by Pui-kuen Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 168-182). / ACKNOWLEDGMENT --- p.I / ABSTRACT --- p.II / TABLE OF CONTENTS --- p.IV / ABBREVIATIONS --- p.X / LIST OF FIGURES --- p.XII / LIST OF TABLES --- p.XIV / Chapter CHAPTER1 --- INTRODUCTION AND BACKGROUND --- p.1 / Chapter 1.1 --- ROLE OF CYTOKINES IN MOUSE EMBRYONIC DEVELOPMENT --- p.1 / Chapter 1.1.1 --- Why mouse model --- p.1 / Chapter 1.1.2 --- Embryonic development of mouse --- p.1 / Chapter 1.1.3 --- An overview of cytokines --- p.4 / Chapter a. --- Classes of cytokines --- p.5 / Chapter i) --- Growth factors --- p.5 / Chapter ii) --- Interleukins --- p.7 / Chapter iii) --- Colony-stimulating factors --- p.9 / Chapter iv) --- Interferons --- p.10 / Chapter v) --- Tumor necrosis factor --- p.11 / Chapter b. --- Cytokine networks --- p.12 / Chapter c. --- Role of cytokines in the whole organism --- p.13 / Chapter 1.1.4 --- Cytokine and receptor gene expression in mouse embryonic development --- p.15 / Chapter a. --- Murine embryonic stem cell model --- p.15 / Chapter b. --- Leukemia Inhibitory Factor (LIF) in mouse embryos --- p.16 / Chapter c. --- IL-6 in mouse embryo --- p.19 / Chapter d. --- Ciliary Neurotrophic Factor (CNTF) in mouse embryo --- p.19 / Chapter e. --- TNF-a and TNF-β in mouse embryos --- p.20 / Chapter f. --- TGF-a in mouse embryos --- p.20 / Chapter g. --- TGF-P in mouse embryos --- p.20 / Chapter h. --- Stem cell factor / c-kit --- p.21 / Chapter i. --- Other cytokines in mouse embryos --- p.22 / Chapter j. --- Cytokine receptors --- p.24 / Chapter 1.2 --- NEUROPOIETIC CYTOKINES --- p.28 / Chapter 1.2.1 --- Family members --- p.28 / Chapter 1.2.2 --- Shared signal transducer gpl30 --- p.29 / Chapter 1.2.3 --- "LIF, CNTF and OSM inhibit differentiation of embryonic stem cells" --- p.31 / Chapter 1.3 --- BIOLOGY OF ONCOSTATIN M (OSM) --- p.33 / Chapter 1.3.1 --- Physical properties of OSM --- p.33 / Chapter 1.3.2 --- Biological activities of OSM --- p.34 / Chapter 1.3.3 --- Molecular aspect of OSM --- p.35 / Chapter 1.4 --- AIMS OF THE STUDY --- p.38 / Chapter CHAPTER2 --- CYTOKINE GFNE EXPRESSION DURING MOUSE EMBRYONIC DEVELOPMENT --- p.40 / Chapter 2.1 --- INTRODUCTION --- p.40 / Chapter 2.1.1 --- Rationale --- p.40 / Chapter 2.1.2 --- Design of primers --- p.43 / Chapter 2.2 --- MATERIALS --- p.44 / Chapter 2.2.1 --- Chemicals and Reagents --- p.44 / Chapter 2.2.2 --- Enzymes --- p.45 / Chapter 2.2.3 --- Buffers --- p.45 / Chapter 2.2.4 --- Solutions --- p.47 / Chapter 2.2.5 --- Probe labeling and detection kits --- p.48 / Chapter 2.2.6 --- Primers and internal probes --- p.49 / Chapter 2.3 --- METHODS --- p.52 / Chapter 2.3.1 --- Preparation of total RNA from mouse embryos at different stages --- p.52 / Chapter a. --- Mice dissection for embryo --- p.52 / Chapter b. --- Guanidinium thiocyanate cell lysate --- p.52 / Chapter c. --- Isolation of RNA by centrifugation through CsCl gradient --- p.53 / Chapter d. --- Spectrophotometric determination of RNA amount --- p.54 / Chapter 2.3.2 --- Preparation of embryo sections --- p.54 / Chapter 2.3.3 --- Primers and internal probes --- p.55 / Chapter 2.3.4 --- Cytokine mRNA Phenotyping by Reverse transcription-Polymerase chain reaction --- p.56 / Chapter a. --- Reverse transcription (First strand cDNA synthesis) --- p.56 / Chapter b. --- Polymerase chain reaction (PCR) --- p.56 / Chapter 2.3.5 --- Analysis of PCR products with agarose gel electrophoresis --- p.57 / Chapter 2.3.6 --- Analysis of PCR products with Southern blotting --- p.58 / Chapter a. --- DNA transfer from gel to nylon membrane --- p.58 / Chapter b. --- Probe labeling --- p.61 / Chapter c. --- Prehybridization --- p.61 / Chapter d. --- Hybridization --- p.62 / Chapter e. --- Detection of DIG-labeled probe --- p.62 / Chapter 2.3.7 --- Cycle titration of PCR and dot blotting of regulatory cytokine mRNA --- p.63 / Chapter a. --- Cycle titration of PCR --- p.63 / Chapter b. --- Dot blotting --- p.63 / Chapter 2.4 --- RESULTS --- p.65 / Chapter 2.4.1 --- Sagittal sections of mouse embryos --- p.65 / Chapter 2.4.2 --- Preparation of total RNA --- p.69 / Chapter 2.4.3 --- Cytokine mRNA phenotyping --- p.71 / Chapter a. --- Southern hybridization for 'no expression' cytokines --- p.74 / Chapter