<p>The existence of immune self-tolerance allows the immune system to mount responses against infectious agents, but not against self-molecular constitutes. Although self-tolerance is a robust phenomenon, in some individuals as well as in experimental models, the self-tolerance breaks down and as a result, a self-destructive autoimmune disease emerges. The underlying mechanisms for the development of autoimmune diseases are not known, but genetic, environmental and immunological factors are suggested to be involved. In this thesis, we used murine mercury-induced autoimmunity to test this suggestion.</p><p>In susceptible mice mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of IgG1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. In contrast, in resistant DBA/2 (H-2^d) mice, none of these characteristics develop after exposure to mercury. By crossing and backcrossing mercury-resistant DBA/2 mice to mercury susceptible strains, we found that the resistance was inherited as a dominant trait in F1 hybrids and that one gene or a cluster of genes located in the H-2 loci determined the resistance to ANolA production, whereas resistance to the other characteristics was found to be controlled by two or three non-H-2 genes.</p><p>We further put forward the “cryptic peptide hypothesis” to investigate whether mercury and another xenobiotic metal use similar pathway(s) to induce the H-2 linked production of ANolA. We found that while mercury stimulated ANolA synthesis in all H-2 susceptible (H-2^s, H-2^q and H-2^f) mouse strains, silver induced only ANolA responses in H-2^s and H-2^q mice, but not in H-2^fmice. Further studies showed that the resistance to silver-induced ANolA production in H-2^fmice was inherited as a dominant trait.</p><p>We next tested the proposition that mercury induces more adverse immunological effects in mouse strains, which are genetically prone to develop autoimmune diseases, using tight-skin 1 mice, an animal model for human Scleroderma. It was found that in this strain, mercury induced a strong immune activation with autoimmune characteristics, but did not accelerate the development of dermal fibrosis, a characteristic in Tsk/1 mice.</p><p>Finally we addressed the Th1/Th2 cross-regulation paradigm by examining if a Th1-type of response could interact with a Th2-type of response if simultaneous induced in susceptible mice. Our findings demonstrated that mercury-induced autoimmunity (Th2-type) and collagen-induced arthritis (CIA) (Th1-type) can interact in a synergistic, antagonistic or additive fashion, depending on at which stage of CIA mercury is administered.</p>

The existence of immune self-tolerance allows the immune system to mount responses against infectious agents, but not against self-molecular constitutes. Although self-tolerance is a robust phenomenon, in some individuals as well as in experimental models, the self-tolerance breaks down and as a result, a self-destructive autoimmune disease emerges. The underlying mechanisms for the development of autoimmune diseases are not known, but genetic, environmental and immunological factors are suggested to be involved. In this thesis, we used murine mercury-induced autoimmunity to test this suggestion. In susceptible mice mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of IgG1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. In contrast, in resistant DBA/2 (H-2d) mice, none of these characteristics develop after exposure to mercury. By crossing and backcrossing mercury-resistant DBA/2 mice to mercury susceptible strains, we found that the resistance was inherited as a dominant trait in F1 hybrids and that one gene or a cluster of genes located in the H-2 loci determined the resistance to ANolA production, whereas resistance to the other characteristics was found to be controlled by two or three non-H-2 genes. We further put forward the “cryptic peptide hypothesis” to investigate whether mercury and another xenobiotic metal use similar pathway(s) to induce the H-2 linked production of ANolA. We found that while mercury stimulated ANolA synthesis in all H-2 susceptible (H-2s, H-2q and H-2f) mouse strains, silver induced only ANolA responses in H-2s and H-2q mice, but not in H-2f mice. Further studies showed that the resistance to silver-induced ANolA production in H-2f mice was inherited as a dominant trait. We next tested the proposition that mercury induces more adverse immunological effects in mouse strains, which are genetically prone to develop autoimmune diseases, using tight-skin 1 mice, an animal model for human Scleroderma. It was found that in this strain, mercury induced a strong immune activation with autoimmune characteristics, but did not accelerate the development of dermal fibrosis, a characteristic in Tsk/1 mice. Finally we addressed the Th1/Th2 cross-regulation paradigm by examining if a Th1-type of response could interact with a Th2-type of response if simultaneous induced in susceptible mice. Our findings demonstrated that mercury-induced autoimmunity (Th2-type) and collagen-induced arthritis (CIA) (Th1-type) can interact in a synergistic, antagonistic or additive fashion, depending on at which stage of CIA mercury is administered.

