Title Mehanizam protulipopolisaharidne imunosti u teških Klebsiella infekcija : doktorski rad Title (english) The mechanism of antilipopolysaccaride immunity in severe Klebsiella infections Author Vanja Vasiljev Mentor Tomislav Rukavina (mentor)Committee member Maja Abram (predsjednik povjerenstva)Committee member Alemka Markotić (član povjerenstva)Committee member Bojan Polić (član povjerenstva)Granter University of Rijeka Defense date and country 2011-02-04, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 616 - Pathology. Clinical medicine Abstract Cilj istraživanja:
Rod Klebsiella predstavlja skupinu od nekoliko iznimno značajnih humanih patogena među kojima najznačajnije mjesto zauzima vrsta K. pneumoniae. K. pneumoniae je čest uzročnik domicilnih a poglavito bolnički stečenih infekcija. Jedan od značajnih problema suvremene medicine predstavlja povećanje otpornosti bakterija, uključujući i K. pneumoniae na antimikrobne lijekove. Stoga je razumljiva i opravdana potreba istraživanja u području alternativnih načina profilakse odnosno liječenja koji su osobito značajni u slučaju teških i po život opasnih infekcija kakve su pneumonija i sepsa. Cilj ovog rada bio je uspostaviti plućnu infekciju s visoko virulentnim sojem K. pneumoniae Caroli, te ispitati stupanj preživljavanja životinja koje su zaštićene protulipopolisaharidnim protutijelima. Kroz eksperimentalni model pneumonije cilj je bio ispitati tijek plućne infekcije te mehanizam djelovanja protulipopolisaharidnih protutijela u zaštićenih životinja na nivou bakterijske diseminacije u periferna tkiva, koncentracije pro- i proupalnih citokina u plućima i plazmi. U sustavnoj infekciji cilj istraživanja je također bio ispitivanje mehanizama djelovanja protulipopolisaharidnih protutijela putem razlike u citokinskom obrascu zaštićenih u odnosu na nezaštićene životinje. Također se ispitala uloga interleukina 17 u sustavnoj infekciji i njegova važnost u protulipopolisaharidnoj imunosti. Cilj ovog rada je i povećati stupanj preživljavanja letalno inficiranih životinja sinergističkom primjenom protulipopolisaharidnih protutijela i antibiotika.
Materijali i metode:
U istraživanju je korišten genetski čisti soj miša BALB/CN. Životinje su intratrahealno i intraperitonealno inokulirane s visoko virulentinim sojem Klebsiella pneumoniae Caroli antigene strukture O1:K2. Zaštićena skupina životinja je profilaktički tretirana protulipopolisaharidnim protutijelima specifičnim za O1 antigen K. pneumoniae (Ru-O1). Praćen je broj bakterija u plućima, jetri i slezeni tijekom plućne infekcije Tkiva su analizirana histološkim i imunohistokemijskim tehnikama. Praćena je koncentracija IL-1β, TNF-α, IFN-γ, IL-6, IL-12 i IL-10 u plućnom tkivu i plazmi intratrahealno inokuliranih miševa, a isti su praćeni i u plazmi sustavno inficiranih životinja.
Uloga IL-17 u sustavnoj infekciji se utvrđivala neutralizacijom štakorskim protumišjim monoklonskim protutijelom. Za određivanje sinergističkog učinka korišten je antibiotik ceftazidim u kombinaciji s Ru-O1 protutijelima.
Rezultati:
Pedesetpostotna letalna doza u eksperimentalnom modelu pneumonije iznosi 30 CFU. Preživljavanje profilaktički tretiranih životinja s Ru-O1 protutijelima nakon letalne plućne infekcije iznosi 33% u odnosu na nezaštićene. Nakon 24 sata dolazi do diseminacije bakterija u krv eksperimentalnih životinja a posljedično i periferne organe. Profilaktički tretirane životinje su u svim ispitivanim terminima imale manji stupanj oštećenja pluća i perifernih organa. Koncentracije citokina u plazmi i plućima zaštićenih životinja pokazale su da 24. sat nakon infekcije iste imaju niže vrijednosti svih ispitivanih proupalnih i upalnih citokina. Kombinacija Ru-O1 protutijela i ceftazidima rezultirala je u povećanju stupnja preživljavanja za dodatnih 30%, a to vrijedi i za sustavnu infekciju. Vrijednosti ispitivanih citokina u plazmi sustavno inficiranih životinja pokazale su da zaštićene životinje imaju niže razine proupalnih citokina 24 sata nakon infekcije. Nasuprot tomu, koncentracija protuupalnog citokina IL-10 bila je viša u zaštićenoj skupini u odnosu na nezaštićenu. Neutralizacijom IL-17 u sustavnoj infekciji pokazano je da IL-17 ima važnu ulogu u početnim fazama sustavne infekcije, te da također da sudjeluje u protulipopolisahardnoj imunosti.