b. --- Consistent' and 'regulatory ´ة cytokines in embryo and placenta --- p.79 / Chapter 2.5 --- DISCUSSION --- p.95 / Chapter 2.5.1 --- Isolation of embryo RNA by guanidinium thiocyanate/ cesium chloride centrifugation --- p.95 / Chapter 2.5.2 --- mRNA Quantitation --- p.96 / Semi-quantitative PCR --- p.98 / Chapter 2.5.3 --- Cytokine mRNA phenotyping by RT-PCR --- p.99 / Chapter a. --- Reverse Transcription --- p.99 / Chapter b. --- GAPDH as a control for normalization --- p.100 / Chapter c. --- PCR for cytokine transcripts --- p.101 / Chapter 2.5.4 --- Cytokines and receptors in embryonic development --- p.103 / Chapter 2.5.4.1 --- Cytokines in hematopoietic development of mouse fetus --- p.104 / Chapter 2.5.4.2 --- Other cytokines --- p.113 / Chapter 2.5.5 --- Expression Pattern in placenta: maternal and fetal communication --- p.116 / Chapter CHAPTER3 --- MOLECULAR ANALYSTS OF MOUSE ONCOSTATIN M --- p.117 / Chapter 3.1 --- INTRODUCTION --- p.117 / Chapter 3.2 --- MATERIALS --- p.121 / Chapter 3.2.1 --- Chemicals and Reagents --- p.121 / Chapter 3.2.2 --- Enzymes --- p.121 / Chapter 3.2.3 --- Buffers --- p.122 / Chapter 3.2.4 --- Solutions --- p.122 / Chapter 3.2.5 --- Culture media --- p.124 / Chapter 3.2.6 --- Competent cell --- p.125 / Chapter 3.2.7 --- DNA materials --- p.125 / Chapter 3.2.8 --- Primers --- p.126 / Chapter 3.3 --- METHODS --- p.127 / Chapter 3.3.1 --- Primers and internal probes --- p.127 / Chapter 3.3.2 --- Cloning of human Oncostatin M exon 2 and exon 3 by PCR --- p.127 / Chapter 3.3.3 --- Subcloning of human OSM exons 2 and 3 into pUC18 --- p.128 / Chapter a. --- Preparation of human OSM exons and plasmid --- p.128 / Chapter i) --- Purification of PCR products --- p.128 / Chapter ii) --- T4 DNA polymerase ´بblunt-end´ة reaction for PCR products --- p.129 / Chapter iii) --- Sma I digestion of pUC18 --- p.129 / Chapter b. --- Ligation --- p.129 / Chapter c. --- Preparation of competent cell --- p.130 / Chapter d. --- Transformation --- p.131 / Chapter e. --- Screening of recombinants by PCR --- p.131 / Chapter f. --- Screening of recombinants by restriction enzyme digestion --- p.132 / Chapter i) --- Preparation of plasmids --- p.132 / Chapter ii) --- Double restriction enzymes digestion of pUC18 --- p.133 / Chapter 3.3.4 --- Verification of the clones of human OSM exons 2 and 3 by cycle sequencing --- p.135 / Chapter 3.3.5 --- Purification of human OSM exons from plasmid for making probe --- p.136 / Chapter 3.3.6 --- Southern blotting --- p.136 / Chapter a. --- Probe making and labeling --- p.136 / Chapter b. --- Preparation of mouse genomic DNAs --- p.137 / Chapter c. --- DNA transfer --- p.138 / Chapter i) --- Digestion of genomic DNA with restriction endonucleases --- p.138 / Chapter ii) --- Gel electrophoresis and DNA blotting --- p.139 / Chapter d. --- Hybridization --- p.139 / Chapter 3.4 --- RESULTS --- p.142 / Chapter 3.4.1 --- Cloning of human OSM exon 2 and exon 3 by PCR --- p.142 / Chapter 3.4.2 --- Subcloning of human OSM exons 2 and 3 into pUC18 --- p.142 / Chapter a. --- Screening of recombinants by PCR --- p.142 / Chapter b. --- Screening of recombinants by restriction enzymes digestion --- p.143 / Chapter 3.4.3 --- Sequence of subcloned exons 2 and3 --- p.147 / Chapter 3.4.4 --- Southern hybridization --- p.149 / Chapter a. --- Genomic DNA preparation --- p.149 / Chapter b. --- Digestion of genomic DNAs --- p.151 / Chapter c. --- Hybridization signal --- p.154 / Chapter 3.5 --- DISCUSSION --- p.158 / Chapter 3.5.1 --- Cross-species hybridization --- p.158 / Chapter 3.5.2 --- Hybridization of human OSM exon fragments against mouse genome --- p.158 / Chapter a. --- hOSM exon 2 as probe --- p.158 / Chapter b. --- hOSM exon 3 as probe --- p.160 / Chapter c. --- Feasibility of using hOSM as probe for fishing out the mOSM gene --- p.160 / Chapter d. --- The cloning of mouse OSM by Yoshimura's group --- p.161 / Chapter CHAPTER4 --- CONCLUSION --- p.162 / Chapter 4.1 --- SUMMARY OF CYTOKINE AND CYTOKINE RECEPTOR GENES EXPRESSION DURING EMBRYONIC DEVELOPMENT --- p.162 / Chapter 4.2 --- FURTHER STUDIES OF THE CYTOKINE ACTIONS ON EMBRYOGENESIS --- p.165 / Chapter 4.3 --- MOLECULAR ANALYSIS OF MOUSE OSM GENE --- p.167 / REFERENCES --- p.168

