Dysregulation of apoptosis during development is a possible mechanism for teratogen-induced birth defects. Neural tube defects (NTDs) are the second most common fetal malformations. Non-specific stimulation of maternal immune system prevents birth defects. This study investigated the role of dysregulated apoptosis in formation of NTDs from two teratogens: valproic acid (VA) and an unknown teratogen found in tap water. Interferon- γ (IFN γ) was used to stimulate maternal immunity to evaluate the role of altered apoptosis in this protective mechanism. Apoptosis was evaluated using flow cytometry, Terminal Transferase dUTP Nick End Labeling (TUNEL) assay and gene expression changes by RT2 Profiler PCR arrays. Additionally, changes in the expression of key signal transduction pathway genes that play a role in development were determined. Increased apoptosis, suggesting involvement in VA teratogenicity, was observed along the neural tube in both normal and abnormal embryos from VA-exposed dams. Increased apoptosis in normal VA-exposed embryos suggests that VA may alter other cellular processes such as cell proliferation and differentiation in addition to apoptosis. Apoptotic percentages in embryos with NTDs from IFNγ+VA dams were similar to controls, which indicated resistance to teratogen-induced apoptosis. In IFNγ+VA-exposed embryos with NTDs, immune stimulation failed to prevent apoptosis. VA initiated both death and survival signaling in the embryos; however, upregulation of the apoptotic genes and down regulation of anti-apoptotic genes of p53 and Bcl2 family tended to shift the balance towards death signaling. This change in gene expression patterns could result in increased apoptosis and NTDs in VA-exposed embryos. Immune stimulation normalized changes in the expression of pro-apoptotic signaling molecules. These results suggest immune stimulation protects embryos from teratogenicity of VA by preventing VA-induced apoptosis. VA altered the hedgehog, Wnt, retinoic acid and fibronectin signaling pathways in embryos with NTDs. These results suggest that VA also disrupted signaling pathways required for various morphogenic events during organogenesis. Immune stimulation normalized the expression of Fn1 and Hspb1 and thus may mediate protection through these signaling pathways. In tap water exposed embryos, no change in apoptotic pattern was observed by flow cytometry, TUNEL assay and RT-PCR. Also, none of the signal transduction pathway genes tested were significantly altered in tap water-exposed embryos. This suggests that apoptosis is not a mechanism for teratogenicity resulting from exposure to the contaminant in tap water. / Ph. D.

Evidence suggests that the neurotropic parasite Toxoplasma gondii may play a role in the development of cognitive impairments. My hypothesis was that congenital exposure to T. gondii would lead to detectable age and sex related differences in behavior and neurotransmitter levels in mice. The neurotransmitter dopamine and commonly used anti-schizophrenic agents were evaluated against T. gondii in human fibroblast cells. Dopamine caused a significant increase in tachyzoite numbers at 250 nM but not 100 nM and the drugs valproic acid, fluphenazine, thioridazine and trifluoperazine inhibited T. gondii development. The effects T. gondii infection had on behavior were examined using a congenital mouse model. Previous work demonstrated maternal immune stimulation (MIS) with interferon gamma (INF-g) resulted in decreased fetal mortality from congenital T. gondii infections; therefore I examined the effects of INF- g treatment of mothers to determine if protection from the behavioral effects of T. gondii occurred in their offspring. No differences in concentrations of neurotransmitters in the brains of congenitally infected mice were observed. I found that mice infected with T. gondii developed adult onset behavior impairments with decreased rate of learning, increased activity and decreased memory, indicating cognitive impairment for male mice and not female mice. My findings support the evidence T. gondii is a factor in the development of cognitive impairments. My results for T. gondii exposed male mice are consistent with the convention that males have more cognitive impairments in the prodromal stage of schizophrenia. MIS with IFN-g had a minimal effect on behavior post sexual maturity but had a greater effect on pre sexual maturity female mice which exhibited difficulties with spatial memory, coordination and the ability to process stimuli. The results indicate the behavior alterations from IFN- g are transient. When MIS is given prior to congenital infection with T. gondii, we detected no behavior deficits in any group of mice, including male mice post sexual maturity. Based on the results of my study, I must reject the hypothesis that neurotransmitter levels are influenced by congenital toxoplasmosis and accept the hypothesis that congenital T. gondii infection caused cognitive impairments in male mice post sexual maturity. / Ph. D.

