<p>Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which T-cells and macrophages invade the islets of Langerhans and selectively destroy the insulin producing β-cells, either directly or through the secretion of e.g. cytokines and nitric oxide (NO). This thesis has studied possible strategies to prevent T1DM. In β-cells and macrophages, NO is produced by inducible nitric oxide synthase (iNOS). </p><p>In the first study, we found that 1400W, a highly selective inhibitor of iNOS could prevent interleukin (IL)-1β induced suppression of rat islet function <i>in vitro</i>, but not diabetes induced by multiple low dose streptozotocin (MLDS), a well established animal model for autoimmune diabetes, <i>in vivo</i>. </p><p>Next, we wanted to test a new type of high affinity blocker of IL-1 action, called IL-1 trap, <i>in vitro</i>. Here we found that an IL-1 trap could prevent the suppressive effects by IL-1β on rat pancreatic islet function. Also, it was sufficient to block the action of IL-1β to prevent islet cell death induced by a combination of IL-1β, tumor necrosis factor-α and interferon-γ.</p><p>In study III, a murine IL-1 trap was found to prolong islet graft survival in the recurrence of disease (ROD) model, a T1DM model that involves syngeneic transplantation of healthy pancreatic islets to diabetic nonobese diabetic mice. Mice treated with IL-1 trap displayed an increased mRNA level of the cytokine IL-4 in isolated spleen cells. This suggests a shift towards Th2-cytokine production, which in part could explain the results. </p><p>Finally, simvastatin an anti-hypercholesterolemic drug that possesses anti-inflammatory properties e.g. by interfering with transendothelial migration of leukocytes to sites of inflammation was studied. We found that the administration of simvastatin could delay, and in some mice prevent, the onset of MLDS-diabetes, and prolong islet graft survival in the ROD model. </p>

Graduation date: 1994

In the healing of cutaneous wounds, paracrine communication between keratinocytes and fibroblasts regulates cell differentiation, proliferation and synthesis of extracellular matrix. Deficient epidermal coverage, as seen in burn-wounds, frequently results in hypertrophic scars. Previous studies suggest that keratinocytes downregulate the production of collagen and profibrotic factors in fibroblasts. We hypothesized that keratinocytes downregulate the expression of the profibrotic factor connective tissue growth factor (CTGF) in fibroblasts, and regulate fibroblast expression of genes important to wound healing. In keratinocyte-fibroblast cocultures, keratinocytes downregulated CTGF mRNA and protein in fibroblasts, through the secretion of interleukin-1 (IL-1) α. Using Affymetrix DNA microarrays, it was demonstrated that factors from keratinocytes regulate the expression of 69 genes important to wound healing. The regulation of 16 of these genes was confirmed by Northern blotting, and IL-1α from keratinocytes regulated all the 16 genes examined. IL-1-mediated CTGF gene regulation was further investigated. Both IL-1 isoforms, α and β, suppressed CTGF expression through an inhibition of CTGF promoter activity. Interestingly, transforming growth factor-β-stimulated Smad phosphorylation was not affected by IL-1. Finally, we hypothesized that CTGF is downregulated in burn wound by split-thickness skin grafting and that the expression of CTGF is suppressed during reepithelialization. The expression of CTGF protein was decreased in successfully skin-grafted wound areas, and increased in open, granulating burn wounds. Moreover, CTGF protein expression was absent beneath the migrating edge of reepithelialization ex vivo. In conclusion, we demonstrate that, in in vitro models, keratinocyte-derived IL-1α regulates the expression of CTGF and other genes with importance to wound healing. Furthermore, it is shown that CTGF expression is suppressed by epidermal wound coverage i burn wounds. These findings may have implications for the understanding of keratinocyte-fibroblast interplay during wound healing and in hypertrophic scar pathogenesis.

