Cannabinoids suppress Th1 immunity in a variety of models including infection with the intracellular pathogen Legionella pneumophila (Lp). To examine the cellular mechanism of this effect, mouse bone marrow-derived dendritic cells (DCs) were studied following infection and drug treatment. DCs produced high levels of IL-12p40 following Lp infection. THC suppressed this cytokine response in a concentration-dependent manner and the endocannabinoids 2-arachidonoyolglycerol and virodhamine less potently suppressed cytokine production. DCs expressed mRNA for cannabinoid receptor 1 (CB1), CB2, and transient receptor potential vanilloid type 1 (TRPV1); furthermore, inhibition of Gi signaling by adding pertussis toxin completely attenuated the suppression induced by low concentrations of THC but not at high concentrations. In addition, the THC suppression was partially attenuated in DC cultures from CB1 and CB2 knockout mice and in cultures from normal mice co-treated with THC and cannabinoid receptor antagonists. Cytokine suppression was not attenuated by pretreatment with the TRPV1 antagonist capsazepine, suggesting that Gi signaling and cannabinoid receptors, but not TRPV1, are involved in THC-induced suppression of DC potential to polarize the development of naïve T cells to be Th1 cells. Besides IL-12, THC suppressed other DC polarizing characteristics such as the expression of MHC class II and co-stimulatory molecules CD86 and CD40, as well as the Notch ligand Delta 4. However, THC treatment did not affect other DC functions such as intracellular killing of Lp and Lp-induced apoptosis. Testing the capacity of THC to suppress DC polarizing function with T cells showed that DCs infected in vitro with Lp were able to immunize mice when injected prior to a lethal Lp infection; however, the immunization potential along with Th1 cytokine production was attenuated by THC treatment of the cells at the time of in vitro infection. In addition, THC-treated and Lp-infected DCs poorly stimulated primed splenic CD4 T cells in culture to produce IFN-gamma (IFN-y); however, this stimulating deficiency was reversed by adding recombinant IL-12p40 protein to the cultures. In conclusion, the data suggest that THC inhibits Th1 polarization by targeting essential DC functions such as IL-12p40 secretion and the maturation and expression of co-stimulatory and polarizing molecules.

The Gram-negative bacterium Legionella pneumophila is an intracellular parasite of aquatic protozoa. It exhibits a distinct dimorphic lifecycle that alternates between vegetative replicative form (RF) and infectious cyst-like form (CLF). Inadvertent inhalation of aerosolized CLFs by immunocompromised individuals leads to an infection in alveolar macrophages causing Legionnaires' disease. To further study a Legionella infection the use of the multicellular organism Caenorhabditis elegans was done. Differential Interference Contrast (DIC) microscopy of live L. pneumophila infected nematodes shows Legionella-containing vacuoles (LCVs) with motile forms. Transmission Electron Microscopy (TEM) defined the ultrastructure of L. pneumophila forms found in the primary infection site of the intestinal lumen and the secondary infection site in the gonadal tissues. These findings suggest the possible intracellular replication cycle of Legionella occurring in the gonadal tissues of the nematode. Providing insight and a plausible evolutionary origin of the ability of L. pneumophila to manipulate the macrophage innate immune system. / October 2014

Drinking water is a potential means for transmission of the opportunistic pathogen Legionella pneumophila. The objective of this research was to document the occurrence of L. pneumophila from source water, partially treated water after conventional water treatment, and distribution water. Water samples were examined for L. pneumophila by direct fluorescent antibody (DFA) techniques and by SYBR Green based real-time multiplex PCR. Primers were designed to amplify a 16S product of 490 bp and a mip product of 290 bp within L. pneumophila. Sensitivity of culture methods and PCR was determined by percent recovery and by using serial dilutions of positive control DNA, respectively. Eighty percent of source water samples were positive for L. pneumophila by real-time PCR versus 100% positive by DFA for L. pneumophila. Twenty percent of GAC filter water samples were positive for L. pneumophila by real-time PCR versus 40% positive by DFA. Distribution water samples yielded mixed results. Twenty percent of biofilms from the distribution system were positive for L. pneumophila by real-time PCR. This project confirms the potential for human infection by L. pneumophila from distribution water within the study area. / Department of Biology

Tübingen, Univ., Diss., 2005.

