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.

Les cytokines sont d'importants médiateurs dans la physiopathologie des lymphomes hodgkiniens (LH). A partir d'une cohorte de 464 patients, nous avons évalué l'impact pronostique de onze SNPs parmi les gènes de cytokines : IL10 (rs1800890, rs1800896, rs1800871, rs1800872), TNFA (rs1800629) ; IL6 (rs1800795) ; IL1B (rs16944) ; ILRN (rs419598) ; INFG (rs2430561) ; IL12 (rs3212227) ; CCL17 (rs223828). Le génotypage du SNP de l'IL12 montre une distribution différente de celle attendue dans la population générale selon le test de Hardy-Weinberg. Ce résultat suggère que les variations génétiques de l'IL12 pourraient être impliquées dans la susceptibilité au LH. Les patients porteurs du génotype IL10-1082AA présentent un taux de rémission complète au traitement initial supérieur aux patients présentant un autre génotype (95% vs. 88% P = .02). Pour les patients de stade avancé III-IV, le taux de survie globale à 6 ans est statistiquement différent entre les génotypes IL10-592AA/CC/AC et IL10-819TT/CC/CT (100%, 94%, 78%, P = .03). Ce résultat est retrouvé pour les patients porteurs de LH n'exprimant pas l'EBV. Pour les LH EBV négatif, le taux de survie sans progression à 6 ans est différent en fonction du génotype du TNFA-308AA/GG/AG (100%, 84%, 68%, P = .03). Il n'a été pas retrouvé de corrélation entre les génotypes et les dosages plasmatiques de l'IL-10, TNFA, IL-1RA, IL-6. Cette étude montre que le ''fond génétique immun'' est important à prendre en considération pour définir le pronostic des patients. Le rôle des SNPs de l'IL10 et du TNFA dans les LH EBV négatif devra être confirmé ainsi que l'influence des variations génétiques de l'IL12 dans la susceptibilité au LH.

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.

<p>The success of implanted medical devices, such as biosensors, is dependent on the immune reaction to the surface of the implanted material. This immune reaction, termed the foreign body reaction, is potentially affected by the physical and chemical properties of the implanted material. Macrophages interact with the surface of the implanted material and secrete intercellular signals, including cytokines and growth factors, which direct the actions of immune cells in the surrounding tissue. The type and quantity of cytokines and growth factors produced by macrophages at an implant surface could be an indicator of the outcome of the foreign body reaction. </p><p>This study investigated the effect of the surface chemistry of an implanted device on the production of cytokines and growth factors. First, microdialysis sampling was characterized as a technique for collecting cytokines and growth factors from the tissue surrounding an implant. Based on this characterization, it was determined that a direct sampling method would be more suitable than microdialysis sampling for determining accurate tissue concentrations of cytokines and growth factors. Second, an in vitro model was developed and utilized to assess cytokine and growth factor production from monocyte/macrophage cultures seeded onto commonly implanted polymeric biomaterials with varying surface chemistries. The materials included in this study were polyethylene (PE), polyurethane (PU), polymethyl methacrylate (PMMA), expanded polytetrafluoroethylene (ePTFE), and a cytotoxic organo-tin polyvinyl chloride (ot-PVC) as a positive control. From this in vitro model, it was determined that the varying surface chemistries of these non-toxic materials, excluding ot-PVC, did not significantly affect the types and quantities of cytokines and growth factors produced. Finally, an in vivo model for evaluating the cytokine and growth factor response to an implanted biomaterial was utilized for comparison with the in vitro findings. In this model, biomaterials were implanted subcutaneously within the lumen of a stainless steel mesh cage. The mesh cage served to create a "pocket" where wound exudate fluid collected within the cage, surrounding the implanted biomaterial. The materials included in this study were PE, PU, and ot-PVC. Cytokines and growth factors produced at the material surface were sampled directly from the exudate fluid. The results from this in vivo study indicate that cytokine and growth factor production were not significantly impacted by the varying surface chemistries of the implanted biomaterials. The in vivo data support the findings from the in vitro model, suggesting that the foreign body reaction proceeds in a similar fashion for each of these non-cytotoxic, polymeric biomaterials with varying surface chemistries.</p> / Dissertation