Alzheimer’s disease is the most common form of dementia. It is a neurodegenerative disorder characterized by extracellular senile plaques and intracellular neurofibrillary tangles. The main constituent of the senile plaques is the neurotoxic β-amyloid peptide. Surrounding the senile plaques are activated astrocytes and microglia, believed to contribute to neurotoxicity through secretion of proinflammatory cytokines, like interleukin-1β and interleukin-6. For many inflammatory actions, including the cytokine induction in glial cells, the transcription factor NFκB plays a key role. This suggests that therapeutical strategies aimed to control the development of Alzheimer’s disease could include administration of drugs that hinder NFκB activation. The major aim of this thesis was to examine the effects of β-amyloid together with interleukin-1β on cytokine expression as well as NFκB activation in glial cells. The possibility to block NFκB activation, and downstream effects like interleukin-6 expression, by using an NFκB decoy was investigated. The possibility to improve the cellular uptake of the decoy by linking it to a cell-penetrating peptide was also investigated. The results obtained provide supportive evidence that inflammatory cytokines are induced by β-amyloid, and that they can indeed potentiate its effects. The results further demonstrate that by blocking NFκB activation, the induction of interleukin-6 expression can be inhibited. By using an improved cellular delivery system, the uptake of the NFκB decoy and hence the downstream cytokine inhibition could be increased. In conclusion, these results demonstrate the possibility to decrease the inflammatory reactions taken place in Alzheimer’s disease brains, which may ultimately lead to a possible way of controlling this disorder.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which T-cells and macrophages invade the islets of Langerhans and selectively destroy the insulin producing β-cells, either directly or through the secretion of e.g. cytokines and nitric oxide (NO). This thesis has studied possible strategies to prevent T1DM. In β-cells and macrophages, NO is produced by inducible nitric oxide synthase (iNOS). In the first study, we found that 1400W, a highly selective inhibitor of iNOS could prevent interleukin (IL)-1β induced suppression of rat islet function in vitro, but not diabetes induced by multiple low dose streptozotocin (MLDS), a well established animal model for autoimmune diabetes, in vivo. Next, we wanted to test a new type of high affinity blocker of IL-1 action, called IL-1 trap, in vitro. Here we found that an IL-1 trap could prevent the suppressive effects by IL-1β on rat pancreatic islet function. Also, it was sufficient to block the action of IL-1β to prevent islet cell death induced by a combination of IL-1β, tumor necrosis factor-α and interferon-γ. In study III, a murine IL-1 trap was found to prolong islet graft survival in the recurrence of disease (ROD) model, a T1DM model that involves syngeneic transplantation of healthy pancreatic islets to diabetic nonobese diabetic mice. Mice treated with IL-1 trap displayed an increased mRNA level of the cytokine IL-4 in isolated spleen cells. This suggests a shift towards Th2-cytokine production, which in part could explain the results. Finally, simvastatin an anti-hypercholesterolemic drug that possesses anti-inflammatory properties e.g. by interfering with transendothelial migration of leukocytes to sites of inflammation was studied. We found that the administration of simvastatin could delay, and in some mice prevent, the onset of MLDS-diabetes, and prolong islet graft survival in the ROD model.

Dental restorative materials interact with their surrounding oral environment. Interaction factors can be release of toxic components and/or effects on biofilm formation and gingiva. In the end of the nineties, a calcium aluminate cement (CAC) was manufactured as a “bioceramic” alternative to resin composite. Dental ceramics are considered to be chemically stable and not to favour dental biofilm formation. Since the influence of aged, resin-bonded ceramic coverages is not fully investigated and the effect of CAC restorations on the dental biofilm formation and gingival response is unknown, those issues were evaluated in this thesis. With or without oral hygiene, in clinical trials including cervical surfaces of CAC, and approximal surfaces of a leucite-reinforced bonded ceramic; biofilm growth, presence of caries-associated bacteria, clinical expressions of gingivitis, the amounts of gingival crevicular fluid (GCF) and its levels of IL-1α, IL-1β and IL-1 ra were investigated in comparison with resin composite and enamel. In addition, the unknown cytotoxic effect of specimens of CAC on fibroblasts was assessed in vitro. With current oral hygiene a similar biofilm formation and gingival response, as evaluated, were observed at sites of CAC, resin composite and enamel. After ceased oral hygiene, more biofilm was assembled on CAC and on resin composite than on enamel. Neither with, nor without oral hygiene, biofilm formation, presence of caries-associated bacteria, clinical gingivitis and the levels of IL-1α, IL-1β and IL-1 ra differed between sites of ceramic, resin composite and enamel. Higher volumes of GCF were collected at ceramic sites compared to enamel. Fresh specimens of CAC showed the lowest cytotoxic effects on fibroblasts compared with three resin composites, zinc phosphate and glass ionomer cements. In conclusion, the low cytotoxic effect of CAC and the limited increase in dental biofilm formation on that material compared with enamel suggest CAC to be a biocompatible dental material with respect to dental biofilm formation, presence of caries-associated microflora and gingival response. This finding, together with the observation that the influence of bonded ceramic on dental biofilm formation, caries-associated microflora and clinical gingivitis was not different from that of enamel, implicates for both CAC restorations and bonded ceramic that the need of oral hygiene and professional oral health care is not reduced.