Lung airway epithelium is the first line of defense against inhaled particulates such as cigarette smoke. Subepithelial dendritic cells (DC) survey the airway epithelial lining and represent the link between innate and adaptive immune response. No study has investigated the effect of cigarette smoke on DC in the presence of epithelial cells (EC). The purpose of this 4x2 factorial design study was to co-culture normal human bronchial epithelial (NHBE) cells with monocyte-derived dendritic cells (MDDC) and examine the effect of cigarette smoke condensate (CSC) and poly I:C stimulation (TLR3 ligand that mimics viral infection) on MDDC homotypic clustering, phagocytosis ability, surface marker expression, DC cytokine response, T cell (TC) proliferation and TC cytokine response. Experiments were performed with MDDC and TC derived from four individual donors. Two planned comparisons (DMSO vehicle control compared to CSC high dose in both the no poly I:C and poly I:C stimulated groups) were analyzed. In MDDC stimulated with CSC, there was a significant increase in homotypic clustering, reduced phagocytosis, increased CD54, increased CD83 and CD86 maturation marker expression. Although no significant changes were observed in MDDC cytokine production, IL10 exhibited a trend to increase with CSC exposure but failed to reach statistical significance. Despite evidence that CSC exposed MDDC are maturing, there was no increase in TC proliferation; however in poly I:C stimulated co-cultures, CSC exposure increased IL2, IL5, IL10 and IL13 expression while non-stimulated co-cultures exhibited an increase in IL10 following CSC exposure. This study indicates that cigarette smoke has the ability to decrease phagocytosis ability of DC and increases co-stimulatory maturation markers without inducing TC proliferation and potentiating a Th2 environment. These findings suggest that DC of smokers may be less likely to mount a normal immune response to invading pathogens thus providing evidence for smoker susceptibility to infection and how DC of smokers may respond to viral infection. In addition, these findings are particularly important in patients with allergic airway disease since increasing Th2 cytokines would increase the risk for disease exacerbation.

Objectives: Three-dimensional (3D) bioprinting is a manufacturing process that incorporates viable cells into a 3D matrix by adding layer upon layer of material. The objectives of this study are to characterize a novel matrix of collagen and hydroxyapatite and to assess the effects of the 3D bioprinting process on cytotoxicity, proliferation rate, and cytokine expression of Homo sapiens palatal mesenchyme (HEPM) cells. Methods: For this, we prepared a 3D matrix of collagen and hydroxyapatite without and with cells. We used light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) to characterize the structure and arrangement of the collagen fibers. We then incubated the matrix with known standards of cytokines to measure adsorption. We assessed the proliferation and viability of HEPM cells in the presence of the 3D construct and after 3D bioprinting. Finally, we assessed the cytotoxicity of this matrix for HEPM cells and assessed its effect on the production of chemokines and cytokines. A one-way fixed effect ANOVA was fit to concentrations of cytokines and pairwise group comparisons were conducted using Tukey’s Honest Significant Differences test (p< 0.05). Results: The matrix was found to contain interwoven strands of collagen and some hydroxyapatite crystals that did not absorb cytokines except for MIP-1a (p< 0.05). The matrix was found to be non-cytotoxic using an AlamarBlue® assay. We found that the cell proliferation rate was lower when seeded on the 3D construct than in 2D culture. We also found that the proliferation rate was very low for the HEPM cells in the 3D bioprinted constructs. In the presence of the 3D construct, the HEPM cells had similar expression profiles of the cytokines measured (P > 0.05 for GM-CSF, IL-6, IL-8, and RANTES). Conclusion: 3D-bioprinting has the potential to be used in dentistry as a novel osteogenic bone grafting material. Here we show that a novel matrix of collagen and hydroxyapatite is non-cytotoxic to HEPM cells and does not induce a proinflammatory response.