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. Topical gene therapy involves administration of the genetic material onto the surface of skin and mucosal membranes. Cationic gemini surfactants (m-s-m, where m represents the carbon atoms in the alkyl tail and s represents the carbon atoms in the spacer) are a novel category of delivery agents with especially high potential for polynucleotides. This is due to their structural versatility, ability to bind and condense DNA, and relatively low toxicity. <p>The objectives were to design, construct and characterize a cationic, non-viral gemini surfactant-based delivery system for an IFN-ã coding plasmid suitable for cutaneous gene therapy and to evaluate this novel therapeutic approach in a Tsk (tight-skin scleroderma) mouse model to determine its clinical feasibility. <p>The delivery systems were characterized by microscopy, dynamic light scattering (DLS), circular dichroism (CD) and small angle X-ray scattering (SAXS). <i>In vitro</i> gene expression was evaluated in PAM 212 keratinocyte culture. The extent of topical delivery of the plasmid using nanoparticle and nanoemulsion formulations was evaluated by measuring IFN-ã levels in CD1, IFN-ã-deficient and Tsk mice. The effect of transgene expression on collagen synthesis was evaluated in Tsk animals by real-time PCR.<p>The <i>in vitro</i> plasmidgeminilipid (PGL) system showed heterogeneous particle size (100-200 nm small particles and 300-600 nm aggregates). Electrostatic interactions between the DNA and PGL systems shifted the negative æ-potential of the DNA (-47 mV) to positive values (30-50 mV). At the same time, condensation of the DNA, and formation of Ø DNA was indicated by the increase of the overall negative signal in the CD spectra, due to the flattening of the 290 nm peak and shift of the 260 nm peak into the negative region in a structure-dependent manner. Lipid organization of the DNADOPE system, in the absence of gemini surfactants, shows hexagonal structure, while addition of gemini surfactant at +/- charge ratio of 10 caused lamellar phase organization. For short spacers (n=3-6), additional Pn3m cubic phase also appear to be present. <p><i> In vitro</i> transfection efficiency in the 12-n-12 series was found to be dependent on the length of the spacer between the two positively charged head groups, with the n=3 spacer showing the highest activity. The PGL systems with 12-3-12 and 12-4-12 led to significantly higher transgene expression compared to the other surfactants of the series. The transfection efficiency significantly correlated with the surface area occupied by one molecule (a). The effect of the tail length influenced the transfection efficiency, with longer tails being associated with higher protein expression. The highest <i>in vitro</i> transfection efficiency was recorded with the 18:1-3-18:1 surfactant (1.4±0.3 ng/5x10E4 cells). <p><i>In vivo</i>, high levels of IFN-ã expression were detected in the skin of animals treated with both nanoparticle (359±239 pg/cm2) and nanoemulsion (607±411 pg/cm2) formulations compared to topical naked DNA (136±125 pg/cm2). IFN-ã levels in the skin of animals injected with 5 ìg DNA were 256±130 pg/cm2. IFN-ã levels in the lymph nodes were higher for the nanoparticle formulation (433±456 pg/animal) compared to nanoemulsion (131±136 pg/animal) suggesting different delivery pathway of the two formulations.<p>IFN-ã expression was at high levels in the skin of Tsk mice after 4-day and 20-day treatments (472±171 and 345±276 pg/cm2). Both 4-day and 20-day treatments reduced the procollagen type I á1 mRNA levels for the topical treatment (64 and 70% reduction) and intradermal injection (58 and 72% reduction). Intercellular adhesion molecule-1 (ICAM-1) was upregulated by 50% in both topically treated and injected animals after 20-day treatment. <p>Here, it has been demonstrated that cationic gemini surfactant-based delivery systems are able to transfect epidermal cells <i>in vivo</i>, and the transgene IFN-ã expression is sufficient to cause significant reduction of collagen in an animal model of scleroderma. It has been shown for the first time that topical gene therapy is a feasible approach for the modulation of excessive collagen synthesis in scleroderma-affected skin.

