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Background: Epidemiological studies suggest that inhalation of carbonaceous particulate matter from increases susceptibility to bacterial pneumonia. In vitro studies report that phagocytosis of carbon black by alveolar macrophages (AM) impairs killing of Streptococcus pneumoniae. We therefore aimed to use a mouse model to test the hypothesis that high levels of carbon loading of AM in vivo increases susceptibility to pneumococcal pneumonia. We also want to develop an air-tissue interface model to assess DNA damage to airway macrophages due to inhalation of manufactured nanoparticals. Methods: Female outbred mice were treated with either intranasal phosphate buffered saline (PBS) or ultrafine carbon black (UF-CB in PBS; 500 μg on day 1 and day 4), and then infected with S. pneumoniae strain D39 on day 5. Survival was assessed over 72 h. The effect of UF-CB on AM carbon loading, airway inflammation, and a urinary marker of pulmonary oxidative stress was assessed in uninfected animals. The human monocyte cell line Mono Mac 6 and primary alveolar macrophages were used to assess DNA damage. To measure DNA damage in macrophages, we used the alkaline Comet assay. After nanoparticle exposure, a total of 100 macrophages were analysed per sample, as n=50 duplicate slides. Tail length, percentage of DNA in the tail of the comet (% tail DNA), tail extent moment and olive tail moment, were calculated for each cell using the Komet Analysis software. Results: Instillation of UF-CB in mice resulted high levels of carbon loading in alveolar macrophages. In uninfected animals, UF-CB treated animals had increased urinary 8-oxodG (P = 0.055), and an increased airway neutrophil differential count (P < 0.01). All PBS-treated mice died within 72 h after infection with S. pneumoniae, whereas morbidity and mortality after infection was reduced in UF-CB treated animals (median survival 48 h vs. 30 h, P < 0.001). At 24 hr post-infection, UF-CB treated mice had lower lung and the blood S. pneumoniae colony forming unit counts, and lower airway levels of keratinocyte-derived chemokine/growth-related oncogene (KC/GRO), interferon gamma( IFN-γ) and other inflammatory cytokines. No increase in DNA damage was observed when cells were placed in the nitrogen gas flow for 10 mins compared with cells placed in air (insert control vs air control).Exposure to nanoparticles from all three metals for 10 min caused a significant increase in DNA damage in human Mono Mac 6 cells compared to insert control. There was no significant difference between DNA damage caused by gold (Au), silver (Ag) and Iron (Fe) nanoparticles. Conclusion: Acute high level loading of AM with ultrafine carbon black particles per se does not increase the susceptibility of mice to pneumococcal infection in vivo. We found significant DNA damage in macrophages cultured for 24 h with doses of up to 10 mg/cm² aerosolized iron, gold or silver nanoparticles.

Complement system is an essential part of innate immune system that plays major role in protection against various pathogens, including Streptococcus pneumoniae. Complement is activated via three pathways; the classical pathway, the alternative pathway and the lectin pathway. Lectin pathway of complement activation is mediated via ficolins, MBL and recently described C-type lectin, CL-11, that recognise a wide range of carbohydrates on microbial surfaces and activate the complement system via MASP-2, the effector enzyme of the lectin pathway of complement activation. A significant role of the lectin pathway has been described previously using MASP-2 deficient mice, with a complete deficiency of the lectin activation pathway specific C3 and C5 converting enzymes C4b2a and C4b2a(3b)n respectively. However, the role of MBL, ficolins and CL-11 in activating lectin pathway against S. pneumoniae has not been fully characterised. In vitro studies demonstrate that ficolin A and CL-11 are the relevant carbohydrate recognition molecules that can activate the lectin pathway of complement on the surface of S. pneumoniae. The protective activity of ficolin A was demonstrated in vivo using ficolin A-/- mice compared to wild-type controls, the ficolin A-/- mice were highly susceptible to pneumococcal infection with higher mortality and higher bacterial burden in both blood and lungs after intranasal infection with S. pneumoniae D39 compared to wild-type controls. These findings imply that the lectin pathway has a significant role in protection against S. pneumoniae infection and highlights the importance of non-MBL mediated lectin pathway activation in the innate host defence against S. pneumoniae. The essential role of the lectin pathway in providing protection against S. pneumoniae was further described by blocking the lectin pathway by i.p. injection of mice with anti-MASP-2 mAb. Mice receiving anti-MASP-2 mAb showed significantly higher mortality after intranasal infection with S. pneumoniae when compared to mice receiving control antibody.