A precious treasure in traditional Chinese medicine (TCM), acupuncture played a vital and irreplaceable role in contributing to people’s health in the thousands of years of Chinese history, and in 2010 was officially added to the “Representative List of the Intangible Cultural Heritage of Humanity” by the United Nations. Because of the side-effects of long-term drug therapy for pain, and the risks of dependency, acupuncture has been widely accepted as one of the most important alternative choice therapies for treating varieties of acute and chronic pain-related disorders. The clinical application and scientific mechanism research of acupuncture have therefore increased intensively in the last few decades. Besides hand acupuncture, other treatment approaches e.g. electroacupuncture (EA) have been widely accepted and applied as an important acupuncture-related technique for acupuncture analgesia (AA) research. The involvement of opioid peptides and receptors in acute AA has been shown via pre-EA application of opioid receptor/peptide antagonists. However, existing publications still cannot illuminate the answer to the following question: how does sustained antinociception happen by EA treatment? The hypothesis of opioid peptide-mediated tonic AA might be able to answer the question. In the first part of this thesis, the institution of a reproducible acupuncture treatment model as well as the endogenous opioid-related mechanisms was demonstrated. An anatomically-based three-dimensional (3D) rat model was established to exhibit a digital true-to-life organism, accurate acupoint position and EA treatment protocol on bilateral acupoint GB-30 Huantiao. The optimal EA treatment protocol (100 Hz, 2-3 mA, 0.1 ms, 20 min) at 0 and 24 h after induction of inflammatory pain by complete Freund’s adjuvant (CFA) on conscious free-moving rats was then established. EA elicited significant sustained mechanical and thermal antinociception up to 144 h. Post-EA application of opioid receptors (mu opioid receptor, MOR; delta opioid receptor, DOR) antagonists naloxone (NLX) and naltrindole (NTI), or opioid peptide antibodies anti-beta-endorphin (anti-END), met-enkephalin (anti-ENK) or -dynorphin A (anti-DYN) could also block this effect at a late phase (96 h) of CFA post-EA, which suggested opioid-dependent tonic analgesia was produced by EA. Meanwhile, EA also reduced paw temperature and volume at 72-144 h post CFA indicating anti-inflammatory effects. Nociceptive thresholds were assessed by paw pressure threshold (Randall-Sellito) or paw withdrawal latency (Hargreaves) and an anti-inflammatory effect was evaluated by measurement of plantar temperature and volume of inflamed paw. The second part of the thesis further suggests the correlation between the chemokine CXCL10 (= interferon-gamma inducible protein 10, IP-10) and opioid peptides in EA-induced antinociception. Based on a comprehensive Cytokine Array of 29 cytokines, targeted cytokines interleukin (IL)-1alpha, interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, interleukin (IL)-13, interferon (IFN)-gamma as well as CXCL10 were selected and quantified by enzyme-linked immunosorbent assay (ELISA), and real time reverse transcription-polymerase chain reaction (RT-PCR) quantification confirmed upregulation of CXCL10 mRNA at both 72 and 96 h. The following hyperalgesic assessment suggested the antinociceptive effect of CXCL10. The double immunostaining localizing opioid peptides with macrophages expressed the evident upregulation of CXCR3-receptor of CXCL10 in EA treated samples as well as the significant upregulation or downregulation of opioid peptides by repeated treatment of CXCL10 or antibody of CXCL10 via behavioral tests and immune staining. Subsequent immunoblotting measurements showed non-alteration of opioid receptor level by EA, indicating that the opioid receptors did not apparently contribute to AA in the present studies. In vitro, CXCL10 did not directly trigger opioid peptide END release from freshly isolated rat macrophages. This might implicate an indirect property of CXCL10 in vitro stimulating the opioid peptide-containing macrophages by requiring additional mediators in inflammatory tissue. In summary, this project intended to explore the peripheral opioid-dependent analgesic mechanisms of acupuncture with a novel 3D treatment rat model and put forward new information to support the pivot role of chemokine CXCL10 in mediating EA-induced tonic antinociception via peripheral opioid peptides. / Als wertvoller Schatz in der traditionellen chinesischen Medizin (TCM) spielt die Akupunktur eine wichtige und unersetzliche Rolle für die Gesundheit