München, Techn. Univ., Diss., 2004.

Universiẗat, Diss., 2004--Köln.

Universiẗat, Diss., 2005--Jena.

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.

<p>Guanylate binding proteins (Gbps) are a family of large GTPases that are highly stimulated by IFNγ and confer resistance to various viral, protozoan, and bacterial pathogens. Following infections of intracellular pathogens, multiple Gbps can localize to pathogen vacuoles and promote the vesiculation and destruction of these structures. While Gbps have also been implicated in pathways independent of vacuolar disruption, their roles in these processes have been less characterized. In this dissertation, I focus on the mechanism of Gbps downstream of vacuolar disruption in order to further elucidate the role of these proteins during immune responses. </p><p> Due to the IFNγ stimulation of caspase-11 pyroptosis, I first addressed the ability of Gbps to promote the non-canonical caspase-11 dependent pathway of pyroptosis. I found that Gbpchr3-/- cells had reduced cell death in response to the vacuolar pathogen, L. pneumophila, and various LPS ligands. Using YFP-Gal3 as a marker for damaged membranes, I showed that there were equivalent levels of damaged pathogen vacuoles between WT and Gbpchr3-/- cells suggesting these proteins promoted pyroptosis independently of vacuolar disruption. Instead, it appears that Gbps modulate the activation of caspase-11 following LPS release into the cytosol. </p><p> The recruitment of Gbps is mediated by multiple host proteins including the Immunity Related GTPases and the autophagy conjugation system. I found in the second study that at least one Gbp, Gbp2, was also recruited to damaged vacuoles through the aid of Galectin-3, a β-galactoside binding protein, as well as the autophagy adaptor protein, p62. As all three proteins were also recruited to sterile damaged vesicles created by hypotonic shock, calcium phosphate precipitates, and lysosomal damage, it suggests Gbps are recruited through a universal mechanism which is independent of PAMP recognition. Interactions between p62, Gbp2, and Gal3 present a model whereby p62 facilitates the recruitment of Gbp2 to damaged membranes through interactions with Galectin-3. Their localization to these sites may subsequently facilitate autophagic degradation of membranes or promote the recruitment of pyroptotic complexes to modulate immune functions although this remains to be elucidated. </p><p> This dissertation examines the less characterized roles of Gbps downstream of vacuolar disruption. By uncovering these alternative pathways, this work provides a foundation to study the variations within the Gbp family and allows for the field to further understand the mechanisms by which they promote cellular immune responses.</p> / Dissertation

If cooling towers are poorly maintained there is a risk of microbial growth such as Legionella which in turn might spread via aerosols and infect humans. This may lead to an outbreak of legionnaires’ disease. The purpose of this study was to highlight the risks of Legionella and cooling towers along with investigating the legal responsibility of businesses and supervision authorities in this regard. The study also investigated whether business should be obligated to register their cooling towers at supervising authorities. The study was partly based on a survey which was sent to Sweden’s 290 municipalities and 21 county administration boards to investigate their knowledge regarding Legionella and cooling towers and if they had inventoried which of their facilities that uses cooling towers. The results of the survey showed that 16% of the municipalities and none of the county administration board had inventoried which of their facilities that uses cooling towers. Half of the municipalities do not have any knowledge if any of their facilities uses cooling towers. Moreover, 45% of the municipalities and 30% of the county administration boards consider that business should register their cooling towers. The results showed that many of Sweden’s municipalities and county administration boards have shortcomings in their knowledge and supervision. Legislation and priorities need to be assessed and regulatory guidance from the Public Health Agency of Sweden is necessary for future progress.