The aim of this project was to investigate the role of complement and lung surfactant protein D in innate immunity to S. pneumoniae.;Pneumolysin, a cytolytic toxin produced by S. pneumoniae, is able to activate the classical complement pathway. The deletion of the ability of pneumolysin to activate complement affected the early growth of pneumococcus in the lungs, the onset of bacteraemia, the histological changes and the recruitment of T lymphocytes into lung tissue during bronchopneumonia.;Lung complement C3 was substantially activated after intranasal infection with wild-type S. pneumoniae in comparison with the isogenic mutant strain unable to produce pneumolysin (PLN-A).;Data presented in this thesis showed that the classical complement pathway plays a critical role in the innate immunity to S. pneumoniae infection. Deficiency in C1q increased the susceptibility to pneumococcal infection and was associated with defects in pneumococcal clearance from lungs and blood, less severe histological changes, recruitment of T cells and a substantial decrease in the activation of complement C3 in the lung.;In vitro studies showed that lung surfactant protein D or its receptor gp-340 is able to bind and agglutinate several strains of S. pneumoniae. Sp-D did not enhance the uptake of pneumococcus by neutrophils. The capsule-type is not a determinant for S. pneumoniae aggregation by Sp-D or gp-340.;Sp-D-deficient mice showed increased susceptibility to pneumococcal infection. Deficiency in Sp-D was associated with decreased pneumococcal clearance in lungs and trachea, early onset and increased levels of bacteraemia. In the infected lung, accumulation of T lymphocytes and more severe inflammation were observed in the absence of Sp-D.

Community-acquired pneumonia (CAP) is a leading cause of adult morbidity and mortality worldwide despite decades of effective antibiotics and vaccination initiatives. There have been no recent significant improvements in outcomes, including 30-day mortality. The bacterium Streptococcus pneumoniae is the most prevalent causative pathogen in CAP, being found in up to half of cases. In September 2006 a childhood pneumococcal vaccine (PCV-7) was introduced, leading to reductions in vaccine-type (VT) pneumococcal disease in infants, with possible additional benefits reported in adults. However, the effect that infant PCV-7 vaccination has on adult disease has to date been inadequately described in a small fraction of patients with invasive CAP, almost exclusively in populations in the US. These issues are explored fully in the literature review, encompassing chapters 1, 2 and 3. New strategies for CAP are therefore required. The outcome of CAP can be improved by a) preventing the disease by vaccination and herd immunity, and b) ameliorating the course of the disease after it has been acquired. This thesis presents a collection of studies that aim to acquire observational data to investigate these two issues. The majority of the included studies are drawn from a two year prospective cohort study of consecutive adults with CAP admitted to a large UK teaching hospital trust between September 2008 and September 2010. After obtaining informed consent, the presence of pneumococcal disease in each participant was established by testing urine samples for pneumococcal capsular polysaccharide, a test which has a high sensitivity and specificity. The urine samples were subsequently tested for pneumococcal serotype. A full record of care processes, investigations, and clinical outcomes was made, and child contact in the month preceding admission was assessed. These methods are described more fully in chapter 4. Chapter 5 presents the data on the pneumococcal serotypes found in the cohort over a two year period, and links them to epidemiological characteristics in the study population. The most prevalent serotypes were 14, 1, 8, 3 and 19A, with VT serotypes less frequent in the second year of the study. Chapter 6 examines the association that infecting serotype has with disease manifestation and patient characteristics. Infection with a serotype not contained within PCV-7 (NVT) was associated with younger and fitter