der Menschen in der über tausendjährigen Geschichte von China und wurde im Jahr 2010 offiziell in das "Weltkulturerbe" der Vereinten Nationen aufgenommen. Aufgrund der Nebenwirkungen von Langzeittherapien zur Schmerzbehandlung und dem Risiko der Abhängigkeit wird Akupunktur weithin als eine wichtigste Alternative für die Behandlung von akuten und chronischen Schmerzen eingesetzt. Die klinische Anwendung und Forschung in der Akupunktur wurden in den letzten Jahrzehnten intensiv vorangetrieben. Neben Handakupunktur gibt es noch andere Behandlungsmöglichkeiten, wie z.B. die Elektroakupunktur (EA). EA ist vor allem eine allgemein akzeptierte und wichtige akupunkturbezogene Technik für die Akupunkturanalgesie (AA) in der Forschung. Die Beteiligung von Opioidpeptiden und Opioidrezeptoren in der akuten AA wurde mittels Anwendung von Opioidrezeptorantagonisten/Opioidpeptidantikörpern appliziert vor EA gezeigt. Nach dem aktuellen Forschungsstand kann man jedoch nicht die Frage beantworten, wie die längerfristige (tonische) Antinozizeption nach EA-Behandlung funktioniert. Mit einer Hypothese zur Opioidpeptid vermittelten tonischen AA könnte man die Frage hierzu beantworten. In der vorliegenden Arbeit wurden in einem Modell der Akupunktur zum ersten Mal endogene Opioid-vermittelte Mechanismen reproduzierbar nachgewiesen. Es wurde ein dreidimensionales (3D) anatomisch-basiertes Rattenmodell entworfen, um am wachen Tier ein genaue Akupunkturbehandlung (EA) an den Akupunkten GB-30 Huantiao beidseitig durchzuführen. Darüber hinaus wurde ein optimiertes Behandlungsprotokoll von EA (100 Hz, 2-3 mA, 0.1 ms, 20 min) bei Ratten 0 und 24 h nach intraplantarer Injektion von komplettem Freunds Adjuvans (CFA) etabliert. Nozizeptive Schwellen wurden mittels Pfotendruckschwelle (Randall-Sellito) oder Pfotenrückzugslatenzzeit (Hargreaves) gemessen und die entzündungshemmende Wirkung durch Messung der Pfotentemperatur und Volumen der entzündeten Pfote ausgewertet. EA bewirkte eine signifikante mechanische und thermische Antinozizeption, welche bis zu 144 h anhielt. Die antinozizeptive Wirkung durch EA war nach Injektion von Opioidrezeptorantagonisten (mu opioid receptor, MOR; delta opioid receptor, DOR) Naloxon (NLX) und Naltrindol (NTI) oder Antikörpern gegen die Opioidpeptide beta-Endorphin (anti-END), Met-Enkephalin (anti-ENK) oder Dynorphin A (anti-DYN) während der späten Entzündungsphase (96 h) mit CFA blockierbar. Dies lässt auf eine durch EA induzierte tonische Analgesie schließen. Darüber hinaus reduzierte EA auch die erhöhte Pfotentemperatur und das erhöhte Pfotenvolumen, welche sehr typisch ist für eine 96-144 h Entzündung mit CFA. Dies spricht für eine entzündungshemmende Wirkung von EA. Nachfolgend wurde die Beteiligung von Opioidpeptiden und dem Chemokin CXCL10 (= Interferon-gamma induziertes Protein 10, IP-10) sowie die Korrelationen zwischen beiden geklärt. Basierend auf umfangreichenden Zytokinarrays mit 29 Zytokinen, wurden die Zytokine Interleukin (IL)-1alpha, Interleukin (IL)-1beta, Tumornekrosefaktor (TNF)-alpha, Interleukin (IL)-4, Interleukin (IL)-13, Interferon (IFN)-gamma und CXCL10 gezielt ausgewählt und im Enzym Immunoassay (ELISA) sowie durch eine Echtzeit Reverse Transkription-Polymerase Kettenreaktion (RT-PCR) quantifiziert. Die Quantifizierung auf mRNA-Ebene zeigte eine Hochregulation von CXCL10 bei 96 h und zum früheren Zeitpunkt von 72 h. Nachfolgende Schmerzschwellenmessungen wiesen auf eine antinozizeptive Wirkung von CXCL10 hin. Eine Doppelimmunfärbung zeigte die Lokalisation von Opioidpeptiden in Makrophagen. Nachweislich wurde die Expression des CXCR3-Rezeptor von CXCL10 in EA behandelten Pfoten erhöht. Ebenso kam es zu einer signifikanten Hochregulation von Opioidpeptiden durch wiederholte Behandlung mit CXCL10 bzw. Herunterregulation der Opioidpeptide nach wiederholter Behandlung mit anti-CXCL10 Antikörper. Dies wurde sowohl in Verhaltenstests als auch in der Immunfärbung zu beobachtet. Immunoblotting zeigte keine Veränderung der Expression von Opioidrezeptoren nach EA, was schließen lässt, dass die Menge an Opioidrezeptoren in den vorliegenden Untersuchungen keine Rolle spielen. Da CXCL10 in vitro keine direkten Effekt auf die Freisetzung des Opioidpeptides beta-END aus frisch isolierten Rattenmakrophagen hat, liegt die Vermutung nahe, dass CXCL10 in vitro eine indirekte Rolle als Mediator zukommt und indirekt die Opioidpeptidfreisetzung aus Makrophagen in entzündlichen Gewebe stimuliert wird. Zusammenfassend wurden in dem hier vorgestellten Projekt die Opioidpeptid-abhängigen analgetischen und peripheren Mechanismen der Akupunktur mit einem neuartigem 3D-Behandlungsmodell der Ratte untersucht und eine Schlüsselrolle des Chemokins CXCL10 bei der Vermittlung der EA-induzierten tonischen Antinozizeption in Abhändigkeit von peripheren Opioidpeptiden. (Translated by Dr. Dagmar Hackel and revised by Priv.-Doz. Dr. Heike Rittner)

