Pneumolysin (PLY) is a proven virulence factor of Streptococcus pneumoniae, deeply involved in the development of pneumococcal invasive diseases. The antibacterial and adjunctive therapies currently used in the management of pneumococcal infection do not address the pneumolysin-induced cytotoxicity. Therefore, even if patients survive the infection, they may suffer from long-term disabilities as a consequence of the exposure to the toxin. This research seeks to address this problem by developing new approaches to the treatment of pneumococcal diseases, based on peptides or small molecules that target pneumolysin and inhibit its activity. The peptides and small molecules were evaluated for their capacity to inhibit the haemolytic activity of pneumolysin in vitro. In total, four peptides and six small molecules were selected for in vivo tests. In vivo experiments were performed using a pneumonia model, where each animal was administered PBS or peptide / small molecule in PBS intranasally, every 6 hours after intranasal infection. One peptide (P1) and three small molecules (SM6, SM10 and SM14) improved significantly the outcome of the treatment group over the control group, when looking at the CFU in the blood, disease sign score and survival comparison. Moreover, two small molecules (SM10 and SM14) tested ex vivo, showed the capacity to abolish pneumolysin cytotoxic effect on the ciliary function of rat ependymal cells. The pneumolysin inhibitors identified in this work were pointed as potential candidates to further testing in drug discovery.

Infection with Streptococcus pneu1110niae is responsible for significant mortality in individuals with a defective humoral response. This diverse group includes patients with the haematological conditions myeloma and chronic lymphocytic leukaemia. Vaccination to prevent pneumococcal infection is recommended by the Department of Health for those individuals in whom disease is likely to be 'more common and/or more dangerous'. The currently recommended 23-valent polysaccharide vaccine has been shown to be poorly immunogenic in patients with myeloma and chronic lymphocytic leukaemia. Responses to vaccination may be improved by conjugation of polysaccharides to immunogenic carrier proteins. Prevenar©, a 7-valent conjugate vaccine has been demonstrated to be immunogenic both in young children and in adults following allogeneic bone marrow transplantation. These groups had both previously been shown to respond poorly to the 23-valent polysaccharide vaccine. A clinical trial was designed to evaluate the use of Prevenar in individuals with myeloma and chronic lymphocytic leukaemia. Serological response to the vaccine was the primary end-point determined by a recently evaluated pneumococcal serotype-specific Bioplex assay. In addition the serological response to the protein carrier was also evaluated using a newly developed assay. The vaccination programme resulted in statistically significant increases in serotype-specific antibody levels. For a proportion of trial participants this represented adequate protection. The immunogenicity of the conjugate vaccine was modest however when compared to the published data in other clinical scenarios. The suboptimal response was most likely to have been related to the deficient cell mediated and humoral immunity observed in this population. More detailed evaluation of these parameters may identify individuals who are more likely to respond to the conjugate vaccine.

There is evidence to suggest that therapy targeted at normalising extravascular lung water volume (EVLW) can improve outcomes from critical illness. Indocyanine green-thermal double indicator dilution (ICG-thermal) is considered the clinical reference standard of EVLW volume measurement but is no longer commercially available. The accuracy and reliability of the only clinically available technology (single-thermal indicator dilution) has been questioned in several studies. This thesis incorporates two clinical studies and one laboratory study designed to assess the measurement of EVLW and intrathoracic blood volumes using a prototype lithium-thermal double indicator dilution technique. The proof of concept study suggested our hypothesis that intrathoracic blood volume (ITBV), which is required for the calculation of EVLW volume, could be determined using lithium indicator