Previous studies show that coronary artery disease (CAD) is a multi-factors and chronic inflammatory disease, and is associated with lipid metabolism. IL-1ra is a naturally occurring anti-inflammatory molecules that block the action of IL-1. However, little is known about the imbalance between IL-1ra and inflammatory mediators in CAD. We attempted to investigate the relationships between inflammatory mediators and serum IL-1ra levels in patients with CAD. In 95 patients with angiographically defined CAD, and 70 healthy controls were studied in a case-control manner. Serum levels of cytokines and the risk factor of CAD were examined. Polymorphisms for IL-1ra gene were detected by PCR, and genotypes and allelic frequencies in both groups were compared. Our major finding include: (1) The risk factors such as elevated BMI, systolic BP, smoking, hypertension, blood glucose, and TG was more frequently found in the CAD group than the control group ( p < 0.001). However, the HDL-C and bilirubin were significantly higher in control group than the CAD group. (2) The relative risk of those in the highest quartile of ratio of LDL-C to HDL-C, TC to HDL-C, and TG to HDL-C were significantly elevated. ( OR = 2.98, p < 0.01; OR = 5.31, p <0.001; OR = 8.43, p < 0.001 respectively) (3) Five different inflammatory markers were significantly elevated including IL-1ra, hs-CRP, IL-6, leukocyte count, and neutrophil percentage between healthy controls and CAD patients. ( p < 0.01) (4) Levels of IL-1ra and other variables such as blood glucose, BMI, TG, IL-6, hs-CRP, and leukocyte count has significantly correlated, and were inversed correlation in bilirubin, and HDL-C in all study subjects. ( p < 0.01) (5) In the multiple logistic regression analysis, adjustment was made for variables. The relative risk of CAD for the highest quartile of IL-1ra, as compared with the lowest quartile, had an Odds ratio 2.57 ( 95% confidence intervals, 1.12 - 5.91, p = 0.026 ) increase in risk for CAD. (6) Similar results were obtained hs-CRP, IL-6 in the highest quartile were increase risk for future CAD. ( OR = 5.86 and 5.79 respectively; p < 0.001) (7) The join effect cytokines of hs-CRP, IL-6, IL-1ra concentrations may play important role in CAD risk. ( OR = 10.19, p < 0.001 ) (8) In addition, IL-1ra allele 2 genotype and allelic frequencies were no significant association with increase in IL-1ra with CAD. In conclusion, we find a significant association of elevated IL-1ra levels in the patients with CAD. Thus, these results support the hypothesis that inflammation, anti-inflammation cytokines and lipoprotein metabolism provide a useful marker for predicting the development of CAD events.

Incidence of dengue fever (DF) has been estimated a 30 fold increase in the past 50 years. Clinical manifestations of DF range from a simple febrile illness with physical soreness to life-threatening dengue hemorrhagic fever (DHF). The need for a better classification of the severity in DEN infections has been proposed to clarify the immunopathogenesis for the prevention and management of serious DEN infections. We attempted to investigate whether different mechanisms involved in the varied manifestations of bleeding tendency and vascular leakage in DF. In a hospital-based study, we first compared clinical features as well as laboratory data including virus load, T helper (Th1/Th2) cytokines, and vascular leakage-related mediators between patients with DHF and DF. Moreover, we defined another class of patients associated with bleeding tendency but not fulfilled with DHF criteria, called DF w/B, for a further comparison. The virus load in blood was not significantly different among DF, DHF and DF w/B. DF patients had a higher Th1 cytokine, IFNr, expression (70.0 ¡Ó 10.7 vs. 33.1 ¡Ó 8.0 vs. 33.0 ¡Ó 7.1 pg/ml; DF vs. DF w/B, p = 0.009; DF vs. DHF, p = 0.002), and both DHF and DF w/B patients had a significantly higher IL-10 levels (14.3 ¡Ó 4.1 vs. 26.2 ¡Ó 3.3 vs. 26.0 ¡Ó 3.5 pg/ml; DF vs. DF w/B, p = 0.023; DF vs. DHF, p = 0.016) than DF patients. Both DHF and DF w/B patients also had a higher rate of secondary dengue infection (DF w/B vs. DHF vs. DF: 50.0%, 74.4% and 14.3%¡A p < 0.001). By contrast, DHF but not DF w/B patients had significantly higher vascular leakage-related mediators: sVCAM-1, PGE2 and TNF£\ levels than DF patients. Patients with DF w/B had a higher platelet counts (DF w/B vs. DHF: 66.0 ¡Ó 8.3 vs. 20.7 ¡Ó 2.1 x109/L, p < 0.001) but lower ALT levels than those with DHF (DF w/B vs. DHF: 56.3 ¡Ó 7.7 and 144.7 ¡Ó 20.5 IU/L). This study provides new insight to different immune mechanisms involved in patients with DF, DF w/B, and DHF. DF involves augmented Th1 reaction, and DF w/B involves altered Th2 reaction, but DHF involves both altered Th2 reaction and augmented vascular insult. Clarification of the immune mechanisms among DF, DFw/B and DHF will facilitate certain specific treatment and prevention of DF patients from varied bleeding tendency and vascular leakage manifestations.