patients, a higher rate of complications such as para-pneumonic effusion, and hypotension at admission. The effect of child contact on pneumococcal disease is reported in chapter 7. Prior contact with a child aged ≤8 years was particularly associated with pneumococcal aetiology, and contact with a PCV-7 vaccinated child independently associated with NVT CAP. The findings from these three chapters are unique in that they relate individual pneumococcal serotype to specific clinical disease patterns, epidemiology and transmission in both invasive and non-invasive pneumococcal CAP for the first time. They show a change in serotype distribution in adults following the introduction of PCV-7 in infants, which is important to inform future vaccine development for both adults and children. Furthermore, different serotypes are associated with different clinical disease patterns, which may have a significant impact on the disease that clinicians see at the “front door” given that the serotype distribution of pneumococcal CAP may be changing. Finally, the link between child vaccination and adult disease provides more direct evidence for the transmission of pneumococci from children to adults as a mechanism for the development of CAP in adults. The second part of this thesis looks at current care processes, and how these might be improved. Chapters 8, 9 and 10 relate to efforts to better predict prognosis, and chapters 11 and 12 with how patents with CAP may be better managed at the “front door”. Symptoms are clearly important to patients, but the role of symptoms in management and outcome is unclear. Chapter 8 presents a study validating a symptom score that has not yet entered routine use, but which is shown to correlate with clinical outcomes, and may be useful in assessing outcome in low severity CAP. The influence that oxygenation status at admission has on outcome is poorly understood. Chapter 9 describes a study showing that whilst hypoxaemia does positively predict adverse outcome, it is not as predictive as existing severity scores. The presence of hypoxaemia may however identify a subset of patients who are classified as low severity by existing severity scoring, but are nevertheless at increased risk of adverse outcome. Severity scoring is the cornerstone of management in adult CAP, and is explored in chapter 10. Current severity scores adequately predict mortality in CAP, but often generate a group of “moderate severity” where appropriate management is often unclear. This study looked at the effect of pre-admission functional status on outcome in conjunction with existing severity scores in this difficult group, and validated a novel severity score for predicting need for escalation of care, SMART-COP. Incorporation of functional status does marginally improve the performance of existing severity scores, but may be of more use as a post-severity score test to identify sub-groups of patients with moderate severity CAP who are at increased risk of death. Chapter 11 looks at the influence that making a prompt diagnosis (rather than prompt treatment with antibiotics, as has previously been studied) has on outcome, using the time between admission and first chest radiograph as a surrogate measure. Whilst an early chest radiograph was not associated with an improvement in mortality, it was associated with a shorter length of hospital stay, and may therefore be regarded as a marker of good quality care. There is current debate as to the role of the speciality physician in the front-door early assessment of patients, and whether early review of patients with CAP may improve outcome compared with management by a non-specialty physician. Chapter 12 looks at the effect that early specialist senior respiratory review has on outcome for adults with CAP, showing a clear benefit on length of hospital stay to early consultant review. In conclusion, this thesis provides an up-to-date picture of the circulating pneumococcal serotypes in non-invasive adult CAP, and correlates infecting serotype to clinical and epidemiological parameters. It also identifies five areas of clinical care where management processes could be improved. By addressing of these aspects the outcome of CAP may be improved in the future.