<p>Although monophosphoryl lipid A was recently approved by the Food and Drug Administration, more vaccine adjuvants are needed to meet the demand for vaccines against new, emerging, and re-emerging diseases. Additionally, characterizing the mechanisms of action of potent vaccine adjuvants is important for moving toward more rational vaccine design based on the careful selection of antigens and adjuvants to stimulate only the desired immune responses. Two experimental vaccine adjuvants, compound 48/80 (C48/80) and IL-1, were evaluated in these studies. The safety and efficacy of the mast cell activator C48/80 was evaluated when used as an adjuvant delivered intradermally (ID) with recombinant anthrax protective antigen (rPA) in comparison with two well-known adjuvants. Mice were vaccinated in the ear pinnae with rPA or rPA + C48/80, CpG oligodeoxynucleotides (CpG), or cholera toxin (CT). All adjuvants induced similar increases in serum anti-rPA IgG and lethal toxin-neutralizing antibodies. C48/80 induced balanced cytokine production (Th1/Th2/Th17) by antigen-restimulated splenocytes, minimal injection site inflammation, and no antigen-specific IgE. Our data demonstrate that C48/80 is a safe and effective adjuvant, when used by the intradermal route, to induce protective antibody and balanced Th1/Th2/Th17 responses. Histological analysis demonstrated that vaccination with C48/80 reduced the number of resident mast cells and induced an injection-site neutrophil influx within 24 hours. Nonetheless, rPA + C48/80 significantly increased antigen-specific IgG titers in mast cell-deficient mice compared to antigen alone, suggesting that C48/80 has mast cell-dependent and mast cell-independent mechanisms of action.</p><p>IL-1alpha and beta have been shown to have strong mucosal adjuvant activities, but little is known about their mechanism of action. Bone marrow chimeric mice were intranasally vaccinated with Bacillus anthracis lethal factor (LF) with or without 4 µg IL-1alpha or a control adjuvant (cholera toxin) to determine if IL-1R1 expression on stromal cells or hematopoietic cells was sufficient for the maximal adjuvant activity of nasally delivered IL-1alpha. IL-1alpha was not active in IL-1R1-deficient (<italic>Il1r1</italic>-/-) mice given <italic>Il1r1</italic>-/- bone marrow, demonstrating that the adjuvant activity of IL-1 was due to the presence of IL-1R1 and not contaminants. Cytokine and chemokine responses induced by vaccination with IL-1alpha were predominantly derived from the stromal cell compartment and included G-CSF, IL-6, IL-13, MCP-1, and KC. Nasal vaccination of <italic>Il1r1</italic>-/- mice given wild-type bone marrow (WT-->KO) and WT-->WT mice with LF + IL-1alpha induced maximal adaptive immune responses, while vaccination of wild-type mice given <italic>Il1r1</italic>-/- bone marrow (KO-->WT) mice resulted in significantly decreased production of LF-specific serum IgG, IgG subclasses, lethal toxin-neutralizing antibodies, and mucosal IgA compared to WT-->KO and WT-->WT mice (p < 0.05). Our results suggest that IL-1R1 expression in the hematopoietic compartment is sufficient for the maximal induction of antigen-specific adaptive immunity after nasal vaccination adjuvanted with IL-1alpha and that while stromal cells are required for maximal adjuvant-induced cytokine production, the adjuvant-induced stromal cell cytokine responses are not required for effective induction of adaptive immunity.</p> / Dissertation

According to the theory of Traditional Chinese Medicine, the main etiology of stroke results from blood stasis. Ligustic Rhizoma (LR), a Chinese herb, is considered to stimulate stasis-dispelling. As Tetramethyl Pyrazine (TMP) is a major component of LR, the aim of the present study is to investigate the effects of TMP on cerebral infarct. We establish an animal model of cerebral infarct by occluding the both common carotid arteries and right middle cerebral artery for 90 minutes, then reperfusing for 24 hrs. Effects of TMP on cerebral infarct are evaluated by the ratio of infarction areas and modified neurologica severity scale (mNSS). In addition, we observe the changes of ED1, tumor necrosis factor-£\ (TNF-£\) and interleukin-1£] (IL-1£]) immuno-reacting cells in the infarction region. The founding indicate that pre-treatment with TMP 100 mg/kg, 120 mg/kg and 140 mg/kg, and post-treatment with TMP 100 mg/kg will decrease the ratio of cerebral infarction area and the neurological deficit. Moreover, pre-treatment TMP 100 mg/kg also decreases ED1, TNF-£\ and IL-1£] immuno-reacting cell. In conclusion, TMP can decrease cerebral infarction area and neurological deficit. The effects of TMP, at least in part, are closely related to microglia, TNF-£\ and IL-1£], suggesting that TMP can be used to treat stroke in human.

