Optimization of biologic therapy in children with inflammatory bowel disease (IBD) using modern biomarkers Abstract to thesis Study programme: Biochemistry and Pathobiochemistry Introduction: In adults, infliximab (IFX) levels correlate with disease activity and antibodies to IFX (ATIs) predict treatment failure. We aimed to determine the association of IFX levels and ATIs with disease activity in paediatric population. Methods: This study was performed as a prospective observational study. We prospectively collected blood, stool, and clinical data from 65 patients (age 10.5-15.1 years) with Crohn's disease (CD) before IFX administration, and measured IFX trough levels, ATIs, and faecal calprotectin levels (CPT). We used multivariate analysis to identify the predictors of IFX levels. IFX and ATIs levels were meassured using ELISA. Results: Lower levels of IFX were associated with ATIs positivity (OR [odds ratio] 0.027, CI [confidence interval] 0.009-0.077). Higher C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR) and CPT levels were found in patients with lower IFX levels. The optimal combination of specificity (50%) and sensitivity (74%) for disease activity was calculated for IFX levels ≥ 1.1 µg/ml using CRP level < 5 mg/l as a marker of laboratory remission. In a model that used CPT ≤...

Bone morphogenetic protein (BMP) signalling plays key roles in skin development and also possesses a potent anti-tumour activity in postnatal skin. To study mechanisms of the tumour-suppressive role of BMPs in the skin, a transgenic (TG) mouse model was utilized, in which a transgenic expression of the BMP antagonist Noggin was targeted to the epidermis and hair follicles (HFs) via Keratin 14 promoter. K14-Noggin mice developed spontaneous HF-derived tumours, which resembled human trichofolliculoma. Initiation of the tumours was associated with a marked increase in cell proliferation and an expansion of the hair follicle stem/early progenitor cells. In addition, the TG mice showed hyperplastic changes in the sebaceous glands and the interfollicular epidermis. The epidermal hyperplasia was associated with an increase in the susceptibility to chemically-induced carcinogenesis and earlier malignant transformation of chemically-induced papillomas. Global gene expression profiling revealed that development of the trichofolliculomas was associated with an increase in the expression of the components of several pro-oncogenic signalling pathways (Wnt, Shh, PDGF, Ras, etc.). Specifically, expression of the Wnt ligands and (¿-catenin/Lef1 markedly increased at the initiation stage of tumour formation. In contrast, expression of components of the Shh pathway was markedly increased in the fully developed tumours, compared to the tumour placodes. Pharmacological treatment of the TG mice with the Wnt and Shh antagonists resulted in the stage-dependent inhibition of the tumour initiation and progression, respectively. Further studies revealed that BMP signalling antagonizes the activity of the Wnt and Shh pathways via distinct mechanisms, which include direct regulation of the expression of the tumour suppressor Wnt inhibitory factor 1 (Wif1) and indirect effects on the Shh expression. Thus, tumour suppressor activity of the BMPs in skin epithelium depends on the local concentrations of Noggin and is mediated, at least in part, via stage-dependent antagonizing of the Wnt and Shh signalling pathways. / University of Bradford, NIH and BBSRC.

Yes / CC-1065, the duocarmycins and yatakemycin are members of a family of ultrapotent antitumour antibiotics that have been the subject of extensive investigations due to their mode of action and potential in the design of new anticancer therapeutics. The natural products and their analogues exert their effects through a sequence selective alkylation of duplex DNA in the minor groove at the N3 of adenine. An understanding of their structure and its effect on biological activity has been derived through chemical synthesis and has also generated new potential lead compounds. These studies form the first section of the review. The desire to progress these compounds to clinic has also led to studies of bioconjugation and prodrug formation and this is discussed in the second section of the review. The combination of synthesis with key biological experiments is a powerful tool to define the requirements for the development of natural products as potential therapeutic agents. The studies described herein form an excellent paradigm for the study and development of other natural products. / EPSRC, Yorkshire Cancer Research, Big C Cancer Research, UCB Pharma

Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.

Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.:Inhaltsverzeichnis I Abbildungsverzeichnis III Tabellenverzeichnis IV Bibliographische Beschreibung V Abkürzungsverzeichnis VII 1 Einleitung 1 1.1 CIK-Zellen (CIK) 3 1.1.1 Merkmale von CIK-Zellen 3 1.1.2 Wirkungsmechanismen von CIK-Zellen 3 1.1.3 Studienlage 4 1.1.4 Bisherige Ansätze zur Verbesserung der Kultivierungsbedingungen 6 1.2 Mesenchymale Stammzellen (MSC) 7 1.2.1 Allgemein 7 1.2.2 Differenzierung von MSC 7 1.2.3 Heterogenität und Einflussfaktoren der MSC - Identitätsproblematik 8 1.2.4 Charakterisierung von MSC 9 1.2.5 Therapeutische Einsatzmöglichkeiten von MSC 11 2 Zielformulierung 15 3 Material und Methoden 16 3.1 Tiere 16 3.2 Materialien 17 3.2.1 Materialien für Zellkultur 17 3.2.2 Materialien für FACS-Analyse 18 3.2.3 Materialien für Zytotoxizitätsassay 19 3.2.4 Materialien für CFU-F-Assay 20 3.3 Methoden 21 3.3.1 Statistische Auswertung 21 3.3.2 Zellkultur 22 3.3.3 FACS (Fluorescence Activated Cell Sorting) 26 3.3.4 Markierung der MSC mit DiD 28 3.3.5 Zytotoxizitätsassay (LDH-Freisetzungsassay) 29 3.3.6 CFU-F-Assay 32 4 Ergebnisse 34 4.1 Beeinflussung der Wachstumskurve 34 4.1.1 Der Wachstumskurvenverlauf von CIK-Zellen (Kontrollen) 34 4.1.2 Der Wachstumskurvenverlauf von CIK-Zellen in der Kokultur mit MSC 35 4.1.3 Der Wachstumskurvenverlauf in MSC-konditioniertem Medium 37 4.1.4 Der Wachstumskurvenverlauf bei Restimulierung an Tag 14 38 4.2 Beeinflussung des Oberflächenphänotyps 40 4.2.1 Der Oberflächenphänotyp von CIK-Zellen 40 4.2.2 Vergleich Oberflächenphänotyp Kontrollen mit kokultivierten CIK 43 4.3 Beeinflussung der Vitalität 46 4.4 Beeinflussung der Zytotoxizität 48 4.5 Identifizierung der MSC 49 4.5.1 Adhärenz an Plastikoberflächen 50 4.5.2 Fibroblastenähnliche Wachstumsmorphologie 50 4.5.3 Wachstum in Colony-Forming-Units 51 4.5.4 Der Oberflächenphänotyp von MSC 53 4.6 Schicksal der MSC in der Kokultur 54 4.6.1 Der Oberflächenphänotyp der adhärenten Zellen nach Kokultur 54 4.6.2 Kokultur mit DiD gelabelten MSC 57 5 Diskussion 59 5.1 Beeinflussung der Wachstumskurve 60 5.1.1 Mechanismen der Beeinflussung des Wachstumskurvenverlaufs 60 5.1.2 Fehlerbetrachtung 68 5.2 Identifizierung der CIK sowie Beeinflussung von Phänotyp und Vitalität 69 5.3 Beeinflussung der Zytotoxizität 70 5.3.1 Vergleich Zytotoxizität Kontrollen mit Kokulturen 70 5.3.2 Fehlerbetrachtung 71 5.4 Identifizierung der MSC 72 6 Schlussfolgerung 75 7 Ausblick 77 8 Zusammenfassung 79 Literaturverzeichnis 83 Danksagung I

Many studies show the ability of gut microbes to modulate the anti-tumour immune response by direct triggering the immune cells or by bacterial metabolites. Interestingly bacteria may even migrate to the tumour tissue and orchestrate the immune response on site. These anti-tumour effects can be improved by the administration of immune checkpoint inhibitors (ICI). Notably, some microbial effects occur only in the presence of ICI. On the contrary, microbiota may also promote tumour growth and negatively impact the effects of ICI therapy. We have disrupted the gut microbiota homeostasis by antibiotics (ATB) to study the effects of gut microbiota on the ICI. This disturbance led surprisingly to reduced tumour growth and enhanced pro-inflammatory immune response not only in the gut but also within the tumour tissue, where especially IFN-γ orchestrated the anti-tumour immune response. Importantly the anti-tumour immune response could be transferred through colonisation of germ-free mice by ATB-changed gut microbiota if concomitantly anti- programmed cell death protein 1 (αPD-1) monoclonal antibody was administrated. These mice had elevated levels of segmented filamentous bacteria (SFB), which induced systemic immune response with increased expression of IL-17 and elevated amounts of Th 17 cells,...