Η Λεγιονέλλωση είναι μια λοιμώδης νόσος που αναγνωρίστηκε το δεύτερο μισό του 20ου αιώνα. Η σοβαρότητα της Νόσου ποικίλει από μια ήπια εμπύρετη ασθένεια (Pontiac πυρετός), μέχρι σοβαρής μορφής πνευμονία (Νόσος των Λεγεωνάριων). Μέχρι στιγμής, το γένος Legionella περιλαμβάνει τουλάχιστον 50 είδη, τα οποία περιλαμβάνουν 70 ξεχωριστές υποομάδες. Συγκεκριμένα η L.pneumophila περιλαμβάνει 16 υποομάδες, τις περισσότερες συγκριτικά με τα άλλα είδη. Το βακτήριο L.pneumophila είναι το πιο συνηθισμένο και επικίνδυνο μέλος της οικογένειας Legionella. Η L.pneumophila serogroup 1 προκαλεί τον μεγαλύτερο αριθμό κρουσμάτων της νόσου στην Ευρώπη και Αμερική. Πρόκειται για ένα υδατογενές παθογόνο βακτήριο που βρίσκεται παντού στο υδάτινο περιβάλλον και αναπτύσσεται σε θερμοκρασίες 20οC-45οC, ενώ η θερμοκρασία των 35οC είναι η ιδανικότερη για την ανάπτυξη της Legionella pneumophila. Η ικανότητα του βακτηρίου να επιβιώνει σε υψηλές θερμοκρασίες, του επιτρέπει να αποικίζει σε τεχνητά υδάτινα συστήματα, τα οποία λειτουργούν σε υψηλότερες θερμοκρασίες από την θερμοκρασία περιβάλλοντος. Μεταδίδεται αερογενώς μέσω των εισπνεόμενων υδατοσταγονιδίων, ενώ μέχρι σήμερα δεν έχει διαπιστωθεί μετάδοση της νόσου από άτομο σε άτομο. Από την στιγμή που το βακτήριο είναι ευρέως διαδεδομένο στο περιβάλλον, μπορεί να εγκατασταθεί και να αναπτυχθεί σε τεχνητά συστήματα νερού, όπως οι πύργοι ψύξης και τα συστήματα ζεστού και κρύου νερού. Το 1986 συγκροτήθηκε η Ευρωπαϊκή Ομάδα Εργασίας για την Νόσο των Λεγεωνάριων (EWGLI: European Working Group for Legionella Infections) και το 1987 υλοποιήθηκε η επιτήρηση των περιπτώσεων της Νόσου των Λεγεωνάριων που συνδέονται με ταξίδια, μέσω του Ευρωπαϊκού Δικτύου Επιτήρησης της Νόσου των Λεγεωνάριων. Στην Ελλάδα, η Νόσος των Λεγεωνάριων αποτελεί νόσημα υποχρεωτικής δήλωσης σε χρονικό διάστημα 24 ωρών από την διάγνωση. Είναι όμως χαρακτηριστικό ότι το ΚΕΕΛΠΝΟ, αναφέρει περιστατικά της Νόσου των Λεγεωνάριων, από το 1998 έως το 2008, ενώ μετά το 2008 δεν παρέχει κάποια δεδομένα για την Νόσο των Λεγεωνάριων. Περιστατικά της Νόσου των Λεγεωνάριων στην Ελλάδα, έχουν αναφερθεί ήδη από το 1982. Κάθε χρόνο δηλώνονται κατά μέσον όρο 13 κρούσματα της Νόσου των Λεγεωνάριων. Ο σκοπός της παρούσας μελέτης ήταν η εξέταση των υδάτινων συστημάτων σε νοσοκομεία και ξενοδοχεία της Νοτιοδυτικής Ελλάδος για την ανίχνευση των ειδών Legionella. Επιπλέον πραγματοποιήθηκε προσπάθεια καταγραφής των κρουσμάτων πνευμονίας και της Νόσου των Λεγεωνάριων που νοσηλεύονταν στα νοσοκομεία, έτσι ώστε να υπάρχει μια γενικότερη εικόνα της παρουσίας του βακτηρίου στην Νοτιοδυτική Ελλάδα. Συνολικά αναλύθηκαν 91 δείγματα νερού από τα υδάτινα συστήματα 8 νοσοκομείων και 25 δείγματα από 9 ξενοδοχεία για το χρονικό διάστημα Μάιος 2008-Δεκέμβριος 2009. Αρχικά πραγματοποιήθηκε ταυτοποίηση του βακτηρίου με την καλλιεργητική μέθοδο (ISO 11731:1998) και στην συνέχεια ακολούθησε απομόνωση του DNA του βακτηρίου. Έπειτα έγινε ταυτοποίηση της Legionella pneumophila με τη Μοριακή Μέθοδο της PCR και τέλος τα θετικά προϊόντα της PCR επιβεβαιώθηκαν με αλληλούχιση που πραγματοποιήθηκε στην Μονάδα Αλληλουχίας του Τμήματος Ανοσολογίας και Ιστοσυμβατότητας της Ιατρικής Σχολής του Πανεπιστημίου Θεσσαλίας. Παράλληλα πραγματοποιήθηκαν φυσικοχημικές αναλύσεις όπως μέτρηση του pH, θερμοκρασία, αγωγιμότητα και μικροβιολογικές αναλύσεις, όπως έλεγχος παρουσίας της Ολικής Μεσόφιλης Χλωρίδας στους 220C και 370C (ISO 6222:1999) και του βακτηρίου Pseudomonas aeruginosa (ISO 16266:2006) . Tο 33% των δειγμάτων νερού που ελήφθησαν από τα Νοσοκομεία της Νοτιοδυτικής Ελλάδας βρέθηκαν θετικά για το βακτήριο L.pneumophila. Όσο αναφορά τα Ξενοδοχεία της περιοχής των Πατρών, στο 36% των δειγμάτων νερού υπήρξε παρουσία του βακτηρίου. Η φυλογενετική ανάλυση έδειξε, πως τα ελληνικά στελέχη που απομονώθηκαν στην παρούσα μελέτη επέδειξαν υψηλή ομολογία με στελέχη L.pneumophila που έχουν χαρακτηριστεί γονοτυπικά με στελέχη της Ιταλίας. Καταγράφηκαν 325 κρούσματα πνευμονίας, εκ των οποίων τα 2 ήταν θετικά για το βακτήριο. Συμπερασματικά η παρούσα μελέτη υποδηλώνει μια συχνή παρουσία του βακτηρίου Legionella pneumophila, στα υδάτινα συστήματα των