Diabetic embryopathy is a major complication of pregnant women with type I diabetes. Immune defects in the pathogenesis of diabetic embryopathy have been suggested. We hypothesized that activated immune system can counteract diabetic effect and result in prevention of diabetic embryopathy. Diabetes was induced in pregnant ICR mice by streptozocin injection. Three different techniques of maternal immune stimulation, complete Freund's adjuvant (CFA), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interferon-gamma (IFN-g), were used to stimulate the maternal immune system. Approximately 50% of fetuses from hyperglycemic (>27 mM/L) dams were malformed, with neural tube defects predominating. Maternal immune stimulation during the time of normoglycemia, i.e. prior to onset of hyperglycemia, was necessary for reducing teratogenic effects associated with hyperglycemia. The immune-stimulated diabetic mice then produced significantly lower numbers of malformed fetuses: CFA 20.9%, GM-CSF 23.3%, IFN-g 13.9%. A gene microarray was then used to examine a selected panel of placental and splenic genes. We hypothesized that a shared profile of placental or splenic gene expression changes may correlate to the reduced birth defect outcome induced by the different immune stimulation procedures. Diabetes did not cause significant changes in placenta or spleen gene expression profile. In placenta, CFA and GM-CSF changed placental gene expression relative to control or diabetes, but differentially affected such genes relative to each other; further, IFN-g did not affect gene expression relative to control or diabetes. Thus no common pattern of improved placental cytokine, cell-cycle, apoptotic, transcription factor, or other gene expression was identified in the immune-stimulated mice. In spleen, all 3 immune activators produced a common altered gene expression profile. The overall gene expression profile after all immune stimulation procedures suggested increased splenocyte activity and cytokine production. The cytokine GM-CSF, in particular, was up-regulated in splenic leukocytes. This cytokine has previously been associated with reduced cleft palate in urethane-exposed mice after immune stimulation, and with reduced limb malformations in cyclophosphamide-treated mice after intra-uterine administration. In contrast, the TGF-beta3 gene was down-regulated in immune-stimulated diabetic mice. This gene was up-regulated in urethane-exposed mice, an effect that may be associated with reduced cleft palate. Thus unlike urethane, TGF-beta3 gene expression did not show a relationship with reduced diabetes-induced birth defects. Taken together, these data prove our hypotheses and suggest that mechanistically diverse forms of immune activation result in protection against diabetes-related teratogenesis, but only if given prior to onset of hyperglycemia. Such immune stimulation in mice may act through systemic immune organs, i.e. spleen, over-riding adverse effects of diabetes on development. / Ph. D.

Ferritin is the major protein responsible for the sequestration, storage and release of intracellular iron. The ferritin protein shell exists as heteropolymers of various combinations of two types of subunits, the H-subunit and L-subunit, a phenomenon that gives rise to the existence of isoferritins. As the roles of the H-subunit and L-subunit differ in the mineralization process, the subunit composition of ferritin will influence the metabolic properties of the assembled ferritin molecules. The primary aim of the present study was to quantitatively measure the expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron in patients with chronic T-helper cell type-1 immune stimulation. A second aim was to investigate the possible role that the expression of the H-subunit and L-subunit of ferritin may have in the establishment and maintenance of an iron transfer block. The study subjects included 48 patients with chronic diseases from the Department of Internal Medicine, Kalafong Hospital and 10 patients with osteoarthritis, scheduled for hip replacement at the Department of Orthopaedics, Pretoria Academic Hospital. Bone marrow and blood samples were collected from each patient. The expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron was quantitatively evaluated by post-embedding immunolocalisation with immunogold transmission electron microscopy. The patients were subdivided into groups with a predominantly T-helper cell type-1 immune reaction (pro-inflammatory) and normal immune status on the basis of C-reactive protein, neopterin and cytokines (INF-γ, TNF-α, Il-1β, Il-6, Il-12, Il-2, Il-8, GM-CSF, Il-4, Il-5, TGF-β and Il-10). The study showed • up-regulation of the H-subunit of ferritin in the bone marrow macrophage in patients