Zaključci:
Pasivna protulipopolisaharidna imunost u plućnoj infekciji pridonjela je boljem ishodu letalno inficiranih životinja. Pred 33%-tnog preživljavanja, uočava se i odgođeno ugibanje letalno inficiranih životinja kao i odgoda diseminacije bakterija u periferna tkiva te smanjena patohistološka oštećenja ispitivanih organa. Pod utjecajem Ru-O1 protutijela dolazi i do modulacije citokinskog odgovora i u sustavnoj i plućnoj Klebsiella infekciji. Stupanj preživljavanja životinja se povećava kombinacijom Ru-O1 protutijela i ceftazidima. Uloga IL-17 u sustavnoj infekciji je izuzetno važna u početnim fazama infekcije kao i u imunosti posredovanoj protu-LPS protutijelima.
Abstract (english) Objective: Bacteria of the genus Klebsiella are well known bacterial pathogens that cause a wide variety of community and hospital acquired infections. Among the genus Klebsiella, K. pneumoniae is the most common bacteria encountered by the physicians worldwide. The high mortality rate of severe infections caused by K. pneumoniae as well as the emerging resistance to antibiotics has centered around the development of different immunologic means for the prevention and/or therapy of these infections. The aim of this study was to establish lung infection by the intratracheal inoculation model with K. pneumoniae Caroli. Throughout the lung model of infection the efficacy of antilipopolysaccharide antibodies (Ru-O1) was determined as well as the dinamic of the bacterial dissemination, cytokine pattern in different time points, patohistological changes in pretreated animals compared with untreated control group. The cytokine pattern was monitored during the lethal systemic infection in pretreated group with Ru-O1 and untreated control group in order to determine the mechanisms of antilipopolysaccharide mediated immunity. The role of interleukin-17 was determined in the systemic Klebsiella infection with and without Ru-O1 antibodies. In order to examine the existence of synergy between Ru-O1 monoclonal antibody and antibiotic the systemic and lung model of infections were used.
Materials and methods:
In all the experiments BALB/CN mice were used. The mice were infected by intratracheal and intraperitoneal route of the infection. The number of bacteria in the lung, liver and spleen was determined at different time points after the infection. The infected organs were analyzed for histopathology by histochemistry and immunohistochemistry methods. The concentrations of the IL-1β, TNF-α, IFN-γ, IL-6, IL-12 and IL-10 were determined in the lung
tissues and plasma of infected mice by EIA. For the neutralization experiments the rat anti mouse IL-17 monoclonal antibody was used. For the examination of sinergistic effect of Ru-O1 antibodies the antibiotic ceftazidime was used.
Results:
The fifty percent lethal dose in experimental lung model was 30 CFU. The survival of Ru-O1 pretreated animals was 33% after lethal lung infection. In protected animals, in all time points, the bacterial dissemination was delayed as well as patohistological findings in examined tissues. Even those animals in pretreated group who died survived a longer period of time in contrast to untreated, control animals. The lung and plasma concentrations of both proinflammatory and antiinflammatory cytokines were lower in the pretreated group 24 hours after the infection. The combination of Ru-O1 antibodies and ceftazidime resulted in 30% higher survival rate in both experimental models. After the systemic infection the concentration of proinflammatory cytokines were higher in the unprotected group compared with protected group of animals. In contrast, the antiinflammatory cytokine IL-10 was higher in the protected animals compared with the unprotected ones. The neutralisation of IL-17 in systemic lethal infection resulted in sudden death of animals in contrast to group pretreated with Ru-O1 antibodies which survived prolonged period of time.
Conclusions:
Passive antilipopolysaccharide immunity in experimental lung infection contributes to the survival of experimental animals, although the survival rate was 33%. Pretreated animals had a delay of bacterial disemination and impaired patohistological changes in contrast to untreated animals. One of the mechanisms of Ru-O1 antibody action was the modulation of the cytokine pattern in pretreated group in contrast to untreated, controls. The survival rate was raised by the combination of the Ru-O1 monoclonal antibodies and ceftazidime in both experimental models. During the systemic Klebsiella infection the role of IL-17 is exceptionally important mainly in the early stages of the infection as well as in the antilipopolysaccaride mediated immunity.
Keywords
Klebsiella pneumoniae
Pneumonija
Sepsa
Lipopolisaharid
Citokini
Protulipopolisaharidna imunost
Ceftazidim
interleukin-17
Keywords (english)
Klebsiella pneumoniae
Pneumonia
Sepsis
Lipopolysaccharide
Cytokine
Antilipopolysaccharide immunity
Ceftazidime
Interleukin-17.
Language croatian URN:NBN urn:nbn:hr:188:239952 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Catalog URL https://libraries.uniri.hr/cgi-bin/unilib.cgi?form=D1121019031 Type of resource Text Extent 131 str; 30 cm File origin Born digital Access conditions Closed access Terms of use Created on 2017-01-19 18:15:50