νοσοκομείων και των ξενοδοχείων. Η έρευνα μας επιβεβαιώνει την ανάγκη τακτικής παρακολούθησης των μικροβιολογικής ποιότητας των υδάτινων συστημάτων των νοσοκομείων και των ξενοδοχείων της Νοτιοδυτικής Ελλάδος. / Legionellosis is an infectious disease that was recognized in the second half of the 20th century. The severity of the disease varies from a mild febrile illness (Pontiac fever) to severe pneumonia (Legionnaires' disease). To date, the genus Legionella comprises at least 50 species, comprising 70 separate subgroups. Specifically, the L.pneumophila includes 16 subgroups, most compared to other species. The bacterium L.pneumophila is the most common and dangerous member of the family of Legionella. L.pneumophila serogroup 1 causes the majority of cases reported in Europe and in the US. Legionella species are aquatic bacteria that are widespread in nature and have been found everywhere in the aquatic environment, developed at temperature 20oC-45oC and 35oC temperature is ideal for growth of Legionella pneumophila. Their tolerance to relatively to high temperatures probably helps them to colonize in artificial water systems that are often above temperatures. Legionellosis is transmitted via airborne aerosols by aspiration and as far there have been no reported cases of inter-human transmission. Once the bacterium is widespread in the environment can be established and developed in artificial water systems such as cooling towers and systems for hot and cold water.In 1986, the European Working Group for legionnaire's disease (EWGLI: European Working Group for Legionella Infections) established and in 1987 was realized the surveillance of cases of the legionnaires' disease associated with travel through the European Network Monitoring legionnaire's disease. In Greece, the Legionnaires' disease is a modifiable disease in period of 24 hours of diagnosis. It is significant that the KEELPNO states cases of Legionnaires ‘disease from 1998 to 2008, but after 2008 does not provide any data on legionnaire's disease. Cases of Legionnaires ‘disease in Greece have been reported since 1982 and every year reported an average of 13 cases of legionnaires' disease. The aim of the present study was to determine the prevalence of Legionella spp. in water systems of hospitals and hotels located in South Western Greece. Furthermore, attempt to record the incidence of pneumonia and legionnaire's disease hospitalized in hospitals, so that there is a general overview of the presence of the bacterium in South Western Greece. A prevalence survey for Legionella spp. by culturing techniques in water distribution systems of eight hospitals (total 91 water samples) and nine hotels (total 25 water samples) occurred in South Western Greece, for the period May 2008-December 2009. Initially carried out identification of the bacterium with the classic culture methods (ISO 1173:1998) and followed by isolation of DNA of the bacterium. Following, was made identification of Legionella pneumophila by molecular methods of PCR and the positive PCR products were confirmed by sequencing conducted in Sequencing Unit of the Department of Immunology and Histocompatibility, School of Medicine, University of Thessaly. In parallel physicochemical analysis carried out, such as residual free chlorine, pH, temperature, conductivity and microbiological analysis such as, Total Count (220C and 370C) and Pseudomonas aeruginosa presence according to ISO 6222:1999 and ISO 16266:2006, respectively. Legionella pneumophila was detected in 33% and 36% of the distribution systems of hospitals and hotels. The phylogenetic analysis showed that Greek strains showed a high homology to L.pneumophila strains isolated during a study of genotypic characterization of Legionella species isolated in Italy. 325 cases of pneumonia were recorded, and 2 of them were positive for the bacterium. In conclusion, our survey results suggest a frequent prevalence of elevated concentrations of Legionella spp. in water systems of hospitals and hotels. Our investigation has confirmed the need to regularly monitor the microbiological condition of water systems in hospitals and hotels in South Western Greece.