with chronic T-helper cell type-1 immune stimulation • no effects for chronic T-helper cell type-1 immune stimulation on the expression of the L-subunit of ferritin in the bone marrow macrophage • no effects for chronic T-helper cell type-1 immune stimulation on the expression of either the H-subunit or L-subunit of ferritin in cells of the bone marrow erythron • a 70% prevalence of iron transfer block in patients with chronic T-helper cell type-1 immune stimulation • up-regulation of the H-subunit of ferritin in the bone marrow macrophage in osteoarthritis patients who had normal T-helper cell type-1 immune activity, but significantly increased TGF-β levels • up-regulation of the H-subunit of ferritin in the patients with iron transfer block • iron availability loses its primary role in the establishment of the circulating red blood profile in conditions with chronic pro-inflammatory activity • indications that the H-subunit and L-subunit of ferritin may play a role in the iron availability for red blood cell haemoglobin production • various correlations in the osteoarthritis patients between the H-subunit and L-subunit of ferritin and different cytokines / Thesis (PhD)--University of Pretoria, 2009. / Physiology / unrestricted

L’œuf est un aliment riche en divers composés dont de nombreuses molécules antimicrobiennes qui sont les effectrices de son système de défense moléculaire innée et complètent l’action des immunoglobulines (IgYs) afin de protéger l’embryon. La composition en IgYs de l’œuf est sous la dépendance notamment des stimulations microbiennes de la poule. L’objectif de notre travail a été d’évaluer l’influence de ces mêmes stimulations sur le système de défense moléculaire innée du blanc d’œuf. Nous avons exploré cette hypothèse en utilisant deux approches expérimentales. La première était basée sur la comparaison de l’activité antibactérienne des blancs d’œufs de poules axéniques, de poules exemptes d’organismes pathogènes spécifiques (EOPS), et de poules conventionnelles. La seconde approche a testé les effets de deux types d’inductions du système immunitaire chez la poule: injection d’un immunostimulant, le lipopolysaccharide bactérien (LPS) et administration oral de souches vaccinales atténuées (virale, bactérienne et parasitaire). Nos résultats montrent que l’activité antibactérienne du blanc d’œuf est augmentée en fonction de la charge microbienne du milieu de vie de la poule, après stimulation de celle-ci par voie intraveineuse avec du LPS ou suite à la vaccination avec des souches atténuées virale ou bactérienne. Néanmoins cette augmentation est modérée de par son amplitude et son spectre antibactérien. Ces résultats suggèrent que les poules peuvent renforcer modérément l’activité antimicrobienne du blanc d’œuf en réponse à des stimuli microbiens de leur milieu et anticiper ainsi les besoins de l’embryon en termes de protection. / The egg is a balanced source of different nutrients and contains a myriad of antibacterial peptides/proteins that ensure its chemical protection. These molecules are a part of its innate molecular defense and, in addition to the maternal immunoglobulins IgY, contribute to the protection of the forming embryo whose development occurs ex utero. It is well documented that yolk immunoglobulin deposition is induced by the environmental microbiome of the hen but no such evidence is available for antimicrobial peptides/proteins. Therefore the aim of this thesis was to assess whether the hen has the ability to stimulate the innate molecular immunity of the egg white when facing a higher environmental microbial load (commensal or pathogenic). To address these questions, we developed two main experimental approaches; the first assessed the impact of the hen environmental microbial load through the comparison of three groups of hens with different immune status:-Germ free, -Specific pathogen free (SPF), and -conventional. The second approach explored the effect of different types of immune stimulation in hens: non-infectious stimulation (systemic injection of bacterial lipopolysaccharide (LPS)); immune stimulation using attenuated live vaccines (Infectious bronchitis virus vaccine, Salmonella enterica Enteritidis vaccine and a complex of Eimeria vaccine). Our results show that the activity of egg white is increased in response to higher microbial environmental charge, after LPS systemic stimulation or after vaccinating hens with live attenuated viral and bacterial strains. However this response is moderate both in its amplitude and microbial spectrum. Altogether, it appears that hens when subjected to immune stimuli, have the ability to reinforce moderately the antibacterial activity of the egg white as an attempt to anticipate the need of protection of their embryos.