Bei der Implantation des Fetus in den Uterus und während der Schwangerschaft kommt es zu einer Interaktion fetaler Trophoblastzellen mit dem mütterlichen Immunsystem. Trotz vieler verschiedener Studien ist bis heute nicht grundlegend geklärt, welche Mechanismen zur immunologischen Akzeptanz des (semi-)allogenen Fetus durch die Mutter beitragen. Zur wechselseitigen Kommunikation zwischen Kind und Mutter tragen Zytokine bei, die von allen immunkompetenten Zellen sezerniert werden können und regulatorisch auf die Immunantwort wirken. Eine Störung im Gleichgewicht der Zytokine kann zu Aborten, Präeklampsie oder anderen Pathologien führen. Eine wichtige Quelle von Zytokinen stellen u.a. die reifen und unreifen dendritischen Zellen (DC), die in der humanen Dezidua nachgewiesen wurden, dar. DC übernehmen als effiziente Antigen präsentierende Zellen eine entscheidende Funktion im Immunsystem und können inflammatorische Immunantworten induzieren. Jedoch spielen sie auch eine wichtige Rolle bei der Vermittlung immunologischer Toleranz. Die menschliche Plazenta weist eine auffällig starke Expression verschiedener humaner endogener Retroviren (HERV) auf. Immunmodulierende Eigenschaften von HERV wurden bereits beschrieben, jedoch nicht die direkte Wirkung von HERV-Proteinen der Plazenta auf Zellen des Immunsystems. Im Rahmen der Arbeit sollte daher die Wirkung des Hüllproteins des HERV-K, das in den Zytotrophoblastenzellen und in den Zellen des extravillösen Trophoblasten nachgewiesen wurde, auf die Zytokinproduktion unreifer (iDC) und reifer DC (mDC) untersucht werden. Als Modellsystem wurden in-vitro aus Monozyten des peripheren Blutes differenzierte DC gewählt. Die DC wurden in unreifem und reifem Zustand mit unterschiedlichen Konzentrationen von HERV-K-Peptiden (rekombinante Proteine (TM05.04 und TM12.12) bzw. Peptide aus 22 Aminosäuren (K120 und K177)) behandelt. Untersucht wurden die Veränderungen der Zytokinspiegel in den Zellkulturüberständen mittels Cytometric Bead Assay und Durchflusszytometrie. Die Messungen zeigten z.T. signifikante Veränderungen der Zytokinproduktion. So wurde die TNF-α-Sekretion der mDC durch K120 und K177 signifikant vermindert. Dieselben Peptide supprimierten ebenfalls signifikant die IL-8-Sekretion der mDC. Jedoch kam es durch alle vier HERV-Peptide (bei drei Peptiden signifikant) zu einer Steigerung der IL-8-Produktion in den iDC. Auch IL-6 wurde von den iDC durch HERV-K mehrheitlich signifikant vermehrt ausgeschüttet. Bzgl. IL-6 ergaben sich jedoch keine signifikanten Veränderungen in den mDC. In allen Ansätzen kam es zu einer konzentrationsabhängigen Stimulation der Sekretion des immunsuppressiven IL-10 (bei je drei Peptiden signifikante Ergebnisse). Keine signifikanten Veränderungen ergaben sich für IL1-β und IL-4. Die Erkenntnis, dass die HERV-Peptide die Zytokinproduktion der DC z.T. signifikant modulieren und diese Veränderung in den unterschiedlichen Reifestadien der DC variieren, lässt vermuten, dass die in der humanen Plazenta exprimierten Proteine einen Einfluss auf den Verlauf einer Schwangerschaft nehmen. Bei einer Schwangerschaft sind extrem fein abgestimmte, komplexe Vorgänge, bei denen viele verschiedene Faktoren eine Rolle spielen, für einen Erfolg vonnöten. Eine Übertragung der in-vitro-Ergebnisse auf in-vivo-Zustände ist nicht leicht zu vollziehen. Die vorliegenden Ergebnisse sprechen jedoch für einen Einfluss des HERV-K auf die Kommunikation zwischen Fetus und Mutter. Inwiefern genau und wie wichtig bzw. essentiell die Expression der HERV-K-Peptide für einen physiologischen Verlauf der Schwangerschaft ist, ob sie einen Einfluss auf Pathologien während der Gestation hat und ob dem HERV-K eine Bedeutung in der humanen Evolution zukommt, kann mit dieser Arbeit nicht geklärt werden. Jedoch gibt diese Arbeit Anlass dafür, den Einfluss des HERV-K auf die menschliche Fortpflanzung weitergehend zu untersuchen. / During the implantation of the fetus in the uterus and during pregnancy fetal trophoblast cells interact with the maternal immune system. Despite a multitude of studies, so far it is not completely clear, which mechanisms contribute to the immunological acceptance of the (semi)allogeneic fetus by the mother. Cytokines are vital to an intact communication between child and mother. They have a significant regulatory influence on the maternal immune response. A disturbance in the balance of cytokines can lead to