hold the prospect of identification and characterization of tumor stem cell populations.

Polyinosinic: polycytidylic acid (pI:C), an interferon (IFN) inducer, was used to investigate the role of IFN in the graft-versus-host reaction (GVHR), induced by injecting parental cells into F1 recipients and assessed for immunosuppression and pathological lesions. / PI:C-treatment of recipients, but not donors, before GVHR-induction suppressed the GVHR, an effect seen only with C57BL/6 (B6) and not A donor cells. Using fluorescein-labelled donor cells, pI:C-treatment was seen to cause a marked decrease in donor cell survival after 2 days. Elimination of donor cells was specific for the B6 donor, was associated with increased natural killer (NK) cell but not macrophage cytotoxic activity, was radioresistant and anti-asialo GM1 sensitive, evidence supporting NK-mediated rejection. / Plotting donor cell recovery against the number of cells injected into variously treated recipients indicated that in unstimulated mice a constant proportion of the injected cells were rejected, pI:C increasing that proportion, suggesting that pI:C changes F1 NK target repertoire. / PI:C-treatment after GVHR-induction increased the severity of the GVHR, especially soon after GVHR-induction, the effect waning afterwards. No strain dependence was observed. / These results demonstrate that IFN/IFN-activated cells play an important role in the regulation and in the immunosuppression/pathogenesis of a GVHR.

The epididymis is the site for the transport, maturation and storage of spermatozoa. It is well established that epididymal structure and function are dependent on the testis for circulating androgens and factors in luminal fluid. Using cDNA microarrays, our objective is to characterize the expression of genes that are regulated over the first week post-orchidectomy in the epididymis of the adult Brown Norway rat. We describe four patterns of gene expression that are simultaneously activated in the epididymis after orchidectomy. We identify potential androgen-dependent genes including glutathione S-transferases and calcium-binding proteins, as well as potential androgen-repressed genes such as glutathione peroxidase-1. The expression of heat shock proteins, cyclins, and apoptosis-associated genes are described in the epididymis after orchidectomy. These results form the first comprehensive analysis of testis-regulated gene expression in the epididymis. This study is the first to demonstrate that gene expression is differentially regulated in the epididymis after orchidectomy.

Current therapies do not prevent the devastating complications associated with type 1 and 2 diabetes. Novel therapies seek to restore a functional beta-cell mass through stimulating endogenous beta-cell mass expansion. Whilst there is considerable evidence that the beta-cell mass is under homeostatic regulation in the normal pancreas, it is unclear if such regulation exists in the context of induced beta-cell mass expansion. The aim of this study was to demonstrate that beta-cell mass expansion resulting from the induction of islet cell neogenesis is subject to long-term homeostatic control in the normoglycemic mouse adult pancreas. / A pentadecapeptide fragment of Islet Neogenesis Associated Protein (INGAP 104-118) was administered daily to adult C57BL/6J mice for 12 weeks. Four animals from the INGAP104-118 treatment group and control group were sacrificed each week. The pancreas was removed from each mouse and stained for insulin. beta-cell mass was calculated as the organ weight multiplied by the percent of insulin+ area of total tissue area. Contrary to our expectations, there was no change in the total beta-cell mass in INGAP104-118-treated animals compared to control. Reanalysis of the stained tissue sections was preformed, and insulin+ structures were classified as being: (1) a duct islet, (2) a cluster of insulin+ cells, or (3) a mature islet. The density (#/mm2) of duct islets, clusters, and total structures in INGAP 104-118-treated animals was significantly increased; conversely, the density of mature islets was significantly decreased. The increase in cluster density suggests that INGAP104-118 induced neogenesis in the pancreas of treated animals. Poisson regression revealed 9th order polynomial time trends in the structure densities. Though these time trends differed between the classes of structures, they were identical in INGAP104-118 and control animals for each class of structure, suggesting an external stimulus was acting equally on both groups. / While this study did not determine if there is homeostatic regulation of induced beta-cell mass expansion, it did reveal important aspects for the design of a future study to address this issue. The definitions for structure classification must be well-established and rates of beta-cell replication should be determined.

Using the rat as a model we tested the hypothesis that pregnancy might lead to an attenuation of the diuretic/natriuretic effects of atrial natriuretic factor (ANF) to allow physiologically required fluid expansion. Our studies revealed that this was indeed the case. Effects of ANF on fluid and sodium excretion as well as on glomerular filtration rate were significantly reduced during pregnancy This decrease in the renal effects of ANF during pregnancy was not associated with any changes in the pharmacokinetics or renal metabolism of ANF. However, binding studies demonstrated a significant decrease in guanylyl cyclase (GC)-linked ANF receptors and a decrease in ANF stimulated cGMP production in glomeruli and papillae of pregnant rats. The ribonuclease protection assay and Western blot analysis revealed that pregnancy caused a significant decrease in GC-A-linked ANF receptors in renal papillae. It is concluded that pregnancy is associated with attenuation of the renal effects of ANF and this is due to a downregulation of GC-A-linked ANF receptors which might reflect physiological adjustment to facilitate fluid/electrolyte expansion.