Zugl.: Giessen, Universiẗat, Diss., 2009.

Würzburg, Universität, Thesis (doctoral), 2001.

Microglia, the resident immune cells of the central nervous system, can become activated following injury, disease, or infection. In vitro, they can be activated by stimuli, which determine the inflammatory phenotype they will develop. In this thesis, stimulating microglia with tumor necrosis factor- and interferon- resulted in classical activation, characterized by proliferation, increased transcription of complement receptor 3 and major histocompatibility class II molecules, and elevated production and transcription of interleukin-1 and nitric oxide. Stimulation with TNF and IFN also changed the intensity of phosphorylated (activated) cyclic adenosine monophosphate response element binding protein immunoreactivity in microglia. Specifically, cells differentiated into populations with high or low pCREB intensity. This was the first example of such a response in microglia and was representative of what occurred in vivo, after ICH. Thus, the characterization of this model will be useful for future studies of this and other intracellular pathways of classically activated microglia.

Understanding the genetic control of gametogenesis is a central goal of developmental biology and is important for treating infertility in humans. An approach to identifying critical genes in mammals is to search for and study homologues of genes known to play key roles in other organisms. In the fly, Drosophila melanogaster, GUS protein is a component of nuage, an electron-dense aggregation in early germ cells, and is required for oocyte development. GUS physically interacts with VASA, an RNA helicase thought to regulate mRNA metabolism. I identified two murine genes, SSB-1 and SSB-4, that are similar to and likely homologues of gus. SSB-1, SSB-4 and GUS each contain two conserved regions, termed the SPRY domain and the SOCS box, respectively. SSB-1 and SSB-4 share about 75% sequence identity and about 70% identity with GUS. Both SSB-1 and SSB-4 RNA and protein were found to be express in mouse ovarian granulosa cells of all stages of folliculogenesis. These cells support oocyte development and also produce steroids. Unexpectedly, SSB-1 and SSB-4 were only weakly or not detectable in oocytes, that contrasts with the expression of GUS in Drosophila oocytes. However, SSB-1 mRNA and protein were expressed in male germ cells; specifically in spermatocytes and spermatids. SSB-1 in spermatids was localized in a specialized structure known as the chromatoid body. Although the function of this structure is not quite clear, it has been compared to nuage, and one of its components is MVH, the murine homologue of VASA. Finally, using RNAi technology, SSB-1 was transiently depleted SSB-1 from a granulosa cell line. These cells showed a transient decrease in expression of the gene encoding P450scc, the rate-limiting enzyme in steroid synthesis. Preliminary results also indicated a decrease in progesterone synthesis. Taken together, these results establish the expression pattern of murine homologues of Drosophila GUS in mouse ovary and testis, reveal it might play function in translation regulation in male spermatogenesis, and identify a potential role in steroidogenesis by ovarian granulosa cells.