miscarriage, preeclampsia or other pathologies. Among others, an important source of cytokines are the mature and immature dendritic cells (DC), which were detected in the human decidua. DC have a crucial function in the immune system: They are efficient antigen presenting cells and are able to induce inflammatory immune responses. However, they also play an important role in the mediation of immunological tolerance. The human placenta shows a remarkably high expression of various human endogenous retroviruses (HERV). Immunomodulating properties of HERV have been described. The direct effect of placental HERV proteins on cells of the immune system, however, has not been analyzed yet. Hence this study examines the effect of the transmembrane envelope protein of HERV-K, which was detected in cytrophoblast and extravillous trophoblast cells, on the cytokine production of human immature (iDC) and mature (mDC) DC. The significant changes in cytokine release suggest that the expression of the HERV-K peptides have an influence on the course of human pregnancy.

It has been recently demonstrated that the cytokine interleukin (IL)-6 is unique among the so called

Lyme borreliosis (LB) is the most common vector-borne disease in the Northern Hemisphere. The infection is caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex, and it is transmitted to humans by ticks. LB is associated with several clinical manifestations, of which erythema migrans (EM) and neuroborreliosis (NB) are the most common inEurope. The course of the disease is usually benign, but can vary between individuals. The underlying pathogenic mechanisms are not fully understood, but the prognosis is probably determined by a complex interplay between the bacteria and the host’s immune response. Previous studies have indicated that a strong initial T helper (Th) 1-response followed by a Th2 response is beneficial for the clinical outcome in LB. The aims of this thesis were to follow the incidence of NB inJönköping County,Sweden, over time, to search for clinical and laboratory markers associated with the risk of developing long-lasting post-treatment symptoms, and to explore the role of the complement system as well as the relative balance between Th-associated cytokine/chemokine responses in LB. The number of NB cases, diagnosed by cerebrospinal fluid (CSF) analysis, increased from 5 to 10/100,000 inhabitants/year in Jönköping County during 2000-2005. Post-treatment symptoms persisting more than 6 months occurred in 13 %, and were associated with higher age, longer-lasting symptoms prior to treatment, higher levels of Borrelia-specific IgG in CSF, and reported symptoms of radiculitis. Facial palsy, headache and fever were frequent manifestations in children, whereas unspecific muscle and joint pain were the most commonly reported symptoms in older patients. Complement activation occurred both locally in the skin in EM and in CSF of NB patients. However, no activation could be detected in blood in NB patients. Elevated levels of C1q, C4 and C3a in CSF, along with correlation between C1q and C3a levels, suggest complement activation via the classical pathway locally in the central nervous system in NB. In vitro experiments with two clinical Borrelia isolates revealed that B. garinii LU59 induced higher complement activation in human plasma compared to B. afzelii K78 that recruited more of complement regulator factor H. To elucidate the role of complement in the phagocytosis process, experiments were performed using whole blood from healthy donors incubated with fluorescence-labelled spirochetes and different complement inhibitors. The results illustrated a central role of complement for phagocytosis of Borrelia spirochetes. We also studied the relative contribution of different Th-associated cytokines/chemokine responses in NB. The results support the notion that early NB is dominated by a Th1 response, eventually accompanied by a Th2 response. IL-17A was increased in CSF in half of the patients with confirmed NB, suggesting a hitherto unknown role of Th17 in NB. In conclusion, the risk of developing long-lasting post-treatment symptoms tend to increase mainly with age and duration of symptoms prior to treatment in NB. The complement system seems to play an important role in host defence to recognize and kill Borrelia spirochetes. However, complement activation in inappropriate sites or to an excessive degree may cause tissue damage, and therefore, the role of complement in relation to disease course needs to be studied further. Likewise, the role of Th17 in LB pathogenesis and host defence should be further evaluated in prospective studies.