dilution, was valid. Peri-operative trends and absolute values of ITBV were consistent with those obtained using ICG-thermal in a similar patient study group. The median absolute value of ITBV measured at baseline using indocyanine green (1417 [±208] ml) was similar to that obtained using lithium indicator dilution 1542 (±601) ml. EVLW volume measured by three indicator dilution techniques was then compared to postmortem gravimetry in porcine models of acute lung injury. Sepsis and acute lung injury were associated with increased EVLW volume, (9.2 [±3.0] ml kg-1), compared to sham operated animals (6.6 [±0.45] ml kg-1) in keeping with previous studies. The Li-thermal (Bias-1.8 [±13.1] ml kg-1) and ICG-thermal (Bias-1.0 [±6.6] ml kg-1) techniques demonstrated acceptable accuracy, but wide limits of agreement suggested poor reliability. 4 The single-thermal technique systematically over-estimated EVLW, with unacceptably wide limits of agreement (Bias +8.5 [±14.5] ml kg-1). In this laboratory investigation, the double indicator methods appeared more reliable than the single-thermal technique. However none could be considered ideal. Results of the final clinical study suggested EVLW volume measurement in man with the Li-thermal method was clearly erroneous (Bias -7.6 [±7.4] ml kg-1) and compared poorly to simultaneous measurements made using the ICG-thermal method (Bias +13.2 [±14.4] ml kg-1). A considerable over-estimation of mean transit time (MTT) when compared to the ICG-thermal technique (Bias 12.8 [±13] sec) was observed, a likely consequence of using an external lithium ion electrode instead of an intra-arterial catheter. Manual analysis of the dilution curves suggested considerable variability when compared to the automated analysis. The poor accuracy of MTT, and consequently ITBV measurements in the clinical study, may partly be due to software analysis of the lithium dilution curves. Thoracic blood volumes derived from measurement of ICG transit time are reliable. However, EVLW calculations based on the thermal indicator transit time are likely to be inaccurate. The findings of these clinical and laboratory investigations demonstrate poor agreement between both the prototype Li-thermal and the single thermal measurements of EVLW volume and the ICG-thermal method. Trans-pulmonary lithium indicator dilution measurements of ITBV and EVLW volume using an external lithium ion electrode are not sufficiently accurate to safely guide clinical interventions in individual patients. Consequently we decided not to further develop the lithium-thermal technique of EVLW volume measurement.

This thesis consists of 3 studies: 1. Determination of the minimal important difference (MID) of the visual analogue scale for dyspnoea (VASD): Determining the MID of the VASD is essential to interpret the results of trials in patients with malignant pleural effusions (MPEs). Patients undergoing a pleural procedure assessed the change in their VASD and the degree of change in their symptoms on a Likert scale. The mean VASD in patients experiencing a ‘small but just worthwhile’ decrease in their symptoms is the MID for the VASD and was found to be 22mm (95% CI 16 - 27mm). 2. Development of a thoracic ultrasound septation score (TUSS): A TUSS is important for objectively assessing the degree of septation within a pleural effusion. An iterative process was used to demonstrate that degree of septation predicts clinical outcome, to identify candidate factors for inclusion in a TUSS and to determine which factors predicted the degree of septation. The final TUSS consisted of an assessment of the degree of homogeneity of septation distribution and number of septations at the most septated area. 3. Effect of an indwelling pleural catheter (IPC) versus standard care for relieving dyspnoea in patients with MPEs: the TIME2 randomised controlled trial (RCT). The objective of this unblinded RCT was to determine whether IPCs are more effective than chest drains and talc pleurodesis at relieving dyspnoea in patients with MPEs. 106 patients were randomised to either IPC or standard care in a 1:1 ratio. The primary outcome was daily VASD over 42 days post intervention. Dyspnoea improved in both groups with no significant difference in mean dyspnoea in the first 42 days (mean score: IPC 25mm (95% CI 19 – 30), standard care 24mm (95% CI 19 – 29)).