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. Topical gene therapy involves administration of the genetic material onto the surface of skin and mucosal membranes. Cationic gemini surfactants (m-s-m, where m represents the carbon atoms in the alkyl tail and s represents the carbon atoms in the spacer) are a novel category of delivery agents with especially high potential for polynucleotides. This is due to their structural versatility, ability to bind and condense DNA, and relatively low toxicity. <p>The objectives were to design, construct and characterize a cationic, non-viral gemini surfactant-based delivery system for an IFN-ã coding plasmid suitable for cutaneous gene therapy and to evaluate this novel therapeutic approach in a Tsk (tight-skin scleroderma) mouse model to determine its clinical feasibility. <p>The delivery systems were characterized by microscopy, dynamic light scattering (DLS), circular dichroism (CD) and small angle X-ray scattering (SAXS). <i>In vitro</i> gene expression was evaluated in PAM 212 keratinocyte culture. The extent of topical delivery of the plasmid using nanoparticle and nanoemulsion formulations was evaluated by measuring IFN-ã levels in CD1, IFN-ã-deficient and Tsk mice. The effect of transgene expression on collagen synthesis was evaluated in Tsk animals by real-time PCR.<p>The <i>in vitro</i> plasmidgeminilipid (PGL) system showed heterogeneous particle size (100-200 nm small particles and 300-600 nm aggregates). Electrostatic interactions between the DNA and PGL systems shifted the negative æ-potential of the DNA (-47 mV) to positive values (30-50 mV). At the same time, condensation of the DNA, and formation of Ø DNA was indicated by the increase of the overall negative signal in the CD spectra, due to the flattening of the 290 nm peak and shift of the 260 nm peak into the negative region in a structure-dependent manner. Lipid organization of the DNADOPE system, in the absence of gemini surfactants, shows hexagonal structure, while addition of gemini surfactant at +/- charge ratio of 10 caused lamellar phase organization. For short spacers (n=3-6), additional Pn3m cubic phase also appear to be present. <p><i> In vitro</i> transfection efficiency in the 12-n-12 series was found to be dependent on the length of the spacer between the two positively charged head groups, with the n=3 spacer showing the highest activity. The PGL systems with 12-3-12 and 12-4-12 led to significantly higher transgene expression compared to the other surfactants of the series. The transfection efficiency significantly correlated with the surface area occupied by one molecule (a). The effect of the tail length influenced the transfection efficiency, with longer tails being associated with higher protein expression. The highest <i>in vitro</i> transfection efficiency was recorded with the 18:1-3-18:1 surfactant (1.4±0.3 ng/5x10E4 cells). <p><i>In vivo</i>, high levels of IFN-ã expression were detected in the skin of animals treated with both nanoparticle (359±239 pg/cm2) and nanoemulsion (607±411 pg/cm2) formulations compared to topical naked DNA (136±125 pg/cm2). IFN-ã levels in the skin of animals injected with 5 ìg DNA were 256±130 pg/cm2. IFN-ã levels in the lymph nodes were higher for the nanoparticle formulation (433±456 pg/animal) compared to nanoemulsion (131±136 pg/animal) suggesting different delivery pathway of the two formulations.<p>IFN-ã expression was at high levels in the skin of Tsk mice after 4-day and 20-day treatments (472±171 and 345±276 pg/cm2). Both 4-day and 20-day treatments reduced the procollagen type I á1 mRNA levels for the topical treatment (64 and 70% reduction) and intradermal injection (58 and 72% reduction). Intercellular adhesion molecule-1 (ICAM-1) was upregulated by 50% in both topically treated and injected animals after 20-day treatment. <p>Here, it has been demonstrated that cationic gemini surfactant-based delivery systems are able to transfect epidermal cells <i>in vivo</i>, and the transgene IFN-ã expression is sufficient to cause significant reduction of collagen in an animal model of scleroderma. It has been shown for the first time that topical gene therapy is a feasible approach for the modulation of excessive collagen synthesis in scleroderma-affected skin.