Immunomodulatory effects of lactic acid bacteria vary with strain and may vary with growth phase and medium. The ability of different lactobacilli strains (Lactobacillus helveticus R0052, L. rhamnosus R0011, L. rhamnosus GG) at different growth phases to modulate macrophage and intestinal epithelial cell cytokine production following a pro-inflammatory challenge was examined. Modulation of cytokine production by human macrophage cell lines (U-937) and intestinal epithelial cells (HT-29) induced by Tumor Necrosis Factor α was assayed by ELISA for interleukin-8 (IL-8). Granulocyte-macrophage colony stimulating factor (GM-CSF) production was assayed by ELISA in the HT-29 cell line. Strain-dependent differences were observed in the ability of viable bacteria and spent de Mann-Rogosa- Sharpe (MRS) broths from log versus stationary growth phase in HT-29 and U-937 cells. Overall, variation in the immunomodulatory activity of these lactic acid bacteria and spent broths reflects not only strain variation but potentially also differences in growth phase and substrate. / UOIT

The pathology of multiple sclerosis (MS) is characterized by an inflammatory mononuclear infiltration in the white matter. There has been converging evidence of the oxidative stress playing a role in the onset and progression of MS. We postulated that the decreasing oxidative stress might help in the management of MS. We know that the induction of phase 2 enzymes decreases the oxidative stress. The experimental allergic encephalomyelitis (EAE) induced in the Lewis rats were used to test this hypothesis. The 24 animals were placed into two groups: 1) those on normal rat chow, 2) those on rat chow containing 7.5 g/kg of tetra-butylhydroxyanisole (BHA), a food preservative. All the animals were administered 100 µg of guinea pig myelin basic protein in their tails to induce EAE and examined daily in a double blinded fashion. On 29th day of the induction, the animals were sacrificed, blood collected for glutathione (GSH) measurements and tissues collected for histology. All the animals, regardless of their diet status, developed symptoms of EAE on different days ranging from tail weakness to hind limb paralysis and all of them reached remission of acute EAE before the 28th day of induction. The non-BHA fed animals developed hind limb weakness in 8 animals and hind limb paralysis in 4 cases, while that of BHA fed group developed tail paralysis in 2, hind limb weakness in 2 and hind limb paralysis in 8 cases. The histology of the non-BHA group correlated well with the clinical symptoms of perivascular mononuclear infiltration. However, the BHA group revealed complete pathological recovery. Animals with BHA in the diet had significantly raised GSH, indicating the induction of phase 2 enzymes. We conclude that dietary phase 2 enzyme inducers show potential therapeutic benefits in EAE and should be examined for this role in MS.

In 1962, Gross and Lapiere described collagenolytic activity in the degradation of tadpole tails during amphibian metamorphosis. This activity was later attributed to a collagenase enzyme belonging to the matrix metalloproteinase family. Over the past 49 years, steady growth in the field of metalloproteinase biology has uncovered that degradation of extracellular matrix components represents only a fraction of the functions performed by these enzymes. The regulatory roles of these enzymes in numerous aspects of mammalian biology remains poorly understood. This thesis investigates the metalloproteinase ADAM17 and its natural inhibitor TIMP3 in acute and chronic inflammation. My work describes the generation of new murine experimental systems of compartmentalized ADAM17 or TIMP3 deficiency and their applications in acute liver inflammation (i.e. fulminant hepatitis and T-cell mediated autoimmune hepatitis) and atopic dermatitis. Loss of Timp3 protected mice against fulminant hepatic failure caused by activation of the death receptor Fas. We determined that TIMP3 simultaneously promotes pro-apoptotic signaling through TNFR1 while suppressing anti-apoptotic EGFR activation in the liver. Mechanistically, we identified that ADAM17 is critical in shedding TNFR1 and EGFR ligands (e.g. Amphiregulin, HB-EGF, TGF) and extended this finding to clinically relevant drug-induced hepatitis. Adult TIMP3 deficient mice also exhibited spontaneous accumulation of CD4+ T cells in the liver. Consequently, polyclonal T cell activation with the lectin Concanavalin A (con A) in a model of autoimmune hepatitis resulted in accelerated liver injury. We identified that this immunopathology relied on TNF bioavailability as mice lacking both Timp3 and Tnf were resistant to con A. Using bone marrow chimeras we established that non-hematopoietic tissues were the physiologically relevant source of TIMP3 in vivo, thereby highlighting an immunosuppressive role for this stromal metalloproteinase inhibitor in cellular immunity. Finally, we investigated epithelial:immune crosstalk in the epidermis by generating tissue-specific ADAM17 deficiency in basal keratinocytes. These mice developed spontaneous inflammatory skin disease that was physiologically consistent with atopic dermatitis. Focused investigation of keratinocyte-specific signaling deregulated by ADAM17 deficiency revealed its requirement for tonic Notch activation, which in turn antagonized transcriptional activity of AP-1 transcription factors on the promoters of epithelial cytokines TSLP and G-CSF. In summary, these works identify cellular mechanisms governing cytokine-mediated communication between epithelial and immune cells to modulate inflammation. The findings that TIMP3 and ADAM17 act as regulators of key inflammatory, proliferative and developmental pathways provide impetus to expand our understanding of this important family of enzymes in mammalian signal transduction.