Η κακοήθης υπεζωκοτική συλλογή (ΚΥΣ), προκαλούμενη από μεταστατικά αδενοκαρκινώματα είτε από πρωτοπαθή μεσοθηλιώματα, αποτελεί μείζον κλινικό πρόβλημα καθώς σηματοδοτεί συστηματική νεοπλασματική διασπορά και χαμηλό προσδόκιμο επιβίωσης. Η τρέχουσα αντιμετώπισή της είναι μη αιτιολογική και συχνά αναποτελεσματική. Ενώ τα νεοπλάσματα που προκαλούν ΚΥΣ φέρουν καλά χαρακτηρισμένες μεταλλάξεις και ο υπεζωκότας θεωρείται προνομιακή ανατομική θέση γονιδιακής θεραπείας, οι μέχρι τώρα μελέτες της αποτελεσματικότητας της υπεζωκοτικής διαγονιδιακής μεταφοράς υπήρξαν αποσπασματικές. ΣΚΟΠΟΣ: Σκοπός της παρούσας μελέτης είναι ο χαρακτηρισμός της έκτασης, της αποτελεσματικότητας και της λειτουργικότητας της διαγονιδιακής μεταφοράς τόσο στο φυσιολογικό υπεζωκοτικό μεσοθήλιο, όσο και και στον πάσχοντα υπεζωκότα με πρωτοπαθείς ή μεταστατικούς όγκους. ΥΛΙΚΑ ΚΑΙ ΜΕΘΟΔΟΙ: Υγιείς ποντικοί φυσικού τύπου C57BL/6 και διαγονιδιακοί ποντικοί-ιχνηλάτες mT.mG έλαβαν ενδοϋπεζωκοτικές εγχύσεις 5 x 108 μολυσματικών μονάδων αδενοϊών τύπου 5 (Ad) που κωδικοποιούν τα γονίδια της λουσιφεράσης (Luc), της πράσινης φθορίζουσας πρωτεΐνης (GFP) και της Cre ρεκομπινάσης. Η αποτελεσματικότητα της διαγονιδιακής μεταφοράς μελετήθηκε με απεικόνιση βιοφωταύγειας/βιοφθορισμού, καθώς και με φθορίζουσα μικροσκοπία. Τα πειράματα επαναλήφθηκαν σε ποντικούς με ΚΥΣ, οι οποίες προκλήθηκαν μέσω υπεζωκοτικής έγχυσης 150.000 κυττάρων συγγενικού μεσοθηλιώματος υπεζωκότα (ΑΕ17) και αδενοκαρκινώματος πνεύμονα (LLC). Προκειμένου να μελετηθεί αν οι αδενοϊοί σε ποιο βαθμό μπορούν να διαμολύνουν κύτταρα in vitro , ανθρώπινες και ποντικίσιες κυτταρικές σειρές , φυσιολογικές και καρκινικές , διαμολύνθηκαν με 10ng/μl Ad-luc και Ad-GFP. AΠΟΤΕΛΕΣΜΑΤΑ: Η θωρακική φωτεινή εκπομπή ποντικών C57BL/6 κορυφώθηκε την πρώτη και υποχώρησε τη δεύτερη εβδομάδα μετά ενδοϋπεζωκοτική έγχυση Ad-Luc . Δύο εβδομάδες μετά από ενδοϋπεζωκοτική χορήγηση Ad-Cre σε ποντικούς mT.mG παρατηρήθηκε διάχυτη εξάλειψη του ερυθρού και εμφάνιση πράσινου φθορίζοντος σήματος σε όλες τις υπεζωκοτικές επιφάνειες, το οποίο εντοπιζόταν κατ’ αποκλειστικότητα στο μεσοθήλιο. Δύο εβδομάδες μετά από έγχυση Ad-Cre σε ποντικούς mT.mG που είχαν αναπτύξει όγκους παρατηρήθηκε πράσινος φθορισμός μαζί με τον κόκκκινο μέσα στους όγκους. Στατιστικώς σημαντική διαμόλυνση παρατηρήθηκε in vitro στα κύτταρα φυσιολογικού μεσοθηλίου, μεσοθηλιώματος ανθρώπου και ποντικού , αδενοκαρκινώματος πνεύμονα και παγκρέατος ποντικού. ΣΥΜΠΕΡΑΣΜΑΤΑ: Η ενδοϋπεζωκοτική έγχυση ανασυνδυασμένων αδενοιών αποτελεί αποτελεσματική μέθοδο διαγονιδιακής μεταφοράς στο φυσιολογικό και παθολογικό υπεζωκοτικό μεσοθήλιο. Ο βαθμός της διαγονιδιακής μεταφοράς επιβεβαιώνεται με τη διαμόλυνση φυσιολογικών και καρκινικών κυτταρικών σειρών in vitro. / --