Osteoarthritis (OA) is a degenerative joint disease which has yet no complete treatment with medication. Doxycycline, a well-known antibiotic, has been shown to prevent matrixmetallopreoteinases-MMPs, indicating potency on OA treatment. However, long term systemic use can cause side effects on other tissues. This study aimed to develop controlled drug delivery systems of doxycycline/doxycycline-chondroitin sulfate (D/D-CS) in the form of PCL microspheres for providing a better and new treatment approach via local application. After optimization studies for size, loading efficiency, surface/structure and release properties, microspheres of low Mw PCL (14 kDa) was decided to be more suitable than those of high Mw (65 kDa). The release profile of former was also more compatible with diffusion model than that of latter. The bio-effectiveness of the microspheres was evaluated with three-dimensional in vitro model / osteoarthritic-rabbit chondrocytes embedded in agarose and subjected to interleukin-1&beta / throughout incubations. In vitro treatments with D/D-CS microspheres showed significant reduction in MMP-13 activity compared with untreated OA controls for 15 and 24-day incubations. Although collagen and GAG analysis results showed no enhancement of synthesis with MS treatments, significant decrease in GAG and collagen release from D/D-CS MS treated groups and from D MS treated ones respectively. Overall evaluations of the efficacy using in vivo rabbit OA model showed better radiographic scores and histological outcomes for D/D-CS MS groups compared to only hyaluronan injected and/or untreated controls in 8 weeks. The ex-vivo biomechanical properties of cartilages demonstrated improved hardness with values comparable to healthy group upon application of D-CS MS.

Interleukin-1 (IL-1) is a prototypic multifunctional cytokine. IL-1 family include interleukin-1 a (IL-1 a), interleukin-1b (IL-1 b) and interleukin-1 receptor antagonist (IL-1 Ra). IL-1 b is the archetypeal pleiotropic cytokine which have been produced by many cells and exerting its biological effects on almost all cell types. IL-1 b is the most potent of known agents that are gastric cytoprotective, antiulcer, antisecretory and an inhibitor of gastric emptying. IL-1 Ra competes with IL-1 b for cell surface receptor occupancy. Host genetic factors that affect interleukin-1 (IL-1) have been reported to influence the susceptibility of Caucasians to gastric cancer. Whether Asians have the same genetic susceptibility remains unclear. In this study, the genetic associations of IL-1B and IL-1RN polymorphisms with gastric cancer and duodenal ulcer in Taiwan were evaluated. Genomic DNA from 140 unrelated Taiwanese patients with gastric adenocarcinoma, 94 with duodenal ulcer and 165 ethically matched healthy controls was typed for polymorphisms at positions ¡V31, -511, and +3954 in the IL-1B gene, and the variable number of tandem repeats polymorphisms in intron 2 of the IL-1RN gene. The allele frequencies of IL-1RN 2R in gastric cancer cases were much higher than those in healthy controls (9% vs. 3%, p = 0.781). The allele frequencies of IL-1B ¡V31, IL-1B ¡V511 and IL-1B +3954 did not differ. An increased risk of the development of intestinal type gastric carcinoma was found in IL-1RN 2R carriers with an odds ratio (OR) of 4.06 (95% confidence interval [CI]: 1.68 ¡V 9.79, p-value=0.085). And another increased risk of the development of diffuse type gastric carcinoma was found in IL-1RN 2R carriers with an odds ratio (OR) of 3.15 (95% confidence interval [CI]: 1.16 ¡V 8.56, p-value=0.061). A significant association was found in IL-1RN 2R/4R genotype and the risk of the development of duodenal ulcer, with an odds ratio (OR) of 2.57 (95% CI: 1.03 ¡V 6.38, p = 0.292). No significant relationship was noted in duodenal ulcer patients with IL-1B genotype examed in this study. Additionally, a synergistic interaction between blood type A and IL-1 RN 2R carriers existed in gastric cancer patients (OR= 4.51; 95% CI: 1.20 ¡V 16.88, p-value=0.516). The synergistic interaction was even stronger between blood type O and IL-1 RN 2R carriers of duodenal ulcer patients (OR= 10.3; 95% CI: 2.10 ¡V 50.61, p-value=0.160). In conclusion, the genetic polymorphisms of IL-1RN 2R and blood type A are associated with the development of gastric cancer. The genetic polymorphisms of IL-1RN 2R and blood type O are associated with the development of duodenal ulcer.